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Placenta and Trophoblast - Methods and Protocols (Vol 1) - M. Soares, J. Hunt (Humana, 2006) WW PDF
Placenta and Trophoblast - Methods and Protocols (Vol 1) - M. Soares, J. Hunt (Humana, 2006) WW PDF
Placenta
and Trophoblast
Methods and Protocols
Volume I
Edited by
Michael J. Soares
Joan S. Hunt
Placenta and Trophoblast
M E T H O D S I N M O L E C U L A R M E D I C I N E™
Placenta and
Trophoblast
Methods and Protocols
Volume 1
Edited by
Michael J. Soares
Institute of Maternal–Fetal Biology
Division of Cancer and Developmental Biology
Department of Pathology and Laboratory Medicine
University of Kansas Medical Center, Kansas City, KS
and
Joan S. Hunt
University Distinguished Professor, Vice Chancellor for Research
Department of Anatomy and Cell Biology
University of Kansas Medical Center, Kansas City, KS
© 2006 Humana Press Inc.
999 Riverview Drive, Suite 208
Totowa, New Jersey 07512
www.humanapress.com
All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in
any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise
without written permission from the Publisher. Methods in Molecular MedicineTM is a trademark of The
Humana Press Inc.
All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not
necessarily reflect the views of the publisher.
Cover illustration: Background: Figure 5 from Chapter 11 (Volume 1), “Mouse Trophoblast Stem Cells”
by J. Quinn et al. Foreground: Figure 4 from Chapter 26 (Volume 1), “Vascular Corrosion Casting of the
Uteroplacental and Fetoplacental Vasculature in Mice” by K. J. Whiteley et al.
eISBN 1-59259-983-4
Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1
Library of Congress Cataloging in Publication Data
Placenta and trophoblast: methods and protocols / edited by Michael J. Soares and Joan S. Hunt.
p. ; cm. — (Methods in molecular medicine ; 121-122)
Includes bibliographical references and index.
ISBN 1-58829-404-8 (alk. paper) — ISBN 1-58829-608-3 (alk. paper)
1. Placenta. 2. Molecular biology.
[DNLM: 1. Placenta. 2. Molecular Biology. WQ 212 P6974 2005] I.
Soares, Michael J. II. Hunt, Joan S. III. Series.
QP281.P5435 2005
612.6’3—dc22 2005006428
Preface
The aim of the two-volume set of Placenta and Trophoblast: Methods
and Protocols is to offer contemporary approaches for studying the biology of
the placenta. The chapters contained herein also address critical features of the
female organ within which the embryo is housed, the uterus, and some aspects
of the embryo–fetus itself, particularly those of common experimental animal
models. In keeping with the organization used effectively in other volumes in
this series, each chapter has a brief introduction followed by a list of required
items, protocols, and notes designed to help the reader perform the experi-
ments without difficulty. In both volumes, sources of supplies are given and
illustrations highlight particular techniques as well as expected outcomes. A
key aspect of these volumes is that the contributors are at the forefronts of their
disciplines, thus ensuring the accuracy and usefulness of the chapters.
Placenta research has progressed rapidly over the past several decades
by taking advantage of the technical advances made in other fields. For example,
the reader will note that many techniques, such as reverse transcriptase poly-
merase chain reaction, northern and western blotting, microarray analyses and in
situ hybridization experiments, are routinely used for dissecting a wide range of
experimental questions. Protein analysis and functional experiments on tissues
and cells that comprise the maternal–fetal interface benefit from studies in endo-
crinology, immunology, and developmental biology. These volumes also
present new ideas on investigating gene imprinting and gene transfer via viral
vectors.
In developing these volumes we encountered the problem of how to
organize the contents so as to be reader-friendly. Our decision was to subdi-
vide in large part by the chronology of pregnancy so that in vivo aspects of
implantation come first, followed by in vitro systems of investigation, then
protocols for phenotypic analyses of placentas of several species. Special tech-
niques mentioned above conclude Volume I. Volume II continues with proto-
cols for studying trophoblast invasion, followed by dissection of how invading
trophoblast cells might be received by uterine immune cells. Returning to the pla-
centa itself, methods for researching trophoblast endocrine and transport functions
are followed by a final series of chapters on how placentas adapt to disease. In this
latter group, two chapters offer help to investigators interested in animal models of
human placental disorders and two address working with the oxygen switches
that program gene expression in early pregnancy, a concept entirely unexplored
v
vi Preface
less than a decade ago. The reader is referred to the Introductions in each of the
two volumes for a more detailed description of the contents.
This project would not have been possible without the contributions of
many individuals. We wish to express our gratitude to the contributing authors
for their time, effort, creativity, and their willingness to share their knowledge
and expertise. Our deep appreciation and gratefulness also goes to Stacy
McClure for her dedicated efforts in maintaining the organization of the manu-
scripts and the correspondence between the editors and the authors. During
this process the publisher has provided us with helpful guidance and instruc-
tion essential for the completion of this effort.
Finally, we hope that these volumes are useful and provide a valuable
resource for both trainees and established scientists striving to advance our
understanding of this unique, entirely essential organ of reproduction.
Michael J. Soares
Joan S. Hunt
Contents
Preface .............................................................................................................. v
Contributors .....................................................................................................xi
Companion Table of Contents for Volume II .................................................. xv
Companion CD-ROM .................................................................................... xix
PART I. INTRODUCTION
1 Placenta and Trophoblast: Methods and Protocols: Overview I
Michael J. Soares and Joan S. Hunt ...................................................... 3
vii
viii Contents
xi
xii Contributors
YAN GU • Center for Food Safety and Applied Nutrition, Food and Drug
Administration, College Park, MD
STUART HANDWERGER • Division of Endocrinology, Cincinnati Children’s
Hospital Medical Center, Cincinnati, OH
KAZUYOSHI HASHIZUME • Department of Veterinary Medicine, Laboratory of
Veterinary Physiology, Iwate University, Morioka City, Iwate, Japan
JOAN S. HUNT • Department of Anatomy and Cell Biology, University of
Kansas Medical Center, Kansas City, KS
BERTHOLD HUPPERTZ • Department of Anatomy II, University Hospital RWTH
Aachen, Aachen, Germany
BETTINA HUSEN • Department of Reproductive Biology, German Primate
Centre, Göttingen, Germany
BING JIANG • Department of Obstetrics & Gynecology, University of Texas
Southwestern Medical Center, Dallas, TX
HAKHYUN KA • Department of Biological Resources & Technology, Yonsei
University, Wonju, Kangwon-Do, South Korea
MARK KIBSCHULL • Institute of Anatomy, University Hospital Essen, University
of Essen-Duisburg, Essen, Germany
SUE KONG • Division of Biomedical Informatics, Cincinnati Children’s
Hospital Medical Center, Cincinnati, OH
TOSHIHIRO KONNO • Institute of Maternal–Fetal Biology, Division of Cancer
& Developmental Biology, Department of Pathology & Laboratory
Medicine, University of Kansas Medical Center, Kansas City, KS
GRACIELA KRIKUN • Department of Obstetrics and Gynecology,
Yale University, New Haven, CT
TILO KUNATH • Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
University of Toronto, Toronto, Ontario, Canada
RUDOLF LEISER • Department of Veterinary Anatomy, Histology
and Embryology, Justus-Liebig-University Giessen, Giessen, Germany
KAI LIEDER • Institute of Physiological Chemistry, Facility of Veterinary
Medicine, University of Leipzig, Leipzig, Germany and Department of
Reproductive Biology, German Primate Centre, Göttingen, Germany
CHARLES LOCKWOOD • Department of Obstetrics and Gynecology, Yale
University, New Haven, CT
CAROLE R. MENDELSON • Departments of Biochemistry and Obstetrics and
Gynecology, University of Texas Southwestern Medical Center, Dallas,
TX
GIL MOR • Department of Obstetrics and Gynecology, Reproductive Immunology
Unit, Yale University School of Medicine, New Haven, CT
Contributors xiii
PART I. INTRODUCTION
1 Placenta and Trophoblast: Methods and Protocols: Overview II
Michael J. Soares and Joan S. Hunt
Index
Companion CD-ROM
This book is accompanied by a CD-ROM that contains all the color
illustrations.
xix
Overview 3
1
Overview I
1. Introduction
The placenta is a specialized pregnancy-specific structure that develops con-
currently with development of the embryo and fetus. From an evolutionary
perspective, the placenta was the essential factor in permitting viviparity, a
reproductive strategy in which fetal development proceeds within the female
reproductive tract. Viviparous species are able to provide greater protection
from environmental risks and can more precisely control the development of
their progeny while they reside in utero. The placenta is comprised of numer-
ous cell types. Among the cell types are specialized epithelioid cells, called
trophoblast, that possess several important functions enabling viviparous devel-
opment (1,2). Trophoblast cells play key roles in protecting the embryo/fetus
from noxious substances, programming maternal support, and preventing mater-
nal immune rejection while at the same time ensuring appropriate bidirectional
nutrient/waste flow required for growth and maturation of the embryo. Although
placenta functions are highly conserved, species-specific elements of placenta
organization and activity are evident. Consequently, placental research has
benefited and will continue to benefit from a comparative approach. Each spe-
cies presents experimentally valuable attributes that can be exploited to better
understand the biology of the placenta and viviparity.
Research on placentas involves not only work on the placenta itself, but also
on the placenta’s maternal home, the uterus. Such studies include incorpora-
tion of experimental strategies beginning with the preparation of the uterus for
embryo implantation and techniques directed at understanding embryo–uter-
ine interactions. Cells involved in this initial interaction are trophoblast arising
from the blastocyst and those associated with the uterine epithelium (3). Tro-
phoblast cells expand in number and organize in species-specific patterns. In
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
some species, trophoblast cells penetrate into the uterine compartment, estab-
lishing intimate relationships with the maternal vasculature, a process referred
to as hemochorial placentation and most commonly found in primates and
rodents (1,4). This type of placentation is also associated with a unique
specialization of the uterine stromal compartment, which is termed
decidualization. Other species exhibit minimal trophoblast invasion resulting
in a segregation of maternal and trophoblast tissues. This type of placentation
is referred to as epitheliochorial or synepitheliochorial and is seen in domesti-
cated animals, including the pig and ruminants (1). A key feature of the uterine
environment in animals possessing this superficial type of placentation is an
extensive development of uterine glands. These structures provide vital nutri-
tive support for the developing embryo and fetus throughout pregnancy in
domesticated species, and also during early stages of gestation in primates.
Thus, placentation is fundamental to creating the milieu in which the embryo
and fetus develop. The quality of the embryonic and fetal environment has last-
ing effects, influencing postnatal health and disease (5,6).
In this volume, the reader is guided through the major experimental strate-
gies and steps required for placenta research on multiple species, focusing on
modifications in the uterus, implantation, the nature of the trophoblast cell lin-
eage, vascular development and genetic analysis.
References
1. Wooding, F. B. P. and Flint, A. P. F. (1994) Placentation. In: Marshall’s Physiol-
ogy of Reproduction, Fourth Edition, Vol. 3 (Lamming, G. E., Ed.), Chapman &
Hall, London: pp. 233–460.
2. Rossant, J. and Cross, J. C. (2002) Extraembryonic lineages. In: Mouse Develop-
ment (Rossant, J., and Tam, P. P. L., Eds.), Academic, San Diego: pp. 155–190.
3. Paria, B. C., Reese, J., Das, S. K., and Dey, S. K. (2002) Deciphering the cross-
talk of implantation: advances and challenges. Science 296, 2185–2188.
4. Georgiades, P., Ferguson-Smith, A. C., and Burton, G. J. (2002) Comparative
developmental anatomy of the murine and human definitive placentae. Placenta
23, 3–19
5. Bateson, P., Barker, D., Clutton-Brock, T., et al. (2004) Developmental plasticity
and human health. Nature 430, 419–421.
6. Gluckman, P. D. and Hanson, M. A. (2004) Living with the past: evolution, de-
velopment, and patterns of disease. Science 305, 1733–1736.
II
METHODS FOR STUDYING EMBRYO IMPLANTATION
AND UTERINE BIOLOGY
2
Methodologies to Study Implantation in Mice
Summary
Pregnancy begins with fertilization of the ovulated oocyte by the sperm. After fertilization,
the egg undergoes time-dependent mitotic division while trying to reach the blastocyst stage
and the uterus for implantation. Uterine preparation for implantation is regulated by coordi-
nated secretions and functions of ovarian sex steroids. The first sign of contact between the
blastocyst and the uterus can be detected experimentally by an intravenous blue dye injection
as early as the end of day 4 or the beginning of day 5 of pregnancy. This blastocyst–uterine
attachment reaction leads to stromal decidual reaction only at sites of implantation. The pro-
cess of implantation can be postponed and reinstated experimentally by manipulating ovarian
estrogen secretion. Stromal decidualization can also be induced experimentally in the hormon-
ally prepared uterus in response to stimuli other than the embryo. Fundamental biological ques-
tions surrounding these essential features of early pregnancy can be addressed through the
application of various techniques and manipulation of this period of early pregnancy. This
chapter describes the routine laboratory methodologies to study the events of early pregnancy,
with special emphasis on the implantation process in mice.
Key Words: Mouse; blastocyst; implantation; ovariectomy; vasectomy; delayed implanta-
tion; embryo transfer.
1. Introduction
The mouse, as one of the most common laboratory animals, is widely used
in basic biological research and could provide useful information that is rel-
evant to human biology. This chapter focuses on some of the procedures for
studying events of early pregnancy in mice. Following mating and fertiliza-
tion, the embryo develops to the blastocyst stage. Attachment of the blastocyst
into the uterine wall is an absolute requirement for further growth and collec-
tion of nutrients from the maternal vasculature. Hence, the implantation pro-
cess is a critical event in the embryo’s life and a central step to the establishment
of placentation and pregnancy.
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
two horns (duplex). After mating, sperm travel through both uterine horns to
reach the site of fertilization. They penetrate the cumulus cells to fertilize eggs.
Usually, more than one sperm enters the perivitelline space. However, only
one sperm penetrates and fertilizes the egg. After fertilization, the zygote
divides mitotically to eventually reach the blastocyst stage. After mating, the
mating stimulus triggers prolactin release from the pituitary, which leads to
the formation of a functional corpus luteum in the ovary, blocking further
ovulation and cyclicity to continue pregnancy.
1.4. Experimental Delay in Implantation
In many animals, implantation is delayed for an extended period, during
which the blastocyst remains in a quiescent state called embryonic diapause
(3,4). Delayed implantation in these animals seems to be a strategic plan for
regulating the time of birth coincident with favorable environmental condi-
tions. In some species, delayed implantation occurs under specific conditions.
Mice show postpartum estrus immediately after parturition. If conception takes
place immediately after parturition, embryos develop into blastocysts, but
remain in a dormant state until the lactational stimuli from suckling pups are
removed. In mice, implantation can be experimentally delayed by removing
the ovarian source of steroids (5). The timing of normal blastocyst implanta-
tion is tightly controlled in mice. Normally, initiation of implantation occurs at
night (2200–2300 h) of day 4 (6). Ovarian steroid hormones are necessary to
prepare the endometrium for the process of implantation. In mice, both ovarian
progesterone and estrogen are required for implantation. The ovary secretes a
small amount of estrogen in addition to progesterone in the morning of day 4
of pregnancy in mice. This preimplantation estrogen secretion is an absolute
requirement for blastocyst activation, preparation of the uterus, and initiation
of the implantation process. Surgical removal of both ovaries in mice before
the preimplantation ovarian estrogen secretion occurs on day 4 leads to delayed
implantation.
1.5. Artificial Decidualization
During normal pregnancy uterine stromal cells first proliferate and then dif-
ferentiate to decidual cells in response to an implanting blastocyst (4). This
process is known as decidualization. Decidualization starts following initia-
tion of blastocyst implantation in mice. The decidua enlarges as the embryo
grows. Decidual cells are characterized by the presence of polyploid nuclei,
and glycogen and lipid in their cytoplasm. As the deciduum grows, it occupies
the uterine lumen at the mesometrial side (dorsal to the embryo). The
antimesometrial decidua is divided into two zones. A thin and dense cellular
zone that immediately surrounds the blastocyst is known as the primary decidual
2. Materials
2.1. Monitoring the Estrous Cycle
2.1.1. Collection of Vaginal Smears
1. Sexually mature female mice (45–50 d old).
2. Clean mouse cage with a wire-top cage cover.
3. Plastic dropper (Fisher Scientific, Hanover Park, IL, cat. no. 13-711-10).
4. Saline (0.9% Sodium chloride solution, Baxter Healthcare Corporation,
Deerfield, IL, cat. no. 281324).
5. Glass slides (Fisher Scientific, cat. no. 12-518-104).
2.9. Euthanasia
2.9.1. Cervical Dislocation
1. Mice.
2. Clean cage with a cage top.
2.9.2 Inhalants
2.9.2.1. CARBON DIOXIDE
1. Carbon dioxide cylinder (local gas supplier).
2. A cage specifically designed for killing mice.
2.9.2.2. ISOFLURANE
1. Isoflurane (Minrad, Inc.).
2. Cotton wool (Absorbent Cotton Co., Inc., Valley Park, MO) or gauze (Kendall
Healthcare Products Co., Mansfield, MA).
3. Bell jar or a scew cap glass container (Fisher Scientific).
3. Methods
3.1. Monitoring the Mouse Estrous Cycle
3.1.1. Collection of Vaginal Smears
1. Grasp the tail of a mouse with the thumb and forefinger of one hand.
2. Place the mouse on the top of the cage cover (wire top). As the mouse attempts to
move forward, quickly grasp the loose skin at the back of the neck using the
thumb and forefinger of the other hand. The head of the mouse will be immobi-
lized, if the skin is held properly.
3. Lift the mouse in your hand and secure the tail between the small finger and the
palm of the same hand.
4. Keep the face of the mouse up and locate the vagina.
5. Fill a plastic dropper with a small amount of saline (0.05 to 0.1 mL 0.9% NaCl)
and insert the tip superficially, but not deeply, into the vagina.
6. Gently squeeze the bulb of the dropper to release saline inside the vagina. Slowly
release the pressure on the bulb to aspirate the vaginal lavage inside the dropper.
3.1.2. Identification of the Stage of the Estrous Cycle
1. Place a drop of aspirated vaginal fluid on a glass slide.
2. Unstained material is observed under a light microscope with 10× and 40× objec-
tive lenses to identify the stage of the estrous cycle.
3. The following criteria are used to identify the specific stage of the cycle:
a. The estrus stage vaginal smear contains anucleated cornified cells (irregular
shaped cells).
b. The metaestrus stage vaginal smear is composed of a mixture of cornified
epithelial cells and leukocytes.
c. At diestrus, the smear contains predominantly leukocytes.
d. At proestrus, the smear shows a predominance of nucleated epithelial cells.
2. If not bred, the same females should be used for breeding 3–4 d in a row because
the pairing with males helps to synchronize the cycle in females. Males copulate
with females at proestrus at around the midpoint of the dark cycle.
3. After a successful mating, the male should be given a rest of 2–3 d.
Table 1
Dating of Early Pregnancy Depending on the Developmental Stage of Preim-
plantation Embryos*
Day of pregnancy Developmental stage of embryos Reproductive tract
*The time of embryonic development may be slow or fast, depending on the strain of mice
and light-dark cycle of an animal facility.
Fig. 1. Schematic representation to show excision of the mouse oviduct. The method
of excision of mouse oviduct is described under Subheading 3.3.2.2.
Fig. 2. Schematic representation to show flushing of the mouse oviduct. The method
of flushing oviducts is described under Subheading 3.3.2.2. The oviduct is flushed to
recover preimplantation embryos.
3. Cut the other end of the horn just below the utero–tubal junction and keep the
uterine horn in a clean moistened tissue paper to absorb blood.
4. Hold one of the uterine horns at the utero–tubal junction and insert the tip of a 27-
gauge needle, with a 3-mL plastic syringe filled with Whitten’s medium attached,
inside the uterine lumen (Fig. 4).
5. Holding the needle and the uterine horn together, push the plunger of the syringe
to flush the uterine luminal contents into a Petri dish. It is important to flush
gently (Fig. 4).
6. Repeat this procedure to excise and flush the other uterine horn.
7. Check uterine flushings under a stereomicroscope for the presence of appropriate
stages of developing embryos.
Fig. 6 (see companion CD for color version). Implantation sites in uterine horns on
day 5 of pregnancy as detected by intravenous Chicago blue B dye injection. The
method of intravenous Chicago Blue B dye injection into tail vein is described under
Subheading 3.3.2.4.
9. Close up the skin with two to three auto clips. The mouse should be wrapped in a
tissue to keep it warm (loss of body heat is common in abdominal surgery) or,
alternatively, placed on a heating pad, and allowed to recover. Animals that are
placed under anesthetic should always be supervised and monitored until fully
awake.
10. Following the operation, the mice are allowed to recover for 2 wk before being
test-bred to confirm their sterility. One or two female mice are placed with the
vasectomized male and are checked for plugs the following morning. Plug-posi-
tive females are sacrificed on day 2, and their oviducts are flushed with saline.
The eggs should be at unfertilized, one-cell stage. The presence of two-cell
embryos would indicate incomplete vasectomy.
8. Attach a serrefine clip to the fat pad (do not to clip the ovary). In the absence of
the ovary in the hormone-treated ovariectomized mice, clip the fat and mesentery
near the oviduct. Try not to touch the uterine horns during this procedure.
9. Locate the tip and gently hold the uterine horn with a pair of forceps approx 1 cm
below the utero–tubal junction (Fig. 7).
10. Five to seven blastocysts must be transferred to each horn. Take up a minute
amount of embryo culture medium (Whitten’s media) in the very tip of the trans-
fer pipet by moving the plunger cap counterclockwise. Next, make a small bubble
by taking up a little air. Then take up some more medium—roughly the same
volume as you hope to transfer the blastocysts in. Take up another bubble, the
same size as before. Then take up blastocysts in the smallest possible volume of
medium, lining them up side by side in the transfer pipet (see Note 8).
11. Once the pipet is loaded and the uterine horn positioned, gently grasp the top of
the uterine horn inside a pair of forceps.
12. While still holding the horn with one hand, use the other hand to gently insert a
26-gauge hypodermic needle through the uterine wall (close to the oviduct) and
into the lumen (Fig. 8). Choose an area of the horn that is relatively devoid of
blood vessels because blood will clot in the tip of the pipet and block it. Remove
Fig. 8 (see companion CD for color version). Insertion of a transfer pipette into the
uterine horn for embryo transfer. The method of blastocyst transfer inside uterine lu-
men is described under Subheading 3.6.4.
the needle and carefully (so as not to lose the site of the hole), without averting
your eyes, pick up the loaded transfer pipet. Gently insert the transfer pipet tip
about 3 mm into the uterine lumen (Fig. 8). Gently release the blastocysts into
the uterus by turning the plunger cap clockwise. Be careful not to allow any air
into the uterus.
13. Once the transfer is complete, quickly rinse the transfer pipet in some Whitten’s
medium and check to see if there were any blastocysts stuck in the transfer pipet.
If there were, transfer these blastocysts again.
14. With the transfer complete, the serrefine clip can now be removed and the uterine
horn gently eased back into the body. Do not touch the uterus, but ease it back by
lifting the edges of the incision in the body wall and allowing the horn to fall
back in, without actually handling it.
15. This procedure is then repeated on the other uterine horn.
16. The incision in the body wall is not sutured. The skin is closed with Michelle
clips—two per incision is usually sufficient. Once surgery is complete, the mouse
is placed in a clean cage and warmed to facilitate recovery (see Note 9).
3.9. Euthanasia
Cervical dislocation is the most common method of killing mice. However,
mice can be killed using inhalants.
3.9.2. Inhalants
3.9.2.1. CARBON DIOXIDE
Carbon dioxide inhalation is the most efficient and acceptable method of
euthanasia.
1. A mouse cage with a solid lid is connected to a carbon dioxide gas cylinder.
2. Place the mice in the cage and cover the cage with a lid.
3. Open the carbon dioxide cylinder and fill the cage with gas.
4. The animals will die within 1–2 min.
3.9.2.2. ISOFLURANE
Isoflurane overdose can also be used to kill mice.
1. A cotton wool or gauze soaked with isoflurane is placed inside a bell jar or a
screw-cap glass container.
2. Place the mouse inside the jar. The mice will die within 1 min (see Note 12).
4. Notes
1. The process of detecting copulatory plugs should be performed gently because
stimulation of the vagina may induce pseudopregnancy.
2. Pregnant mice usually do not breed when placed with male mice. There is no
other visual or noninvasive method for definitive identification of early preg-
nancy.
3. Abdominal distention is apparent in most mice by day 8 or later depending on the
litter size and degree of swelling of the implantation sites.
4. This is a relatively simple procedure but requires practice. If the needle is inside
the vein, injection will be smooth. If the syringe plunger does not move smoothly
and resistance is felt while injecting or swelling around the injection site occurs,
withdraw the needle and try again slightly above the first injection site (proximal
to the body). It is always advisable to start injecting from the tip of the tail. After
several attempts, it is advisable to change the needle because the tip becomes
blunt.
5. The purpose of describing delayed implantation is that this model provides a
powerful tool to examine steroid hormone regulation of uterine and embryonic
changes with respect to embryo–uterine interactions during implantation.
6. The pseudopregnant female will display the hormonal profile of a normal preg-
nant female for several days after mating. The hormonal milieu of pseudopreg-
nancy begins to differ from pregnancy after 7 to 8 d as a result of the absence of
a developing embryo inside the uterus.
7. Injection of too much oil inside the lumen may migrate to the other uterine horn
and cause decidualization. Prominent swelling of the uterus will indicate the
extent of stromal cell decidualization in response to the artificial stimulus.
Swelling of the uterus due to decidualization will be visible 48 h after the oil
injection. The intraluminal oil injection to a pseudopregnant mouse uterus on day
4 afternoons (1300–1400 h) will yield the best results. If ovariectomized mice are
used, animals should be exposed to progesterone (SC injection) for at least 48 h
(daily injection of 2 mg progesterone per 0.1 mL sesame seed oil per day for 2 d)
before the injection of oil inside the uterine lumen. These animals must also be
maintained with daily progesterone injection after the induction of decidualization.
8. Loading blastocysts into the transfer pipet will take some practice. If it is likely
to take more than a few minutes to load the transfer pipet, then do not expose the
uterine horn until the pipet has been loaded. This prevents drying out and further
trauma to the uterine horn. Alternatively, the uterine horn, ovary, and so on may
be moistened repeatedly with a sterile cotton bud and saline.
9. Animal welfare guidelines recommend that all vertebrates undergoing procedures
that might cause more than momentary pain or distress be treated with analge-
sics, unless it can be scientifically justified that the treatment will interfere with
the experimental procedure. Analgesics should be given immediately after the
surgery. A simple skin incision may only require 24 h of analgesic treatment.
Rodents subjected to abdominal surgery or similar procedures normally require
analgesic for the first 12 h. It is not appropriate to wait until signs of pain or
distress are demonstrated before administering analgesics. In rodents, the signs
of pain following surgery are manifested as decreases in food and water con-
sumption. Consult your veterinarian and animal care committee for specific post-
operative care in your institute, because these procedures are based on
institutional rules and regulations.
10. The Avertin stock solution is light-sensitive and hydroscopic. Keep Avertin in a
dark bottle at room temperature. The Avertin stock solution is quite stable at
room temperature
11. It will take about 3–5 min for the mouse to become fully anesthetized (lack of toe-
pinch reflex). An additional 0.1–0.2 mL can be administered if required. The mouse
will remain anesthetized for approx 15–20 min and recover within 30–60 min.
Keep the mouse warm during recovery. The effective dosage is dependent upon
the weight of the mouse.
12. Isoflurane should be used in a fume hood to minimize the risk of exposure to the
gas by the operator.
Acknowledgments
We gratefully acknowledge Dr. S. K. Dey for helpful discussions and expert
advice. This work was supported by National Institutes of Health (NIH) grant
HD 42636, HD 40193, HD 44741 and HD 37394 to B.C.P.
References
1. Allen, E. (1922) The estrous cycle in the mouse. Am. J. Anat. 30, 297–371.
2. Snell, G. D., Fekete, E., Hummel, K. P., and Law, L. W. (1940) The relation of
mating, ovulation and estrous smear in the house mouse to time of day. Anat. Rec.
76, 39–54.
3. Carson, D. D., Bagchi, I., Dey, S. K., et al. (2000) Embryo implantation. Dev.
Biol. 223, 217–237.
4. Dey, S. K. (1996) Implantation. In: Reproductive Endocrinology, Surgery and
Technology (Adashi, E. Y., Rock, J. A., and Rosenwaks, Z, Eds.), Lippincott-
Raven, New York: pp. 421–434.
5. Yoshinaga, K. and Adams, C. E. (1966) Delayed implantation in spayed, proges-
terone treated adult mouse. J. Reprod. Fert. 12, 593–595.
6. Das, S. K., Wang, X. N., Paria, B. C., et al. (1994) Heparin-binding EGF like
growth factor gene is induced in the mouse uterus temporally by blastocysts solely
at the site of its apposition: a possible ligand for interaction with blastocyst EGF-
receptor in implantation. Development 120, 1071–1083.
7. Psychoyos, A. (1973) Endocrine control of egg implantation. In: Handbook of
Physiology, Williams and Wilkins, Baltimore, MD: pp.187–215.
8. Paria, B. C., Huet-Hudson, Y. M., and Dey, S. K. (1993) Blastocyst’s state of
activity determines the “window” of implantation in the mouse receptive uterus.
Proc. Natl. Acad. Sci. USA 90, 10,159–10,162.
9. Paria, B. C., Tan, J., Lubahn, D. B., Dey, S. K., and Das, S. K. (1999) Uterine
decidual response occurs in estrogen receptor-α-deficient mice. Endocrinology
140, 2704–2710.
10. Wordinger, R. J., Jackson, F. L., and Morrill, A. (1986) Implantation, deciduoma
formation and live births in mast cell-deficient mice (W/Wv). J. Reprod. Fertil.
77, 471–476.
3
Blastocyst Culture
D. Randall Armant
Summary
Experimental models of blastocyst development based on in vitro culture have played a
prominent role in advancing our understanding of peri-implantation development, a process
that is relatively inaccessible in vivo. Blastocyst culture provides a robust approach for exam-
ining embryonic interactions with the microenvironment under highly controlled conditions.
Major events that occur in utero can be followed in vitro, including blastocyst expansion, hatch-
ing, and adhesion to extracellular matrices. This chapter will describe a method for obtaining
and culturing mouse blastocysts. Morphological changes that occur during blastocyst culture
will be discussed and related to the corresponding development in utero. Finally, quantita-
tive assays will be detailed for monitoring peri-implanatation development of the trophoblast
in vitro.
Key Words: Blastocyst; mouse; trophoblast; embryo culture; hatching; implantation; attach-
ment reaction; adhesion; migration; fibronectin binding assay; outgrowth; microspheres; image
analysis; morphometry; extracellular matrix; fibronectin; Matrigel; collagen gel.
1. Introduction
Preimplantation development in mice takes place between embryonic days
(E) 0.0 and E 3.5, resulting in the formation of a blastocyst. As with all pre-
implantation stages, the blastocyst can complete development outside of the
female reproductive tract under the direction of an endogenous developmen-
tal program. The early stages of peri-implantation development are recapitu-
lated in vitro, including blastocyst expansion, cell proliferation, and hatching
from the zona pellucida. After hatching, interactions with the uterus that occur
in vivo from E 4.0 to E 6.5 can be simulated by following the attachment of the
blastocyst to the culture plate and, if an appropriate extracellular matrix (ECM)
is provided, monitoring the migratory or invasive activity of trophoblast cells.
The maternal milieu plays a central role in augmenting blastocyst develop-
ment. Blastocyst culture provides an experimental system for investigating
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
35
Fig. 1. Formation of the mouse blastocyst. At the eight-cell stage, the embryo com-
pacts to produce a morula (A). After the next cell division, the embryo begins to pro-
duce a cavity or blastocoel and forms a blastocyst by the 32-cell stage (B). Two
populations of cells emerge during blastocyst formation. The inside cells of the morula
(white) become the inner cell mass (ICM), a group of pluripotent stem cells eccentri-
cally positioned within the blastocyst. Cells in the outer layer of the morula (gray)
form the first epithelium, the trophectoderm, which differentiates into the trophoblast
lineages of the placenta. The blastocyst expands by accumulating additional fluid
within the blastocoel and hatches free of the zona pellucida (C). In the mouse, polar
trophectoderm cells contacting the ICM proliferate, while the remaining mural tro-
phectoderm cells differentiate into trophoblast giant cells.
chapter are met. Blastocyst differentiation in vitro from gestation day (GD) 4
to GD 7 (see Note 1) is slow, as assessed by activities that are believed to
correspond to developmental events occurring in utero (Table 1). However,
the introduction of growth factors normally provided by the uterus can acceler-
ate in vitro development significantly (8).
1.2.1. Expansion and Hatching
Developing blastocysts accumulate fluid, expanding their circumference as
the blastocoel volume increases (Fig. 1C). Expansion of the blastocoel can be
monitored by morphometric measurement and proceeds linearly from GD 4 to
Table 1
Comparison of Blastocyst Developmenta
Time in vitro
Developmental events (in vitro counterpart) Time in utero Morning of:
that of blastocysts collected from the uterus on gestation day (GD) 4 at 0900 h and cultured in
serum-free medium. In vitro, blastocysts attach nonspecifically to plates on GD 6. They adhere
specifically to surfaces or microspheres coated with extracellular matrix (ECM) proteins on GD
7. Invasive activity is assessed from GD 7 onward by monitoring trophoblast outgrowth on ECM.
bDiscussed in this chapter as fibronectin-binding activity
Fig. 2. Differentiation of the blastocyst in vitro. The blastocyst expands and hatches
by squeezing through a tear in the zona pellucida (A). Around the time that the tropho-
blast becomes adhesion competent, the blastocyst collapses (B). An empty zona to the
left clearly shows the opening formed during hatching. The earliest sign of trophoblast
outgrowth is the appearance of spreading cells near the base of the embryo (arrow-
heads; C). During the next 24 to 72 h, the field of migrating trophoblast cells sur-
rounding the embryo increases in area (D–F). The clump of cells at the center of the
outgrowth consists of remnants of the ICM and undifferentiated trophoblast cells.
cells must become adhesive at their apical surfaces, dissociate cell–cell junc-
tions, spread, and begin to migrate. The time between acquisition of adhesion
competence and our ability to detect migrating trophoblast cells posed a seri-
ous technical limitation for experimentally assessing blastocyst development.
We were able to monitor early signs of adhesive activity on the apical surface
of trophoblast cells by decorating intact blastocysts with fluorescent
microspheres coated with adhesive ECM proteins (18) (Fig. 3). Coating
microspheres with a proteolytic fragment of fibronectin containing the central
cell adhesion-promoting domain (FN-110), we demonstrated that binding was
specific and dependent on appropriate integrins. The onset of fibronectin-
binding activity correlated with trophoblast outgrowth on plates coated with
fibronectin, demonstrating a physiological relationship between the two experi-
mental approaches.
The fibronectin-binding assay, quantified using fluorescence microscopy
and image analysis, has proven highly useful in experimental designs to test
agents that delay (31) or accelerate (8) the rate of blastocyst development.
Using this approach, we found that adhesion to fibronectin at the apical sur-
face of the trophoblast is upregulated after an initial exposure to fibronectin for
1–3 h (18) (Fig. 3). Furthermore, fibronectin was shown to ligate integrins
localized at the apical surface of the trophectoderm, activating intracellular
1 mg/mL in PBS and stored frozen in small aliquots that are slowly thawed on ice
before dilution to the appropriate final concentration.
15. Peptides for coating plates: FN-110 (08-103, Upstate Biotech, Lake Placid, NY),
a 110 kDa proteolytic fragment of fibronectin with adhesive activity, and
GRGDSP (03-34-0035, Calbiochem), a synthetic peptide containing the critical
sequence that mediates adhesion in the cell binding domain of fibronectin. Both
peptides are prepared and stored as in item 14.
16. Inverted microscope with fluorescence detection system (DM-IRB, Leica,
Wetzlar, Germany), interfaced through a B/W CCD digital camera (Orca,
Hamamatsu Photonics K.K., Hamamatsu City, Japan) with computer-based image
analysis software (Simple PCI, Compix Inc., Canberry Township, PA), or a com-
parable system from other manufacturers.
17. Fluorescent polystyrene microspheres (Bangs Laboratories, Fishers, IN) approx
1 µm in diameter (see Note 7).
18. Heparitinase (from Flavobacterium heparinum; E.C. 4.2.2.8; 100704-1, Seikagaku
America, East Falmouth, MA) prepared at 0.1 U/mL in Hank’s balanced salt solu-
tion (Invitrogen).
3. Methods
3.1. Blastocyst Production and Culture
3.1.1. Superovulation and Mating
1. Female mice are superovulated by injection of 5 IU PMSG, followed 44–48 h
later with 5 IU of hCG. Injections of 0.1 mL are given intraperitoneally between
1200 and 1600 h (see Note 8).
2. Immediately after the hCG injection, each female is paired for mating overnight
with a stud male.
3. The females are checked the next morning for a vaginal plug (solidified semen at
the entrance to the vagina) by lifting the tail and probing with a small spatula or
blunt rod. Plugs must be checked early, as they may fall out by the late morning.
4. The pregnant female mice are then separated from males and may be housed in
groups until the desired stage of pregnancy is reached. Nonpregnant animals may
be re-used for superovulation and mating after resting them for 2–3 wk.
5. Embryos can also be obtained without superovulation by monitoring the estrus
cycle of each female to determine the appropriate time for mating (35).
2. Pregnant mice are euthanized by cervical dislocation, the body is doused with
70% ethanol, and a small ventral incision is made with surgical scissors in the
abdominal skin. The skin is then grasped on either side of the incision and pulled
apart to expose the underlying body wall. The peritoneal cavity is opened using
fine scissors to make a lateral incision through the ventral abdominal wall at a
level approximating the top of the rear legs. After pushing the intestines upward
to expose the reproductive tract, grasp the junction between the oviduct and the
left uterine horn with fine forceps. Free as much fat and membrane from the
uterine horn as possible.
3. Cut the uterotubal junction on the side of the forceps towards the oviduct. A
second cut just above the junction of the two uterine horns will free the left horn.
Place the uterine horn into M2 medium and repeat the procedure with the right
uterine horn.
4. After collection, each uterine horn is flushed with 1 mL of M2 medium using a
syringe needle inserted into the lumen of the horn at the uterotubal junction. The
junction wall is held tightly to the needle with forceps as medium is gently released
from the syringe into the lumen, exiting at the opposite end into an empty Petri
dish. Collection of embryos is made easier by preventing the medium from reach-
ing the wall of the Petri dish.
5. Using a stereomicroscope, the medium is scanned for blastocysts, which are col-
lected by mouth-operated micropipet (see Note 11).
6. Embryos are transferred to microdrop cultures and separated from debris or
contaminating epithelial cells by transfer through at least three more drops of
medium. They can then be transferred to new microdrop cultures or harvested
for other purposes.
bile on the culture plate surface. A fibronectin binding assay can be used to
directly measure the adhesion competence of trophoblast cells, demonstrating
adhesive activity on the apical surface prior to the onset of outgrowth (18).
Blastocyst outgrowth on ECM is representative of the period of trophoblast
invasion and may be quantified either by determining the percentage of blasto-
cysts with migrating trophoblast cells, or by measuring the area occupied by
trophoblast cells as they migrate outward. Later, we provide protocols for assess-
ing these measures of blastocyst differentiation during development in vitro.
Normally, blastocysts are cultured on BSA-coated surfaces until GD 5,
allowing them to hatch from the zona pellucida and approach the attachment
competent stage. Most embryos will hatch independently, but those that fail
should be freed of the zona by repeatedly drawing the blastocyst in and out of
a micropipet drawn to a diameter slightly smaller than that of the embryo.
Hatched or dezonaed blastocysts are best cultured singly in small microdrops
to avoid their aggregation. It is convenient to arrange the microdrops in a cir-
cular pattern on the Petri dish for easy scanning when assessing their progress.
Once embryos are added to culture medium, minimize their exposure to ambi-
ent room conditions by rapidly returning them to the incubator after each
observation.
3.2.1. Blastocyst Attachment
Attachment is determined by swirling or tapping the culture plate while
observing the embryo through a stereomicroscope. Unattached blastocysts
will move relative to debris or marks in the plastic, whereas attached embryos
remain firmly in place. Removing the zona pellucida prematurely does not alter
the timing of blastocyst attachment during culture (24). However, delayed hatch-
ing will obscure detection of attachment and subsequent outgrowth. Therefore,
it is expedient to mechanically remove zonae from any blastocysts that have
not hatched by the end of GD 5.
Whereas attachment is transient in medium containing only BSA, it is con-
tinuous with trophoblast adhesion and outgrowth on surfaces coated with ECM
components or in serum-supplemented medium. The time between the onset of
attachment and the beginning of outgrowth can be as long as 12–24 h, although
supplementation with serum or growth factors can shorten it to less than 1 h.
3.2.2. Trophoblast Outgrowth
To form a blastocyst outgrowth (Fig. 2), trophoblast cells must adhere to an
ECM that can be provided by supplementing the medium with serum, which
contains fibronectin and vitronectin. Alternatively, blastocysts will outgrow in
a defined serum-free medium when cultured either on polystyrene surfaces
coated with individual ECM proteins or on gels composed of purified collagen
or a complex basement membrane (Matrigel).
3. After the progression of outgrowth area expansion has been established in a time
study, subsequent experiments may be conducted by choosing one time point
that is within the linear range of increasing outgrowth area (see Note 12).
3.2.4. Measurement of Fibronectin-Binding Activity
Although the binding assay described here is intended for measuring cell
adhesion to fibronectin, a similar approach can be adapted for other proteins
that mediate trophoblast adhesion. For large proteins, it is best whenever pos-
sible to coat polystyrene microspheres with a small active fragment of the pro-
tein to increase ligand density. We have coated fluorescent microspheres with
proteolytic fragments containing the cell binding domains of fibronectin,
laminin, and entactin to assess the adhesive activity of developing blastocysts.
The fibronectin-binding assay is performed in three steps.
1. It is necessary to remove charged heparan sulfate from the surface of the tropho-
blast to prevent nonspecific binding of microspheres through electrostatic inter-
actions.
2. Fibronectin-binding activity at the apical surface of the blastocyst must be
upregulated through exposure to soluble or immobilized fibronectin for reasons
discussed under Subheading 1.2.3.
3. Blastocysts are incubated with fibronectin-coated microspheres and high affinity
binding is assessed.
Procedures for each of these steps will be detailed, along with protocols for
preparing fibronectin-coated microspheres and quantifying microsphere bind-
ing to blastocysts.
3.2.4.1.PREPARATION OF MICROSPHERES
1. Fluorescent microspheres (1.0-µm diameter) are supplied as 2.5% solutions and
must be washed to remove surfactants.
2. Centrifuge 200 µL of the microspheres suspension in a 0.6-mL tube at 10,000g
for 1 min.
3. Remove the supernatant, add 200 µL sterile PBS and vortex to resuspend.
4. Repeat steps 2 and 3 twice.
5. Centrifuge the suspension at 10,000g for 1 min.
6. Remove the supernatant and add 200 µL sterile PBS containing 144 µg/mL FN-110.
7. Agitate the suspension at room temperature for 24 h on a vortex mixer set to its
lowest speed.
8. Repeat steps 2 and 3 three times.
9. Remove the supernatant and add 200 µL sterile PBS containing 1 mg/mL α1-acid
glycoprotein as a blocking step (see Note 13).
10. Repeat step 7.
11. Repeat steps 2 and 3 three times.
12. Remove the supernatant and add 200 µL PBS containing 10 mg/mL BSA (PBS/
BSA).
incubator for observation too frequently, which could compromise their viabil-
ity and alter development.
The rates of blastocyst expansion, hatching, attachment, and outgrowth are
all determined by repetitive observation as embryos develop in culture, scoring
the percentage of embryos that have achieved the respective endpoint. Choose
a specific time during development to initiate the experiment, designated as
0 h. Examination of the embryos three to five times daily will usually pro-
vide robust data. A total N of 30 to 50 embryos in each experimental group
usually provides sufficient power for statistical analysis. It is preferable to obtain
overall percentages by pooling data from all experiments rather than to calculate
percentages for each individual experiment and then average the percentages.
Probit analysis, available in the SPSS statistical software package (SPSS Inc.,
Chicago, IL), is used to calculate a T50 (the time when half of the embryos are
scored positively) for each experimental group and generate confidence inter-
vals (CIs) to indicate whether there are statistically significant differences
between groups. The T50 is a measure of developmental rate, where faster
progression results in a lower T50 value. If their 95% CIs do not overlap, T50s
are statistically different (p < 0.05).
Outgrowth area and fibronectin-binding activity are measured once or twice
daily. Values are pooled from all experiments and the data for each experimen-
tal group is subjected to analysis of variance. A total N of 15 to 30 embryos
in each experimental group usually provides sufficient power for statistical
analysis.
4. Notes
1. Fertilization and commencement of embryonic development occur within a few
hours of ovulation, around midnight after pairing mice for mating. Therefore, in
vivo development is estimated to begin at midnight, which is designated E 0.0.
Zygotes can be collected from the oviduct the following morning at E 0.5,
whereas blastocysts are collected from the uterus 3 d later at E 3.5. A different
system is used to track in vitro development after embryos are removed from the
reproductive tract. The day after mating is designated GD 1. For example, if
blastocysts are collected from pregnant dams on E 3.5, development in vitro from
that time forth begins with GD 4. The distinction between developmental time in
vivo and in vitro is important because the rate is highly variable in vitro, depend-
ing on culture conditions, and does not necessarily correspond to the stage attained
at that time in vivo (Table 1).
2. Other mouse strains may be used. Males of F1 strains, such as B6SJLF1/J, are
particularly good studs that successfully mate at rates in excess of 80%.
3. Other mouse strains may be used. Outbred female mice are easily induced to
superovulate. Hogan et al. (35) provide a list of inbred and F1 strains that are
high and low ovulators.
4. We use Ham’s F-10 medium for blastocyst culture, but many other medium for-
mulations work well. The principal requirements for complete blastocyst differ-
entiation in culture are the inclusion of amino acids (23,25,40). Other complex
media (e.g., CMRL 1066, Dulbecco’s modified Eagle’s medium [DMEM], RPMI
1620) are satisfactory. We have no experience in the production of trophoblast
outgrowths with the newer media developed specifically for preimplantation blas-
tocyst culture. Those supplemented with amino acids, such as KSOM+AA (11),
are likely to work well.
5. M2 is useful for embryo collection because it is a HEPES-buffered medium that
will maintain pH at ambient CO2 levels.
6. Microdrop culture (see Note 10) is performed in a nonhumidified incubator. Hu-
midification causes moisture to collect on the surface of the oil, eventually drip-
ping through the oil and diluting the culture medium.
7. Microspheres are available in various fluorescent colors to match most fluores-
cence detection systems. They are also available conjugated to reactive groups
for covalent coupling to proteins. Alternate commercial sources of this product
include Molecular Probes (Eugene, OR) and Polysciences (Warrington, PA).
8. Mice should be housed in an approved animal care facility with a regulated light-
ing cycle. For the protocol described here, lights were set to turn on at 0700 h and
turn off at 2100 h (14 h light/10 h dark). If blastocyst collection is desired at a
time of day other than morning, the light cycle and injection schedule should be
adjusted accordingly.
9. It is also possible to generate blastocysts by initiating in vitro development at
earlier preimplantation stages, collecting embryos from the oviduct (35). Gener-
ally, embryo production diminishes with gestational age, but earlier initiation of
in vitro development delays blastocyst formation. Removing blastocysts from
the uterus is technically less challenging than flushing oviducts. Both approaches
will generate embryos that can be cultured through the peri-implantation stages.
10. Some comments on the microdrop culture, as described by Hogan et al. (35), are
provided. Drops (2–10 µL) of embryo culture medium are arrayed on a Petri
dish. If a tissue culture-treated plate is used, the drops tend to run together. The
plate is then flooded with water-extracted mineral oil to cover the drops and pre-
vent their evaporation. At the outset, plates should be marked on the underside
with a laboratory pen to keep track of individual embryos or groups that will be
cultured on the plate.
11. Micropipets (see p. 134 of ref. 35) are made by heating a glass Pasteur pipet
about half way up the narrow end over a Bunsen burner. Once the glass is fluid,
move the pipet out of the flame and quickly pull a thin strand of glass, which
should remain hollow. Break it to create an opening that has a diameter 25–50%
larger than a blastocyst. Latex hosing with a mouth piece is attached with a cot-
ton filter to the pipet to control suction. While observing embryos through a ste-
reomicroscope, place the pipet in medium or through oil into a microdrop until it
rests next to an embryo. Controlling the suction, draw the embryo just into the
end of the pipet. Removing the pipet from the medium stops the suction. Once it
is inserted into another microdrop, create pressure to deliver the embryo. Take
care not to draw the embryo in too far or to expel all of the medium in the pipet,
as bubbles will be produced. Turn the dish to position the drop on the side oppo-
site your hand so the pipet can be held at a shallow angle. With a little practice,
this technique can be quickly mastered. Microspheres of 50–100 µm diameter
are useful for learning the technique without using valuable embryos.
12. The area occupied by the blastocyst prior to outgrowth should be taken into con-
sideration in analyzing outgrowth area. For example, the average area of blasto-
cysts before beginning outgrowth could be determined and subtracted from
measurements obtained during outgrowth to calculate the increase in outgrowth
area. This will provide a more accurate basis for comparing experimental groups.
13. The strong negative charge of α1-acid glycoprotein conferred by multiple sialic
acid moieties reduces nonspecific interactions of the microspheres with nega-
tively charged cell surfaces.
14. Adhesion incompetent blastocysts will produce low levels of fibronectin-binding
activity that are unaffected by prior upregulation with fibronectin or by competi-
tion with soluble fibronectin during incubation with microspheres. By calculat-
ing the difference in fibronectin-binding activity between embryos upregulated
with fibronectin and those exposed only to BSA or embryos assayed in the pres-
ence of competing fibronectin (∆ fibronectin-binding activity), blastocysts from
different stages of development are most accurately assessed for adhesion com-
petence.
15. Prior to adding embryos, it may be necessary to resuspend microspheres that
have settled during the chilling process using a micropipette to mix the medium.
The microsphere suspension will obscure your view of the embryos, making it
difficult to locate them after the 30-min incubation. This problem may be re-
duced by placing the embryos in the shallow portion of the drop, near its edge.
Medium drawn from the top of the drop can be blown over the embryos to assist
in their recovery.
16. For an eight-bit imaging program, grey level will range from 0 (black) to 255
(white). By setting the program to determine the average grey level (not the total
grey level), it will not matter if the area you are tracing over each embryo varies.
Acknowledgments
This research was supported by National Institutes of Health grants HD
36764 and AA12057. The author thanks Dr. Zitao Liu, Dr. Jun Wang and Mr.
Po Jen Chiang for preparing images of mouse embryos that were used for illus-
trations.
References
1. Johnson, M. H. and Ziomek, C. A. (1983) Cell interactions influence the fate of
mouse blastomeres undergoing the transition from the 16- to the 32-cell stage.
Dev. Biol. 95, 211–218.
33. Rout, U. K., Wang, J., Paria, B. C., and Armant, D. R. (2004) alpha5beta1,
alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regu-
late adhesion and migration of differentiating mouse trophoblast cells. Dev. Biol.
268, 135–151.
34. Wang, J. and Armant, D. R. (2002) Integrin-mediated adhesion and signaling dur-
ing blastocyst implantation. Cells Tissues Organs 172, 190–201.
35. Hogan, B. L., Beddington, R. S., Constantini, F., and Lacy, E. (1994) Manipulat-
ing the Mouse Embryo. A Laboratory Manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY.
36. Lopata, A., Kohlman, D. J., Bowes, L. G., and Watkins, W. B. (1995) Culture of
marmoset blastocysts on matrigel: a model of differentiation during the implanta-
tion period. Anat. Rec. 241, 469–486.
37. Kliman, H. J. and Feinberg, R. F. (1990) Human trophoblast-extracellular matrix
(ECM) interactions in vitro: ECM thickness modulates morphology and pro-
teolytic activity. Proc. Natl. Acad. Sci. USA 87, 3057–3061.
38. Yelian, F. D., Yang, Y., Hirata, J. D., Schultz, J. F., and Armant, D. R. (1995)
Molecular interactions between fibronectin and integrins during mouse blastocyst
outgrowth. Mol. Reprod. Dev. 41, 435–448.
39. Armant, D. R. (1991) Cell interactions with laminin and its proteolytic fragments
during outgrowth of mouse primary trophoblast cells. Biol. Reprod. 45, 664–672.
40. Martin, P. M., Sutherland, A. E., and Van Winkle, L. J. (2003) Amino acid trans-
port regulates blastocyst implantation. Biol. Reprod. 69, 1101–1108.
4
Isolation of Hormone Responsive Uterine Stromal Cells
An In Vitro Model for Stromal Cell Proliferation and Differentiation
Virginia Rider
Summary
The female sex hormones estrogen and progesterone stimulate proliferation and differentia-
tion of human and rodent uterine cells. The purpose of this chapter is to provide a method for
isolating hormone-responsive rat uterine stromal cell lines that can be used to study steroid
control of the cell cycle. Uteri from ovariectomized rats are differentially digested with trypsin
to separate epithelial and stromal cells. The stromal cells are cultured in a standard growth
medium containing 10% fetal bovine serum. After several passages, the purity of the stromal
cell lines is determined using immunocytochemistry. Cell proliferation is studied by culturing
the stromal cells in serum-free medium containing sex steroids and other mitogens. Cell cycle
progression is assessed by flow cytometry, 3H-thymidine and BrdU incorporation, whereas
proliferation is monitored using the MTT assay. Cell cycle regulators are visualized by North-
ern and Western blotting whereas cyclin–cyclin-dependent kinase activity is monitored using
immune complex kinase assays. Uterine stromal cell lines isolated using the methods reported
in this chapter provide a suitable model system to investigate the signal transduction events that
stimulate hormone-dependent control of the cell cycle.
Key Words: Uterus; stromal cells; decidua; cell cycle; estrogen; progesterone.
1. Introduction
The growth and function of any tissue is dependent on regulated prolifera-
tion and differentiation of its cellular components. Within endocrine target tis-
sues of the reproductive tract, hormones exert specific temporal, spatial, and
interactive effects. Estrogens are associated generally with cell proliferation in
uterine and breast tissues, whereas progesterone is considered more as the hor-
mone promoting cellular differentiation in these organs. However, progester-
one is a potent mitogen for stromal cells in the uterus and the lobuloalveolar
cells in the mammary gland (1). The preeminence of progesterone in female
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
57
reproduction has been highlighted by studies from mice lacking the progester-
one receptor by targeted mutagenesis (2). The progesterone receptor “knock-
out” mouse exhibits abnormalities in all aspects of reproduction including sexual
behavior, mammary gland development, ovulation, and implantation (2).
The rodent uterus is a well-characterized model system for studying the hor-
monal control of uterine cell proliferation and differentiation (3–8). Under the
influence of estrogen at days 2 and 3 post coitum, the luminal and glandular
epithelial cells proliferate (3,8). At day 4 of pregnancy in the rat, proliferation
switches from epithelial to stromal compartments (5). Stromal cells do not
divide without progesterone, and proliferation is blocked by progesterone
antibodies and progesterone receptor antagonists (8,9). During normal preg-
nancy, the uterine stromal cells proliferate and differentiate (decidualize) into
decidual cells (11). The stromal cells located at the antimesometrial region of
the endometrium are the first to show signs of differentiation. These cells
uncouple DNA replication from cytokinesis and become polyploid (11,12).
The differentiation of the uterine stroma spreads from cells located in the
antimesometrial and periluminal regions outwards towards the myometrium
(13). Decidualization of stromal cells is more restricted to the periluminal
region in the rat compared with the mouse (13).
Because the decidual cells located in the antimesometrial region of the uterus
express different proteins than the mesometrial cells, it is possible that these
two cell populations arise from different progenitors. However, stromal cells
isolated from these two regions and placed in culture lose their differentiated
gene expression (14). This suggests that stromal cells arise from a single stem
cell population and differences in gene expression are due to positional effects
within the endometrium rather than inherent genetic differences. This is an
important concept because it suggests that the mesometrial and antimesometrial
stromal cells share the same lineage, but differences in phenotype occur because
localized effects are exerted on stromal cells depending on their position within
the endometrium. If this interpretation is correct, then it should be possible to
stimulate either the mesometrial or antimesometrial differentiation programs
when the appropriate signal transduction cascades involved have been iden-
tified.
Major progress in understanding the control of the cell cycle has come from
proliferative studies of cells in model culture systems (reviewed in refs. 15–
18). It is our view that conceptual advancement about steroid mediated prolif-
eration and differentiation will be generated using cell culture systems that
recapitulate key hormone-dependent control of cell cycle events. Moreover, it
seems essential to clearly define steroid-dependent effects on the proliferative
cycle vs the differentiation pathway. Although these two events are likely
intertwined, there must be important signals that direct stromal cells from a
2. Materials
2.1. Culture Media
1. Standard growth medium. Medium 199 culture medium supplemented with
Earle’s salts and L-glutamine (Fisher Scientific, Hanover Park, IL) containing
100 U/mL penicillin (Sigma), 100 µg/mL streptomycin (Sigma), and 10%
heat-inactivated fetal bovine serum (FBS; Sigma) (see Note 1). Medium is
stored at 4°C.
2. Serum-free medium. Dulbecco’s modified Eagle’s medium, phenol red-free
(Gibco, Grand Island, NY) in a 3:1 mixture with MCDB-105 (Sigma) containing
supplements including 100 U/mL penicillin (Sigma), 100 µg/mL streptomycin
(Sigma), 5 µg/mL bovine insulin (Sigma), 10 µg/mL human transferrin (Sigma),
50 µg/mL ascorbic acid (Sigma), and 1 mg/mL reagent grade bovine serum albu-
min (Sigma). The medium is sterilized by filtration and stored at 4°C.
3. Freezing medium. Medium 199 culture medium supplemented with Earle’s salts
and L-glutamine (Fisher Scientific, Hanover Park, IL) containing 100 U/mL peni-
cillin (Sigma), 100 µg/mL streptomycin (Sigma), 10% dimethlysulfoxide
(DMSO; Sigma) and 20% heat-inactivated FBS (Sigma). The medium is stored
at 4°C.
4. Trypan blue (0.4%). Trypan blue powder (Sigma) is dissolved in phosphate-buff-
ered saline (PBS) and sterilized by filtration. The solution is stored at 22°C. It is
diluted 1:1 with the cell sample to be counted.
3. Methods
3.1. Cell Isolation
1. Sexually mature rats (150–175 g body weight) are ovariectomized and rested for
10 ds prior to stromal cell isolation.
2. Uterine horns are removed under anesthesia and trimmed of fat and mesentery.
The tissue is kept on ice.
3. The uterine horns are slit open longitudinally under a dissecting microscope. The
uterine horns from each rat are placed in a 60-mm culture dish (Fisher) contain-
ing 3.0 mL of tissue dissociation medium.
4. The tissue is incubated at 37°C for 35 min. The dish is vortexed at low speed for 10 s.
5. The tissue dissociation medium is removed and replaced with 2.0 mL of fresh
dissociation medium.
6. The uterine horns are incubated at 37°C for 60 min. The dish is vortexed at low
speed for 10 s.
7. The dissociation medium containing the cells is transferred to a 15-mL sterile
culture tube containing 0.1 vol of FBS. The uteri are washed with dissociation
medium and the wash is added to the sterile tube.
8. The medium containing the dissociated cells is centrifuged at 500g for 5 min.
9. The supernatant is discarded.
10. The cell pellet is suspended in 3.0 mL of standard growth medium.
11. When all of the uteri have been processed, the cell suspensions are combined and
centrifuged at 500g for 5 min.
12. The supernatant is discarded and the cell pellet is suspended in 10 mL of standard
growth medium. The cells are seeded on two 60-mm dishes, 5 mL each.
13. The cells are cultured in standard growth medium in an atmosphere of 5% CO2
and 95% air in a humidified chamber at 37°C (see Note 3).
14. Purity of the cells should be assessed using immunocytochemical analysis with
vimentin, desmin, and cytokeratin antibodies (32) (see Note 4).
15. Estrogen receptor transcripts can be measured using reverse transcription and
polymerase chain amplification (28). Progesterone (25) and estrogen (Fig. 1)
receptor proteins are detected by Western immunoblotting.
16. The response to mitogenic agents, including sex steroids, can be assessed by
flow cytometry (26,27) and the MTT assay (25). Entry into DNA replication is
assessed by 3H-thymidine incorporation (26) and BrdU incorporation (29).
17. Temporal expression of cell cycle regulators can be monitored by Northern and
Western blotting. Activity of cyclin–cyclin-dependent kinases can be monitored
using immune complex kinase assays (27).
Fig. 1. Rat uterine stromal cell lines express estrogen receptor (ER)-α protein. Uter-
ine stromal cells (UIII, passage 22) were cultured in medium 199 containing 10% fetal
bovine serum as detailed elsewhere (26). Cell extracts were prepared (27) and size
fractionated by sodium dodecyl sulfate-polyacrylamide gel (10%) electrophoresis. As
a positive control for ER-αexpression, extract from T47D breast cancer cells (34) was
electrophoresed on the same gel. The proteins were transferred to a nitrocellulose
membrane by standard methods (27). The membrane was reacted with a mouse ER-
αantibody (1:500 dilution, AER611, NeoMarkers, Freemont, CA) and an anti-mouse
secondary antibody (1:25,000 dilution). The blot was incubated with the SuperSignal
West Femto Maximum Sensitivity Substrate kit (Pierce, Rockford, IL) and exposed to
X-ray film for 1 min. The size of the major reactive species shown by the arrow at Mr
65,000 was determined from molecular size standards (BioRad, Hercules, CA). This
protein is consistent with the size for ER-α. In the absence of primary antibody the
major reactive protein was not detected (data not shown). Lane 1: Rat uterine stromal
cell extract. Lane 2: T47D breast cancer cell extract. Arrows on the left indicate the
position of the molecular size standards.
3. The cells are centrifuged at 500g for 5 min and the supernatant is carefully
removed from the resulting cell pellet.
4. The cells are suspended in 1 mL of freezing medium and the suspension is placed
into a cryovial.
5. The cryovial is kept at –80°C for at least 8 h before freezing the cells in liquid
nitrogen (see Note 8).
6. To thaw the cells, the cryovial is removed from the liquid nitrogen and quickly
placed in a water bath at 37°C. The thawed cells are transferred into a T75 flask
containing 10 mL of growth medium with 20% FBS (see Note 9).
7. The day after thawing the cells, the medium containing 20% serum is removed
and replaced by normal growth medium (10% FBS).
4. Notes
1. We have used heat-inactivated FBS from a variety of distributors. The cells
respond well in all of the FBS samples we have tested. Once the cell lines are
established, the cells grow rapidly and the medium should be changed as the
color shifts from red to orange. Confluent cells should be split and seeded into
new flasks or frozen and kept in liquid nitrogen. We do not maintain cells in
continuous culture because the hormonal responsiveness is better if they are fro-
zen down between experiments. The cells should not be allowed to overgrow.
2. Stromal cells have been stimulated to proliferate with both progesterone (25) and
medroxy progesterone acetate (27). Because the endpoint of the assays (prolif-
eration) occurs within 48 h after stimulation, there is no obvious difference in the
proliferative response to these two hormones.
3. This is the most critical aspect of the isolation procedure. During the first few
days of culture, there will be substantial cell death. As the cells begin to grow, it
is tempting to split and seed them into culture flasks. However, it is important to
maintain the cells for approx 10 d in the original 60-mm dishes or until they are
confluent. If the cells are split too soon, they will die. The cells should be seeded
into a T25 (25 cm2) flask when first split. If the density of the cells is too low
upon transfer, they will die.
4. After the fourth passage, the stromal cell lines will stabilize and growth will be
rapid. The purity of the cells can be assessed using antibodies that distinguish
stromal from epithelial cells. We have not assessed purity prior to the fourth
passage. The advantage of obtaining stromal cell lines over freshly isolated stro-
mal cells is that the cell lines are more homogeneous. Regardless of the care
taken in the isolation procedure, the initial cell isolates will be contaminated with
nonstromal cells (endothelial cells, immune cells, glandular epithelial cells).
During the first weeks after isolation, and for the first few passages, there is
considerable cell death. The cells that remain express stromal cell markers exclu-
sively (25).
5. Stromal cells adhere tightly to the surface of the culture flasks. It generally requires
5 min for the cells to detach but this should be monitored under the microscope.
Cells left in trypsin too long will incur damage to their membranes and die.
6. It is important to split the cells before overgrowth occurs. In general, there will
be between 3–5 × 106 cells per T75 flask. For routine passaging, we split the cells
1:2 and culture in 10 mL of normal growth medium per flask.
7. For freezing the cells, it is optimal to have between 2–5 × 106 total cells. The
cells are suspended in 1 mL of freezing medium. When the cells are thawed, the
frozen cells are split into two T75 flasks (0.5 mL each) in a total volume of 10 mL.
8. The basic procedure is to always freeze the cells slowly and thaw rapidly. Cells
will maintain viability at –80°C for several days. However, they will not be
viable stored at that temperature for long periods of time. Their viability (>90
%) is excellent when stored in liquid nitrogen.
9. The viability of the frozen cells is better when they are cultured initially in me-
dium containing 20%, rather than 10%, FBS. However, this medium is not opti-
mal for cell growth and maintenance. The medium containing 20% FBS is replaced
with medium containing 10% FBS 24 h after thawing the cells.
10. For each individual experiment a sufficient number of cells are propagated such
that cells within a given experiment are from the same passage.
11. The consistency in cell number in each well for a given experiment is critical for
measuring significant differences among treatments. Plating density is another
crucial factor. If the cells overgrow in the serum-free medium because the initial
plating density is too high they will undergo apoptosis in response to mitogenic
agents. If too few cells are plated, they will die in the serum-free medium. We
find that once the optimal number of cells is determined, the mitogenic response
is consistent, regardless of passage number.
12. With the rat uterine stromal cell lines, the percentage of cells in G1 phase after
72 h of serum-starvation is approx 70%. When cells are serum starved for 48 h,
only about 60% are at G1 phase. The greater the percentage of cells in G1 phase,
at the start of the experiment, the greater the synchronous response to mitogenic
agents.
13. Cell number is critical for determining the amount of time samples are incubated
with the MTT reagent. The MTT assay relies on the conversion of MTT into a
formazan product by the activity of mitochondrial dehydrogenases (33). The
relationship between cell number and absorbance should be determined in pilot
studies. If the cells are incubated with MTT reagent for too long, the assay is not
valid because linearity between cell number and absorbance is lost. Under the
conditions described here for stromal cell proliferation assays we find the 30–
60 min incubation time is optimal.
Acknowledgments
The author would like to thank Oliver Flieger, Marta Piva, Stephanie Jones,
Bruce Kimler and William Justice for contributing to the methods used to isolate
and characterize endocrine-dependent stromal cell proliferation. This research is
supported by the National Science Foundation (NSF IBN-0091504).
References
1. Clarke, C. L. and Sutherland, R. L. (1990) Progestin regulation of cellular prolif-
eration. Endocr. Rev. 11, 266–301.
2. Lyndon, J. P., DeMayo, F. J., Funk, C. R., et al. (1995) Mice lacking progesterone
receptor exhibit pleiotropic reproductive abnormalities. Genes Dev. 9, 2266–2278.
3. Finn, C. A. and Martin, L. (1967) Patterns of cell division in the mouse uterus
during early pregnancy. J. Endocrinol. 39, 593–597.
4. Quarmby, V. E. and Korach, K. S. (1984) The influence of 17β estradiol on pat-
terns of cell division in the uterus. Endocrinology 114, 694–702.
5. Rider, V. and Psychoyos, A. (1994) Inhibition of progesterone receptor func-
tion results in loss of basic fibroblast growth factor expression and stromal cell
proliferation during uterine remodeling in the pregnant rat. J. Endocrinol. 140,
239–249.
6. Galassi, L. (1968) Autoradiographic study of the decidual cell reaction in the rat.
Dev. Biol. 17, 75–84.
7. Martin, L. and Finn, C. A. (1968) Hormonal regulation of cell division in epithe-
lial and connective tissues of the mouse uterus. J. Endocrinol. 4, 1363–1371.
8. Cullingford, R. W. and Pollard, J. W. (1988) RU486 completely inhibits the
action of progesterone on cell proliferation in the mouse uterus. J. Reprod. Fert.
83, 909–914.
9. Rider, V., Wang, M-Y., Finn, C. and Heap, R. B. (1986) Antifertility effect of pas-
sive immunization against progesterone is influenced by genotype. J. Endocrinol.
108, 117–121.
10. Finn, C. A. (1971) The biology of decidual cells. Adv. Reprod. Physiol. 5, 1–26.
11. Moulton, B. C. and Koenig, B. B. (1984) Uterine deoxyribonucleic acid synthesis
during preimplantation in precursors of stromal cell differentiation during
decidualization. Endocrinology 115, 1203–1307.
12. McConnel, K. N., Sillar, R. G., Young, B. D., and Green, B. (1982) Ploidy and
progesterone-receptor distribution in flow sorted deciduomal nuclei. Mol. Cell.
Endocrinol. 25, 99–104.
13. Krehbiel, R. H. (1937) Cytological studies of the decidual reaction in the rat
during early pregnancy and in the production of deciduomata. Physiol. Zool. 10,
213–241.
14. Gu, Y. and Gibori, G. (1995) Isolation, culture and characterization of the two
cell subpopulations forming the rat decidua: differential gene expression for
activin, follistatin and decidual-related prolactin protein. Endocrinology 136,
2451–2458.
15. Musgrove, E. A. and Sutherland, R. L. (1994) Cell cycle control by steroid hor-
mones. Cancer Biol. 5, 381–389.
16. Pardee, A. B. (1989) G1 events and regulation of cell proliferation. Science 246,
603–608.
17. Sherr, C. J. (1993) Mammalian G1 cyclins. Cell 73, 1059–1065.
18. Mani, S.K., Julian, J., Lampelo, S., and Glasser, S.R. (1992) Initiation and main-
tenance of in vitro decidualization are independent of hormonal sensitization in
vivo. Biol. Reprod. 47, 785–799.
19. Fukamachi, H. and McLachlan, J. A. (1991) Proliferation and differentiation of
mouse uterine epithelial cells in primary serum-free culture: estradiol-17 beta sup-
presses uterine epithelial proliferation cultured on a basement membrane-like sub-
stratum. In Vitro Cell Dev. Biol. 27A, 907–913.
20. Jacobs, L. L., Sehgal, P. B., Julian, J., and Carson D. D. (1992) Secretion and
hormonal regulation of interleukin-6 production by mouse uterine stromal and
polarized epithelial cells cultured in vitro. Endocrinology 131, 1037–1046.
21. Whitworth, C. M., Mulholland, J., Dunn, R. C., and Glasser S. R. (1994) Growth
factor effects on endometrial epithelial cell differentiation and protein synthesis
in vitro. Fertil. Steril. 61, 91–96.
22. Rider, V., Piva, M., Cohen, M. E., and Carlone, D. L. (1995) Alternative splicing
and differential targeting of fibroblast growth factor receptor 1 in the pregnant rat
uterus. Endocrinology 136, 3137–3145.
23. Rider, V., Carlone, D. L., and Foster, R.T. (1997) Oestrogen and progesterone
control basic fibroblast growth factor messenger RNA in the rat uterus. J.
Endocrinol. 154, 75–84.
24. Rider, V., Carlone, D. L., Witrock, D., Cai, C., and Oliver, N. (1992) Uterine
fibronectin content and localization are modulated during implantation. Dev.
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uterine stromal cell proliferation is progesterone dependent. Biol. Reprod. 55,
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interactions in uterine stromal cells. Biol. Reprod. 59, 464–469.
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rat uterine stromal cells through G1 phase of the cell cycle requires progesterone-
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factors differentially regulate the growth and differentiation of cultured human
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Normal rat uterine stromal cells in continuous culture: characterization and
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381–391.
5
Rat Decidual Cell Cultures
Summary
Pregnancy requires profound reorganization of the different tissues forming the uterus.
Growth and differentiation of the uterine endometrial cells give rise to the decidual tissue, a
transitory organ, which plays a key role in fetal survival. In this chapter, we describe a tech-
nique for the dispersion and the separation of the two different decidual cell subpopulations
with high yield and viability. We also detail a cell culture method, which allows the mainte-
nance of the function and life span of these highly purified decidual cells when cultured either
separately or in a co-culture system.
Key Words: Rat; pseudopregnancy; decidualization; enzymatic tissue dispersion;
antimesometrial and mesometrial decidual cells; cell culture.
1. Introduction
A marked response to implantation and pregnancy in rodents and primates
is the growth and transformation of the uterine endometrial stromal cells known
as decidualization. In humans, decidualization normally occurs with each men-
strual cycle, and the formation of the decidual tissue depends primarily on lev-
els of progesterone and estradiol in the circulation. However, in other species,
including rodents, decidualization requires, in addition to adequate levels of
these hormones, an exogenous trigger, which may be either the contact of the
blastocyst with the endometrium or artificial stimulation at the luminal surface
of uterine horns. Decidualization of the endometrial stroma, induced by either
the blastocyst in pregnant rats or by artificial stimuli in pseudopregnant rats,
gives rise to at least two major cell populations located in opposite sides of the
uterus. The cells that decidualize in the antimesometrial region (opposite to
where blood vessels gain access to the uterus) become more extensively differ-
entiated than the cells in the mesometrial region, which undergo only limited
differentiation. These two decidual cell populations differ not only in their
morphology, but also by the genes they express and the putative roles they play
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
69
2. Materials
1. Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, WI).
2. Hank’s balanced salt solution (HBSS) without Ca2+ and Mg2+.
3. Fluorescein diacetate (FDA).
4. Trypan blue.
5. Nylon mesh.
6. Oxygen tank.
7. Peristaltic pump (Pharmacia, Peapack, NJ).
8. In-line surge suppressor (Cole Parmer, Chicago, IL).
9. Water-jacketed Cellstir (Wheaton Scientific, Millville, NJ).
10. Elutriator (JE-6B rotor with a Sanderson chamber).
11. 10- or 20-mL syringe.
12. Bubble trap.
13. Three-way connecters.
14. Connecting rubber tubes.
15. Cell Culture Inserts with Cyclopore membrane (Falcon Plasticware, BD Bio-
sciences, Bedford, MA ).
16. Enzyme Dispersion Solution: RPMI-1640 without glutamine containing collage-
nase type I (50 U/mL), dispase (2.4 U/mL), and deoxyribonuclease (200 U/mL).
17. HBSS Elutriation Buffer: HBSS with 25 mM HEPES and 0.1% bovine serum
albumin (BSA), pH 7.4.
18. Decidual cell culture medium: RPMI-1640 supplemented with 10% fetal bovine
serum (FBS), 2X antibiotic-antimycotic, 1X glutamine, 1X nonessential amino
acids, 1X sodium pyruvate, and 0.5% D-glucose.
3. Methods
3.1. Animals
3.1.1. Pseudopregnancy
The decidual tissue described here is collected from pseudopregnant female rats.
1. Rats are housed in a controlled environmental temperature (22°C) and kept under
a photoperiod of 14 h of light and 10 h of darkness. Rat chow and water are
provided ad libitum.
2. To generate rats pseudopregnant, young Sprague-Dawley female rats at proestrus
are mated with vasectomized males. The day a vaginal plug is found is desig-
nated as day 1 of pseudopregnancy (see Note 1).
Cellstir at 35°C for 45–60 min. Generally, 3–4 g of pooled decidual tissues per
100- to 150-mL enzyme solution should give a complete dispersion. However, in
case of an incomplete dispersion occurring after the first incubation, as a result of
either the presence of excessive tissue or aged enzymes used or other reasons, the
undissolved tissues are allowed to settle for 1 min.
2. The supernatant containing dispersed cells is collected and kept at 4°C.
3. Fresh enzyme solution is added again and the incubation is repeated as described
above.
4. Such dispersed decidual cells are filtered through a nylon mesh to remove any
undissolved tissue debris and centrifuged at 200g for 5 min at 4°C.
5. Cell pellets are gently resuspended in 10 mL HBSS Elutriation Buffer and are
kept in room temperature until elutriation.
Table 1
Settings of Peristaltic Pump Flow Rate and Elutriator Speed
Fractions Revolution Pump flow rate Volume
of elutriation of elutriator (rpm) (mL/min) (mL) Resulting contents
slowly injected into the elutriator through the air bubble trap using a 10- or 20-mL
syringe, as shown in the elutriator’s menu. Sometimes a blockage in the elutriation
system can occur during the injection as a result of either a fast injection or over-
load of excessive cells in the system. The blockage can be seen as a sharp and
constant increase of the chamber pressure and visible cell build-up in the cham-
ber. The pressure and cell build-up in the elutriator’s chamber must be monitored
constantly. If an appropriate amount of cells are slowly injected into the elutriation
system, the chamber pressure showing in the pressure gage should not rise.
2. Elutriation is carried out at the room temperature.
3. Four 200-mL fractions are collected using different pump flow rate and revolu-
tion parameters as shown in Table 1.
4. Each fraction is collected in four 50-mL conical centrifuge tubes and contains
different cell subpopulations.
5. As shown in Table 1, highly purified mesometrial decidual cells are collected in
fraction 2, and antimesometrial decidual cells are primarily collected in fraction 4.
6. Cells from the same fractions are pooled together, washed twice with HBSS
elutriation buffer, and finally resuspended in the Decidual Cell Culture Medium.
7. Cell viability is determined by the trypan blue dye exclusion (7) and/or fluores-
cein diacetate staining method (8), approx 80% in general (see Notes 5–7).
Fig. 1 (see companion CD for color version). Elutriated and cultured decidual cells.
Decidual tissues were obtained from day-9 pseudopregnant rats. Antimesometrial and
mesometrial decidual cells were enzymatically dispersed, separated by elutriation, and
stained with fluorescein diacetate in a suspension (upper panel). Upper left: small
mesometrial decidual cells (~10–15 µm in diameter). Upper right: large antimeso-
metrial decidual cells (~30 µm or greater, depending on their differentiation stage).
Elutriated decidual cells were then seeded for 24 h and their distinct morphology shown
in the lower panel. Lower left: mesometrial cells are small and mostly binucleated,
contain less lipid droplets, and remain undifferentiated, with a fibroblast-like appear-
ance in culture. Lower right: antimesometrial decidual cells are large and
polynucleated, with a syncytial-like appearance, and are rich in lipid droplets (red or
dark black dots).
Fig. 2. The co-culture system for antimesometrial and mesometrial decidual cells.
Upper panel: antimesometrial cells are seeded onto a cell culture insert and mesome-
trial cells are seeded into a well of six-well culture plate. Lower panel: a reversed
co-culture arrangement of panel A.
approx 5-cm2 area) and another type, e.g., mesometrial decidual cells (3 × 106
viable cells), is plated into wells of an appropriate culture plate (see Note 9).
2. The inserts are then placed into the wells of the plate as shown in Fig. 2, and the
culture medium (~2.5 mL) is added to the wells to reach and maintain a level
equal to that in the insert.
3. The arrangement of cell types in this co-culture and the viable cell density in
each portion of the co-culture should be determined according to the design of
each experiment.
4. Cells should be incubated at 37°C under an atmosphere of 5% CO2-95% air for
16–18 h to allow attachment.
5. The cells are washed at least three times with FBS-free culture medium prior to
appropriate treatments or experiments (9).
6. Microscopic observation should be performed at this point to check the cell
attachment for those cells seeded in the wells.
7. At the end of a culture, decidual cells can be easily scraped from both the inserts
and wells for extraction of proteins or nucleic acids. If it is desired, the cells can
also be dissolved enzymatically or chemically (see Note 10).
4. Notes
1. Pseudopregnancy can be also induced by physical stimulation of the vagina of
rats on estrus using a glass rod. In ovariectomized female rodents, pseudopreg-
nancy can also be induced by a sequential progesterone and estradiol treatment.
However, the decidualization reaction in such induced pseudopregnant females
generally is not as strong as that seen in females mated with infertile males.
2. The decidualization can also be induced by intrauterine injection of oil. How-
ever, this method generally does not induce a strong decidualization reaction in
uterine endometrium as seen by the surgical scratching procedure.
3. There is a narrow window for the induction of decidualization. On day 5 of
pseudopregnancy and/or pregnancy, the uterine endometrium of the female rat
becomes very sensitive to a physical stimulation. Therefore, the surgical proce-
8. The number of yielded cells per gram of decidual tissue varies, depending on the
pseudopregnancy stage of an animal. In general, at the peak of decidual reaction
(day 9 of pseudopregnancy), one gram of decidual tissue can yield approx 3 × 106
antimesometrial and 6 × 106 mesometrial decidual cells.
9. The cell plating density should be considered according to the experimental
design, especially when a co-culture system is employed. As shown in Fig. 1,
the size of an antimesometrial cell is much larger than that of a mesometrial cell,
but on the other hand, the number of antimesometrial cells yield per gram of
decidual tissue is much fewer than mesometrial cells. Therefore, one must decide
whether a same plating density for both cell types should be used or it is neces-
sary. It is also worthy to note that if both cell subpopulations are plated equally
with a high density in culture, it may result in a confluence stage for
antimesometrial cells even just after the attachment period, but not for mesome-
trial cells.
10. Cultured decidual cells also show dynamic changes in their function as seen in
decidual tissue in vivo. However, it is not clear yet whether these functional
changes observed in vitro completely reflect and/or parallel that in vivo. For
example, the expression of prolactin receptor mRNA in decidual mesometrial
cells from day-9 pseudopregnant rats is abundant during the first 24-h culture. It
starts to decline at 48 h, and completely disappears after 72 h in cultured mesome-
trial cells.
References
1. O’Shea, J. D., Kleinfeld, R. G., and Morrow, H. A. (1983) Ultrastructure of
decidualization in the pseudopregnant rat. Am. J. Anat. 166, 271–298.
2. Gibori, G. (1994) The decidual hormones and their role in pregnancy recognition,
in Endocrinology of Embryo-Endometrium Interactions (Glasser, S.R.,
Mulholland, J., and Psychoyos, A., eds.). Plenum, New York: pp.217–221.
3. Gu, Y. and Gibori, G. (1999) Deciduoma, in Encyclopedia of Reproduction
(Knobil, E. and Neill, J.D., eds). Academic, San Diego: pp. 836–842.
4. Fitz, T. A., Mayan, M. H., Sawyer, H. R., and Niswender, G. D. (1982) Character-
ization of two steroidogenic cell types in the ovine corpus luteum. Biol. Reprod.
27, 703–711.
5. Nelson, S. E., McLean, M. P., Jayatilak, P. G., and Gibori, G. (1992) Isolation,
characterization, and culture of cell subpopulation forming the pregnant rat cor-
pus luteum. Endocrinology 130, 954–966.
6. Nelson, S. E. and Gibori, G. (1993) Dispersion, separation and culture of different
cell population of the rat corpus luteum, in Methods in Reproduction Toxicology
(Chapin, R. E. and Heidel, J., eds.). Academic, New York: pp 340–359.
7. Tennant, J. R. (1964) Evaluation of the trypan blue technique for determination of
cell viability. Transplantation 2, 685–694.
8. Rotman, B. and Papermaster, B. W. (1966) Membrane properties of living mam-
malian cells as studies by enzymatic hydrolysis of flourogenic esters. Proc. Natl.
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cell subpopulations forming the rat decidua: differential gene expression for
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2451–2458.
10. Gu, Y., Soares, M. J., Srivastava, R. K., and Gibori, G. (1994) Expression of
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1427.
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progesterone receptors in the endometrium, myometrium and metrial gland of the
rat during the decidualization process. Endocrinology 114, 1627–1634.
6
The Immortalization of Human Endometrial Cells
Summary
The loss of replicative potential with each cell division has been attributed to the progres-
sive shortening of telomeres. This “mitotic clock” occurs because most normal human cells are
telomerase-negative. Telomerase is a multicomponent enzyme that prevents loss of telomeric
DNA associated with normal cell division. Transfection of cells with vectors expressing the
catalytic subunit of human telomerase (hTERT) is often sufficient for immortalization. In this
article, we will address this approach in the establishment of immortalized endometrial cells
and its value in facilitating in vitro studies.
Key Words: Uterus; endometrium; endometrial cells; stromal cells; glandular epithelial
cells; endothelial cells; immortalization; telomerase.
1. Introduction
Scarcity of human tissue and the inability to passage and maintain cells in
culture for long periods of time makes immortalization of primary cells an
ideal research tool. Unfortunately, the process of immortalization often results
in abnormal karyotypes and aberrant functional characteristics. To avoid the
latter drawback, several laboratories, including our own, have introduced
telomerase into cultured primary cells. Telomerase is a multicomponent
enzyme that comprises a template RNA plus an essential catalytic protein
subunit (human telomerase [hTERT]) (1). This method results in immortaliza-
tion of many target cells by preventing the normal shortening of telomeres
observed in adult somatic cells during mitosis. Telomeres are specialized DNA/
protein structures, which are located at the ends of eukaryotic chromosomes.
They contain tandemly repeated DNA sequences, which have a role in main-
taining the chromosomes during cell division by serving as the templates for
telomerase (2).
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Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
79
2. Materials
1. Antibiotic and antimycotic (ABAM; Gibco , Grand Island, NY).
2. ITS+(tm) Premix (Becton-Dickinson/Collaborative Research, Bedford, MA).
3. Glutamine (Gibco).
4. Hank’s balanced salt solution (HBSS) (Gibco).
5. Stripped calf serum (SCS) (Gemini, Woodland, CA).
6. Type I collagenase (Worthington, Lakewood, NJ).
7. Basal medium (BM): Dulbecco’s modified Eagle’s medium (DMEM) + Nutrient
Mixture F-12 HAM (Sigma, St. Louis, MO) supplemented with NaHCO3 (1.2 g/L),
10 mL ABAM/L, 10 mL/L ITS+, 100 mL SCS, and 50 mL/L glutamine adjusted to
pH 7.3 and filtered through a 0.2-µm sieve (see Note 1).
8. 45 mesh stainless-steel sieve (Newark Wire-Cloth Co.).
9. Human hTERT-expressing cell line: pA317 hTERT plus puromycin resistance
(Geron Corp. Menlo Park, CA).
10. Medium for initial growth of pA317 cell line: DMEM (high-glucose with L-gluta-
mine) +10% fetal bovine serum + ABAM.
11. Polybrene (Sequabrene, Sigma).
12. TRAPeze enzyme-linked immunosorbent assay (ELISA) Detection Kit
(Chemicon International, Inc., Temecula, CA)
3. Methods
3.1. Human Endometrial Stromal Cell Isolation and Cell Culture
After obtaining written informed consent and institutional approval, early
secretory endometria from reproductive age women are obtained from hyster-
ectomies for benign conditions (e.g., myomas). The endometrium is collected
and transported in BM to a sterile laminar flow hood and processed as follows:
1. The tissue is washed with HBSS. After most of the blood has been removed, the
tissue is finely minced in 10 mL HBSS and spun for 10 min at 4°C at 500g.
2. Approximately 300 mg of the wet pellet is resuspended in 10 mL of Hams F10 +
10% SCS containing 0.25% type I collagenase for 90 min in a vigorously shaking
water bath at 37°C (see Note 2).
3. In a sterile laminar hood, the digestate is filtered through a 45 mesh stainless
steel sieve to remove the glands. The supernatant is spun as described above and
the pellet is resuspended in DMEM + 10% SCS.
4. Cells are seeded in polystyrene plastic cell culture flasks. After 40 min, the
medium is changed to remove any floating material.
5. The HESCs are grown to confluence in BM+ at 37°C in a standard humidified
95% air/5% CO2 incubator.
6. The next day, discard the transfection medium and add BM.
7. After a 48 h incubation, trypsinize the cells and culture them under selection
media (BM plus puromycin [800 ng/mL]) (see Note 4).
4. Notes
1. To avoid nonspecific estrogenic effects, DMEM must not contain phenol red.
2. During the 90 min collagenase digestion, the tubes are vortexed vigorously to
break up the clumped fragments.
3. Passage 1:10 when the flask reaches 80–90% confluence using Trypsin-ethyl-
enediamine tetraacetic acid (EDTA); do not allow the cells to become over
confluent.
4. Typical doses of puromycin used for selection are between 0.5 and 2.5 µg/mL.
Because each cell has a different sensitivity to puromycin, testing the sensitivity
prior to the transfection process is recommended. For this, treat the cells with
increasing concentrations of puromycin for 24 or 48 h and determine cell viabil-
ity by trypan blue, MTT, or any other cell viability assay.
Acknowledgments
We would like to acknowledge Mizanur Rahman, MD, Rebeca Caze, MS,
Ayesha Alvero, MD., Seth Guller, PhD, Frederick Schatz, PhD, Eva Sapi, PhD,
Paula Aldo, and Mazin Qumsiyeh, MD for their input in this project. This work
was supported in part by grants from the National Institutes of Health: RO1
HD33937-06 (CJL) and RO1 HL70004-01A1 (CJL).
References
1. Condon, J., Yin, S., Mayhew, B., et al. (2002) Telomerase immortalization of
human myometrial cells. Biol. Reprod. 7, 506–514.
2. Emrich, T., Chang, S.-Y., Karl, G., Panzinger, B., and Santini, C. (2002) Quanti-
tative detection of telomerase components by real-time, online RT-PCR analysis
with the LightCycler, in Methods in Molecular Biology, Vol. 191: Telomeres and
Telomerases: Methods and Protocols (Double, J. A. and Thompson, M. J., eds.).
Humana, Totowa, NJ: pp. 99–108.
3. Toouli, C. D., Huschtscha, L. I., Neumann, A. A., et al. (2002) Comparison of
human mammary epithelial cells immortalized by simian virus 40 T-Antigen or
by the telomerase catalytic subunit. Oncogene 21, 128–139.
4. Chiu, C. P. and Harley, C. B. (1997) Replicative senescence and cell immortality:
the role of telomeres and telomerase. Proc. Soc. Exp. Biol. Med. 214, 99–106.
5. Weinrich, S. L., Pruzan, R., Ma, L., et al. (1997). Reconstitution of human
telomerase with the template RNA component hTR and the catalytic protein sub-
unit hTRT. Nat. Genet. 17, 498–502
6. Bibby, M. C. (2002) Introduction to telomeres and telomerase, in Methods in
Molecular Biology, Vol. 191: Telomeres and Telomerases: Methods and Proto-
cols (Double, J. A. and Thompson, M. J., eds.). Humana, Totowa, NJ: pp. 1–12.
7. Kyo, S., Nakamura, M., Kiyono, T., et al. (2003) Successful immortalization of
endometrial glandular cells with normal structural and functional characteristics.
Am. J. Pathol. 163, 2259–2269.
8. Krikun, G., Mor, G., Alvero, A., et al.. (2004) A novel immortalized human en-
dometrial stromal cell line with normal progestational response. Endocrinology
145, 2291–2296.
7
Sheep Uterine Gland Knockout (UGKO) Model
Summary
Endometrial gland development is a postnatal event in the ovine uterus that can be inhibited
epigenetically by chronic exposure of ewe lambs to a synthetic progestin after birth. The uterus
of neonatally progestinized ewes lack endometrial glands and display a uterine gland knockout
(UGKO) phenotype. Progestin ablation of endometrial gland development is specific, because
it does not affect development of extra-uterine reproductive tract structures or the hypotha-
lamic–pituitary–ovarian axis. The UGKO ewe is a useful model for study of uterine develop-
ment and the role of endometrial glands in uterine function during the estrous cycle and
pregnancy. UGKO ewes exhibit altered estrous cycles due to the inability of the uterus to pro-
duce luteolytic pulses of prostaglandin F2α. UGKO ewes are infertile, and blastocysts hatch
normally but fail to survive or elongate during early pregnancy. This pregnancy defect is pri-
marily due to the absence of endometrial glands and their secretions rather than alterations in
expression of either anti-adhesive or adhesive molecules on the endometrial epithelium.
Genomics and proteomics are being used to identify specific components of histotroph that are
absent or diminished in the UGKO ewe and will serve as markers of endometrial function and
uterine receptivity.
Key Words: Sheep; uterus; endometrium; gland; histotroph; epigenetic; steroid; progester-
one; neonate; pregnancy; implantation; conceptus; defect; embryo loss.
1. Introduction
The bicornuate ovine uterus consists of two uterine horns connected by a
short uterine body. The uterine wall can be divided functionally into the
endometrium and myometrium. The adult endometrium of ruminants
(sheep, cattle, and goats) consists of two epithelial cell types (luminal epithe-
lium [LE] and glandular epithelium [GE]), stratified stromal compartments that
include a densely organized adluminal zone of fibroblastic cells (stratum
compactum) extending into a more loosely organized zone in the deeper or
basal endometrium (stratum spongiosum), blood vessels, and immune cells.
Grossly, the adult ovine endometrium is divided into raised, aglandular
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
85
Fig. 1. Histological comparison of day-14 pregnant uteri from control and uterine
gland knockout (UGKO) ewes. The uteri were fixed in paraformaldehyde, sectioned
(5 µm), and stained with hematoxylin and eosin. (A) Control uterus that contained two
normal day 14 conceptuses. (B) UGKO uterus that failed to support conceptus devel-
opment. (C) UGKO uterus that contained only a single tubular conceptus. (D) UGKO
uterus that contained a single fragile filamentous conceptus. The UGKO phenotype
was produced by exposing neonatal ewe lambs to a 19-norprogestin from birth for 8
wk. Legend: L, lumenal epithelium; G, glandular epithelium; S, stroma; M, myo-
metrium (original magnification ×166).
whereas myometrial width and morphology are not different from normal ewes
(18). The specific targeting of only the uterine endometrium by the progestin
exposure makes it an attractive model with which to study mechanisms regu-
lating endometrial organization and gland morphogenesis, also termed
adenogenesis, in the neonate, as well as the functional role of endometrial
glands in the adult.
Recent studies of the UGKO ewe model revealed an essential role for
endometrial glands and their secretions in normal estrous cycles and in peri-
implantation conceptus survival and growth. Mature UGKO ewes are unable
to exhibit normal estrous cycles as a result of insufficient production of
2. Materials
2.1. Animals
Newborn female sheep (Ovis aries).
3. Methods
1. The skin in the area of implant administration is sheared and disinfected with
betadine followed by an alcohol scrub.
2. A sterile scalpel blade is used to make a small incision through the skin in the
periscapular area.
3. The progestin implant is inserted subcutaneously in the periscapular area of ewe
lambs within 12 h of birth. The progestin inhibits differentiation and develop-
ment of the endometrial glandular epithelial cells from the luminal epithelium in
the uterus of the neonatal ewe. Progestin ablation of endometrial adenogenesis is
permanent and produces the UGKO phenotype. Any delay in progestin implant
administration may not be 100% effective to inhibit endometrial gland differ-
entiation. Unpublished observations indicate that administration of the proges-
tin implants on day 7 after birth does not inhibit endometrial gland development
(C. A. Gray and T. E. Spencer, unpublished observations).
4. If the Synchromate B implant is used, a new implant must be administered to the
ewes on PND 14, 28, and 42 to ensure exposure to the progestin from birth to at
least PND 56 (see Note 1).
5. Implanted ewe lambs are maintained according to standard animal husbandry
practices until they reach puberty.
6. After puberty, the UGKO ewes will exhibit altered estrous cycles of 17 to 43 d in
length. Luteolysis and behavioral estrus can be induced in UGKO ewes using
exogenous PGF2α (Lutalyse®, Kalamazoo, MI).
7. The uterus should be removed and assessed to confirm the absence of endome-
trial glands (see Note 2). A general marker of endometrial glandular epithelium
in the ovine uterus is expression of the long and short forms of the prolactin
receptor gene (30,31). The mRNA for the prolactin receptor is expressed only in
the endometrial glandular epithelium of the uterus of postnatal, cyclic, pregnant,
and postpartum ewes (30,31,35). During early pregnancy, expression of the
progesterone receptor is lost from the endometrial glands between days 11 and
17 post mating (day 0 = mating) (36). Loss of the PR is accompanied by the onset
of OPN and UTMP gene expression in the endometrial glandular epithelium
(29,30). The expression of OPN and UTMP genes are indicative of terminal dif-
ferentiation of the endometrial glands of pregnancy, because their expression is
abrogated after parturition in the postpartum uterus (35). Interestingly, the termi-
nally differentiated endometrial glands of pregnancy are devoid of detectable PR
gene expression (37), but the PR returns in the endometrial glands after parturi-
tion, concomitant with the loss of OPN and UTMP gene expression (35).
4. Notes
1. Exposure of neonatal ewes to a progestin for 8, 16, or 32 weeks prevented endome-
trial adenogenesis and produced the UGKO phenotype in adult ewes (17).
2. Three endometrial phenotypes are consistently observed in norgestomet-treated
ewes: (1) no glands; (2) slight glandular invaginations into the stroma; and (3)
limited numbers of cyst- or glandlike structures in the stroma (17) (see Fig. 1).
Most neonatally progestinized ewes exhibit the first phenotype as adults. The
uterus of individual sheep only exhibit one of the phenotypes, because the pheno-
type is homogenous within a horn. The different endometrial phenotypes do not
appear to result from genetic differences in responsiveness to neonatal progestins,
but rather are due to the timing of progestin implant administration. The implant
must be administered very soon after birth to inhibit the program of endometrial
gland differentiation and development. Another potential cause of the differing
endometrial phenotypes is infection at the site of implant administration, which
decreases systemic delivery of the progestin. No specific differences in cyclicity
have been observed in ewes with one of the endometrial phenotypes compared
with the other phenotypes. However, ewes whose uterus exhibits limited num-
bers of cyst- or glandlike structures may possess the ability to nurture the con-
ceptus to a more advanced stage of development (e.g., fragile filamentous) as
compared to ewes with the other phenotypes (e.g., no conceptus or tubular,
growth-retarded conceptus) (22). Regardless of the endometrial phenotypes, all
three types of UGKO ewes are not capable of sustaining pregnancy much past
day 14 and exhibit a peri-implantation type of pregnancy defect (21,22).
Acknowledgments
The authors would like to thank Frank F. Bartol, Fuller W. Bazer, and Kristin
M. Taylor, as well as former graduate students in the Bazer/Spencer Labora-
tory, for their participation in the development of the sheep UGKO model.
This work was supported by grants from the United States Department of
Agriculture (USDA) (98-35203-6322 & 2001-02259).
References
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3. Bazer, F. W. (1975) Uterine protein secretions: relationship to development of the
conceptus. J. Anim. Sci. 41, 1376–1382.
4. Martal, J., Chene, N., Camous, S., et al. (1997) Recent developments and potenti-
alities for reducing embryo mortality in ruminants: the role of IFN-tau and other
cytokines in early pregnancy. Reprod. Fertil. Dev. 9, 355–380.
5. Kane, M. T., Morgan, P. M., and Coonan, C. (1997) Peptide growth factors and
preimplantation development. Hum. Reprod. Update 3, 137–157.
6. Bell, S. C. (1988) Secretory endometrial/decidual proteins and their function in
early pregnancy. J. Reprod. Fertil. Suppl. 36, 109–125.
7. Beier, H. M. (2000) The discovery of uteroglobin and its significance for repro-
ductive biology and endocrinology. Ann. N.Y. Acad. Sci. 923, 9–24.
8. Carson, D. D., Bagchi, I., Dey, S. K., et al. (2000) Embryo implantation. Dev.
Biol. 223, 217–237.
9. Gray, C. A., Bartol, F. F., Tarleton, B. J., et al. (2001) Developmental biology of
uterine glands. Biol. Reprod. 65, 1311–1323.
10. Burton, G. J., Watson, A. L., Hempstock, J., Skepper, J. N., and Jauniaux, E.
(2002) Uterine glands provide histiotrophic nutrition for the human fetus during
the first trimester of pregnancy. J. Clin. Endocrinol. Metabol. 87, 2954–2959.
11. Roberts, R. M. and Bazer, F. W. (1988) The functions of uterine secretions. J. Reprod.
Fertil. 82, 875–892.
12. Fazleabas, A. T., Hild-Petito, S., and Verhage, H. G. (1994) Secretory proteins
and growth factors of the baboon (Papio anubis) uterus: potential roles in preg-
nancy. Cell. Biol. Int. 18, 1145–1153.
13. Bartol, F. F., Wiley, A. A., Floyd, J. G., et al. (1999) Uterine differentiation as a
foundation for subsequent fertility. J Reprod. Fertil. Suppl. 54, 287–302
14. Wiley, A. A., Bartol, F. F., and Barron, D. H. (1987) Histogenesis of the ovine
uterus. J. Anim. Sci. 64, 1262–1269.
15. Bartol, F. F., Wiley, A. A., Coleman, D. A., Wolfe, D. F., and Riddell, M. G.
(1988) Ovine uterine morphogenesis: effects of age and progestin administra-
tion and withdrawal on neonatal endometrial development and DNA synthesis.
J. Anim. Sci. 66, 3000–3009.
16. Spencer, T. E., Stagg, A. G., Joyce, M. M., et al. (1999) Discovery and character-
ization of endometrial epithelial messenger ribonucleic acids using the ovine uter-
ine gland knockout model. Endocrinology 140, 4070–4080.
17. Gray, C., Bartol, F. F., Taylor, K. M., et al. (2000) Endometrial glands are required
for preimplantation conceptus elongation and survival. Biol. Reprod. 62, 448–456.
18. Gray, C. A., Bazer, F. W., and Spencer, T. E. (2001) Effects of neonatal progestin
exposure on female reproductive tract structure and function in the adult ewe.
Biol. Reprod. 64, 797–804.
19. Gray, C. A., Taylor, K. M., Bazer, F. W., and Spencer, T. E. (2000) Mechanisms
regulating norgestomet inhibition of endometrial gland morphogenesis in the neo-
natal ovine uterus. Mol. Reprod. Dev. 57, 67–78.
20. Spencer, T. E., and Bazer, F. W. (2002) Biology of progesterone action during
pregnancy recognition and maintenance of pregnancy. Front. Biosci. 7, d1879–
d1898.
21. Gray, C. A., Taylor, K. M., Ramsey, W. S., et al. (2001) Endometrial glands are
required for preimplantation conceptus elongation and survival. Biol. Reprod. 64,
1608–1613.
22. Gray, C. A., Burghardt, R. C., Johnson, G. A., Bazer, F. W., and Spencer, T. E.
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23. Guillomot, M. (1995) Cellular interactions during implantation in domestic rumi-
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25. Burghardt, R. C., Johnson, G. A., Jaeger, L. A., et al. (2002) Integrins and extra-
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33. Carpenter, K. D., Gray, C. A., Bryan, T. M., Welsh, T. H., Jr., and Spencer, T. E.
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8
A Baboon Model for Inducing Endometriosis
Asgerally T. Fazleabas
Summary
Endometriosis is a disease that is associated with severe pelvic pain and is a major cause of
infertility in women. It is an enigmatic disease whose etiology and pathophysiology has been
studied to a limited extent. The events associated with the establishment of the disease and
mechanisms associated with infertility are difficult to assess in a systemic manner in women. In
order to understand the early and progressive events associated with the establishment of the
disease, we have developed a baboon model in which the disease can be induced. This induc-
tion manifests itself in a manner that recapitulates the spontaneous disease. The advantage of
the induced model is that the progressive changes in both the ectopic and eutopic endometrium
can be studied in a nonhuman primate model at specific times during the menstrual cycle and as
the disease process continues.
Key Words: Baboon; Papio anubis; endometriosis; uterus; endometrium.
1. Introduction
Endometriosis is defined as the presence of endometrium-like tissue outside
of the uterine cavity. It is one of the most common causes of infertility and
chronic pelvic pain and affects 1 in 10 women in the reproductive-age group
(1). This incidence increases up to 30% in patients with infertility and up to
45% in patients with chronic pelvic pain (2). Endometriosis is an estrogen-
dependent gynecological condition. According to Sampson’s theory (3), frag-
ments of menstrual endometrium are refluxed through the fallopian tubes into
the peritoneal cavity, then attach to and grow on peritoneal surfaces. However,
the fundamental mechanisms by which menstrual endometrium adheres, pro-
liferates, and establishes a functional vasculature in an ectopic site remain to
be elucidated. We propose that endometriosis develops in two distinct phases.
Phase I is invasive and dependent on ovarian steroids. Phase II, which is the
active phase of the disease, is characterized by endogenous estrogen biosyn-
thesis.
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
95
The baboon is a valuable and clinically relevant model with which to study
the etiology and consequences to fertility of this enigmatic disease (4). Experi-
mental evidence indicates that intrapelvic injections of menstrual endometrium
can induce endometriosis in this primate, thereby supporting the basic tenets of
the Sampson’s hypothesis (3). Furthermore, because hormonal manipulations
are possible, we can also address the role that ovarian and extra-ovarian hor-
mones play in the establishment and maintenance of this disease.
2. Materials
1. Laproscopic equipment with video and photographic accessories.
2. Unimar Pipelle (Cooper Surgical Inc., Shelton, CT)
3. Methods
3.1. Induction of Disease
The experimentally induced baboon endometriosis model was first estab-
lished by D’Hooghe et al. (5). We have modified the original procedure, which
is described in this chapter and in refs. 6 and 7).
Menstrual endometrium (approx 1 g) is harvested on day 2 of menses using
a Unimar Pipelle just prior to laparoscopy. The Pipelle is inserted through the
external cervical os into the uterine lumen. The cervical os is visualized by
dilating the vagina using a pediatric speculum. Menstrual fluid and tissue is
aspirated into the Pipelle by rotating it gently within the lumen and applying
suction aspiration. Usually, two Pipelles filled with fluid and tissue is suffi-
cient for inoculation and the induction of endometriosis (see Notes 1 and 2).
The peritoneal cavity and reproductive tract is visualized by laparoscopy
and the absence of any lesions or adhesions is documented by video recording
(see Note 3). Under laparoscopic guidance, the menstrual tissue in the Pipelle
is deposited at three sites: the pouch of Douglas, the broad ligament adjacent to
the oviducts, and on the uterus. At the subsequent menses, the animals undergo
a second laparoscopy and endometrial re-seeding at the same ectopic sites.
Following the second seeding, and the third menses, laparoscopy is performed
to evaluate the extent of endometriosis (6). Additional laparoscopies are per-
formed at periodic intervals every 3 mo following the initial inoculation (see
Note 4). Figure 1A shows a blue lesion documented by video recording and
the corresponding histological appearance of the lesions. The presence of glands
and stroma at this ectopic site that was seeded using menstrual endometrium
meets the classical pathological definition of endometriosis (Fig. 1B,C).
Following video documentation, endometriotic lesions and portions of nor-
mal peritoneum can be biopsied using a harmonic scalpel under laparoscopic
guidance. If both the eutopic and ectopic tissue is required, an endometriectomy
Fig. 1. (A) Video micrograph of a blue lesion on the peritoneum. (B,C) Histologi-
cal sections of endometriotic lesions showing the presence of both glands and stroma.
4. Notes
1. Baboons, unlike rhesus macaques, usually have a straight cervix without exten-
sive folds. Occasionally, there are baboons whose cervixes are difficult to navi-
gate with the Pipelle. It is prudent to evaluate the animals in a mock cycle either
during menses or in the proliferative stage of the menstrual cycle to evaluate the
accessibility of the uterine cavity.
2. To study direct hormonal effects on lesion formation, the ovariectomized baboon
model could be utilized. Using the steroid hormone replacement regimen described
in ref. 9, a progesterone withdrawal bleed is induced following 28 d of estradiol
and progesterone treatment. The baboons usually have a menstrual bleed 48 h
following progesterone withdrawal. Menstrual tissue collected from these ani-
mals can be used for inoculation to induce endometriosis. Steroid hormone or
control treatments could then be initiated in the ovariectomized animals.
3. Baboons are also afflicted with spontaneous endometriosis, and this disease is
progressive throughout the animal’s lifespan (8). Therefore, it is critical that prior
to the induction of endometriosis using the induced model, a thorough
laparoscopic evaluation be performed to rule out spontaneous disease in the study
animals.
4. For control studies, we inoculate the peritoneal cavity with endometrial tissue
obtained at the mid secretory phrase of the menstrual cycle. In our experience,
this tissue does not attach to the peritoneum and form active endometriotic
lesions, as do tissues from the menstrual effluent (6).
Acknowledgments
The expert assistance of the Veterinary Staff at the Biological Resources
Laboratory and the technical assistance of Ms. Allison Brudney is gratefully
acknowledged. These studies have been supported by National Institutes of
Health (NIH) grant HD40093.
References
1. Eskenazi, B. and Warner, M. L. (1997) Epidemiology of endometriosis. Obstet.
Gynecol. Clin. North Am. 24, 235–258.
2. Gruppo italiano. (1994) Prevalence and anatomical distribution of endometriosis in
women with selected gynaecological conditions: results from a multicentric Italian
study. Gruppo italiano per lo studio dell’endometriosi. Hum. Reprod. 9, 1158–1162.
3. Sampson, J. A. (1927) Peritoneal endometriosis due to menstrual dissemination of
endometrial tissue into the peritoneal cavity. Am. J. Obstet. Gynecol. 14, 422–469.
4. D’Hooghe, T. M. (1997) Clinical relevance of the baboon as a model for the study
of endometriosis. Fertil. Steril. 68, 613–625.
5. D’Hooghe, T. M., Bambra, C. S., Raeymaekers, B. M., De Jonge, I., Lauweryns,
J. M. and Koninckx, P. R. (1995) Intrapelvic injection of menstrual endometrium
causes endometriosis in baboons (Papio cynocephalus and Papio anubis). Am. J.
Obstet. Gynecol. 173, 125–134.
6. Fazleabas, A. T., Brudney, A., Gurates, B., Chai, D., and Bulun, S. E. (2002)
A primate model for endometriosis, in Proceedings of a NIH Workshop on
Endometriosis: Emerging Research and Intervention Strategies (Yoshinaga,
K. and Parrot, E., eds.). Ann. N. Y. Acad. Sci. 955, 308–317.
7. Fazleabas, A. T., Brudney, A., Chai, D., Langoi, D., and Bulun, S. E. (2003) Ste-
roid receptor and aromatase expression in baboon endometriotic lesions. Fertil.
Steril. 80(Suppl 2), 820–827.
8. D’Hooghe, T. M., Bambra, C. S., Raeymaekers, B. M., and Koninckx, P. R. (1996)
Serial laparoscopies over 30 months show that endometriosis in captive baboons
(Papio anubis, Papio cynocephalus) is a progressive disease. Fertil. Steril. 65,
645–649.
9. Fazleabas, A. T., Miller, J. B., and Verhage, H. G. (1988) Synthesis of estrogen
and progesterone dependent proteins by the baboon (Papio anubis) endometrium.
Biol. Reprod. 39, 729–736.
9
A Baboon Model for Simulating Pregnancy
Asgerally T. Fazleabas
Summary
Estrogen and progesterone secreted by the corpus luteum regulate the function of the uterine
endometrium in preparation for pregnancy. Embryonic signals superimposed on this steroid
hormone-primed uterus further modulate the uterine environment to make it conducive to preg-
nancy. Understanding the signaling mechanisms that initiate the embryonic–maternal dialog in
humans is not feasible. In an effort to elucidate the role of chorionic gonadotropin as a mediator
of endometrial function in addition to its luteotrophic role, we have developed a simulated
pregnant model in the baboon. Infusion of chorionic gonadotropin in a manner that mimics
blastocyst transit induces major changes in the morphology and secretory activity of the
endometrium. This model provides a method by which the function of various embryonic
factors on endometrial can be tested in an in vivo model.
Key Words: Baboon; Papio anubis; pregnancy; chorionic gonadotropin; implantation.
1. Introduction
The establishment of pregnancy requires a synchronous interaction between
the embryo and the endometrium. These interactions are necessary to both pro-
long corpus luteum function and modulate the uterine environment to permit a
normal embryo to attach and invade a receptive uterine endometrium (1–4). In
ruminants and pigs, embryo signals act in a paracrine manner on the uterine
endometrium to inhibit the pulsatile release of the luteolytic factor, prostaglan-
din F2α. In contrast, the primate embryonic signal, chorionic gonadotropin
(CG), has a direct luteotrophic effect on the corpus luteum. The presence of
luteinizing hormone (LH)/CG receptors have been documented to the present
in the primate endometrium (5,6), and in vitro stimulation of both epithelial
and stromal endometrial cells activates signal transduction and gene transcrip-
tion (7–11). To determine if CG further modulates the estrogen and progester-
one primed receptive endometrium in vivo, we have developed a nonhuman
primate simulated pregnant model. The baboon (Papio anubis) was used as the
nonhuman primate of choice.
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
101
2. Materials
1. V4 Polyvinyl Cannula (BioLab Products, Lake Havasu City, AZ).
2. Dow Corning silastic capsules (Dow Corning, Midland, MI).
3. Silastic medical adhesive Type A (Dow Corning, Midland, MI).
4. Alzet osmotic minipump 2ML1 (Alza Corp, Palo Alto, CA).
5. Recombinant human CG (hCG; Serono Pharmaceuticals).
6. Microsurgical dissection instruments.
7. 4.0 nonabsorbable suture on a tapered needle.
8. Coat-a-Count Estradiol Assay kit (Diagnostic Products Corporation, Los Ange-
les, CA).
3. Methods
Ovulation is monitored in normally cycling female baboons by measuring
serum estradiol levels beginning 7 d following the first day of visible menses.
Day 1 of ovulation is designated to be 48 h following the estradiol surge.
3.1. Placement of Cannula
On day 6 post ovulation, baboons are sedated with Ketamide hydrochloride
(10 mg/kg) and transferred to intravenous administration of Thiopentol. The
animals are maintained on a surgical plane of anesthesia with isofluorane and
oxygen.
Under sterile operating conditions, the oviducts are exteriorized following a
mid-ventral incision. Using a pair of microdissection scissors, an initial cut is
made through the mesosalpinx of the oviduct in a region of the isthmus with no
convolutions. Care must be taken not to cut through the entire mesosalpinx.
The oviductal lumen is visualized using microdissection forceps to ensure that
the lumen is open. The polyvinyl cannula with a medical adhesive bead attached
1–2 cm from the tip is inserted into the oviductal lumen (Fig. 1). Generally, the
cannula will extend approx 1 cm into the oviductal lumen. The cannula is
sutured in place with 4.0 nonabsorbable suture and the cut edges of the
mesosalpinx are pulled over the adhesive bead and sutured in place (Fig. 1).
Once the cannula is secure in the oviduct, a small subcutaneous flank inci-
sion is made on the side of the body wall that the cannula is attached. The body
wall is punctured using blunt dissection with a hemostat and the polyvinyl
cannula is exteriorized. The open end of the cannula is attached to the Alzet
minipump (Fig. 1).
3.2. Preparation of Alzet Minipump
The 2ML1 Alzet pump holds 2 mL of fluid and lasts for 7 d with a flow rate
of 10 µL/h. For hCG infusion, the solution containing 1.25 IU/10 µL is made
up in a 2.5-mL volume. The Alzet minipump is primed in sterile saline over-
night at 37°C. For control studies, the hCG is heat-inactivated by boiling the
solution for 30 min. The manufacturer provides detailed information for filling
and preparing the osmotic minipump.
The appropriately primed minipump is inserted into the open end of the
cannula. Prior to insertion, the dead space within the cannula is gently primed
with part of the balance 0.5 mL of solution. A 21-gauge stub adapter needle
attached to a 1-cc syringe is inserted into the cannula and the solution is gently
infused to fill up the dead space. The cannula is then attached to the metal
prong on the Alzet pump and held in place by 2.0 silk sutures. The pump is
then inserted into a subcutaneous pocket and the infusion is initiated (Fig. 1).
3.3. Harvesting of Endometrial Tissues
Endometrial tissue is harvested on either day 10 or day 14 post ovulation.
Day 10 corresponds to the approximate day of implantation in the baboon and
day 14 corresponds to the earliest time point at which an implantation site can
be readily identified (12). The 24-h infusion rate of 30 IU hCG is equivalent to
the amount of baboon CG secreted by dispersed baboon trophoblast cells cul-
tured in vitro (13). The 4-d infusion period (days 6–10 post ovulation) corre-
sponds to the window of time when the blastocyst is present within the uterine
cavity and is associated with the initial phases of attachment and invasion.
Thus, this simulated pregnant model mimics the intrauterine hormonal milieu
associated with blastocyst transit and attachment in the baboon (see Notes 1
and 2).
The uterus is carefully exteriorized following a mid-ventral incision (Fig. 2B).
The myometrium is gently injected with 2 mL of vasopressin (20 IU/mL) using
a 3-cc syringe and a 25-gauge needle. This constricts the blood vessels in the
myometrium and decreases the bleeding during the myometrial incision. Using
a number 11 scalpel blade, a longitudinal incision is made through the myo-
metrium until the functionalis tissue is exposed (Fig. 2D). The functionalis is
gently peeled away from the basalis using the pointed end of a metal weighing
spatula (Fig. 2E,F). This procedure is termed an endometriectomy. Following
removal of the endometrial tissue, the myometrial incision is closed by con-
tinuous suture using 4.0 Vicryl and a small tapered needle (Fig. 2G). Prior to
closing the myometrial incision, the uterine cavity is flushed extensively with
warm sterile saline to minimize bleeding and adhesions.
If the corpus luteum is required for analysis, a small incision is made at the
site of the ovarian stigma using a number 15 scalpel blade. The corpus luteum
is gently teased out of the ovary using a small curved hemostat. The incision is
closed with 4.0 Vicryl suture.
Following the endometriectomy and lutectomy, the peritoneal cavity is
extensively flushed with warm saline until there is minimal blood in the flush.
This extensive flushing prevents significant adhesion formation and permits
the multiple use of these animals as permitted by the Institutional Animal Care
and Use Committee (IACUC) guidelines.
105
8/29/05, 11:18 AM
Fig. 2. Step-by-step illustration of the endometriectomy procedure in the baboon to harvest endometrial tissues.
105
106 Fazleabas
The peritoneum and fascia are closed using 2.0 Vicryl on a tapered needle
using a simple interrupted suture technique. The subcutaneous tissue is closed
with a 3.0 Vicryl simple continuous suture line and the skin is then closed with
3.0 Vicryl with a continuous subcuticular suture (Fig. 2H). This is a subdermal
suture that ensures that the skin suture will not be loosened by the animal.
The Alzet minipump that has been placed subcutaneously is then removed
following a subcutaneous incision. The tip of the cannula attached to the pump
is cut and it is anchored to the body wall using a 0.5-cm piece of silastic tubing
and a 2.0 silk suture. This provides a landmark for locating the cannula for the
next infusion (see Note 3). The skin incision is closed using 3.0 Vicryl and a
subdermal suture. The amount of solution left in the pump is measured by
aspirating the fluid using a 25-gauge needle provided by the manufacturer.
This provides assurance that the expected volume of hormone has been infused
and that the pump has functioned appropriately (see Notes 4 and 5).
3.5. Analysis of Endometrial Tissues
The endometrial tissues obtained by endometriectomy are transported to the
laboratory in ice-cold Ca2+/Mg2+-free Hank’s balanced salt solution (see Note
6). The tissue is carefully dissected under a microscope under sterile condi-
tions so that the luminal surface is exposed. Using a sharp, single-edged blade,
portions of the tissue are fixed in the appropriate fixatives for histological and
immunocytochemical analyses or for in situ hybridization (9). Tissues can also
be snap frozen in liquid nitrogen for RNA extraction (9) or subjected to enzy-
matic digestion for stromal and epithelial cell isolation (14,15). In general,
following CG stimulation, the amount of functionalis tissue harvested is approx
500–700 g. Figure 3 provides a composite example of the response of the
baboon endometrium to CG stimulation during the window of receptivity (9).
None of the changes are evident if the hCG had been heat inactivated prior to
infusion (9).
107
4. Notes
1. The cannulated animals could serve as their own controls. Surgeries could be
scheduled on day 10 or 14 post ovulation of a regular menstrual cycle. Endome-
trial tissues can be obtained by endometriectomy in the absence of CG stimula-
tion, which reduces between animal variability with or without treatment. If this
procedure is selected, the cannula is inserted into the oviduct after the
endometriectomy. The open end of the cannula is exteriorized through the flank
incision and sutured to the body wall using a 0.5-cm piece of silastic tubing. The
animals are rested for three consecutive menstrual cycles prior to the next sur-
gery. For CG treatment, a small incision is made above the silastic anchor and the
cannula is gently extruded from under the skin. The cannula is flushed with the
treatment solution and inserted into the metal tip of the Alzet minipump. The
pump is placed in the subcutaneous pocket and the treatment initiated on day 6
post ovulation. These manipulations do not require an incision into the animal’s
body cavity at the initiation of treatment.
2. To determine if the effects are directly on the uterine endometrium, the treatment
paradigm could be done in ovariectomized animals following hormone replace-
ment (16). The cannula is placed into the oviduct at the time of ovariectomy and
anchored to the body wall. CG stimulation can be initiated following the sequen-
tial treatment with estrogen and progesterone implants to mimic hormonal
changes during the menstrual cycle (9,16,17).
3. Over a period of time, the cannula that is placed subcutaneously for easy access
becomes brittle. This appears to be primarily to the result of a granulation reac-
tion. Because it is less pliable, insertion into the metal post on the Alzet osmotic
minipump can be more difficult. This can be overcome by expanding the open
end of the cannula with 21-gauge stub adapter or by using the tip of the dissecting
scissors to expand the top of the tubing. Once inserted into the pump, the cannula
is secured using 2.0 silk ties.
4. The procedure describes infusion of CG to determine the role of the major pri-
mate embryonic signal on uterine receptivity. However, this cannulation proce-
dure can be used for infusing a variety of other hormones or growth factors in the
presence or absence of CG to determine individual or synergistic effects of these
factors on endometrial function during the window of receptivity.
5. Stimulation with CG via the Alzet pump and cannula is only effective upto 14 or
15 days post ovulation. Longer infusions do not provide sufficient luteotrophic
stimulation to maintain the corpus luteum. If long-term treatment with CG (up to
day 18 post ovulation) is required, then the animals can be given hCG injections
twice daily for up to 12 d, beginning on day 6 post ovulation. This regimen mim-
ics hormonal and endometrial changes that are comparable to the initial stages of
pregnancy. Details of this procedure have been previously published (17).
6. In both cycling and CG treated animals the uterine lumen can be flushed at the
time of surgery to obtain uterine flushings. Following exteriorization of the
uterus, the assisting surgeon clamps the cervix and the oviducts with his fingers.
Five to ten milliliters of Ca2+/Mg2+-free Hands Buffered saline is aspirated into a
Acknowledgments
The expert assistance of the Veterinary Staff at the Biological Resources
Laboratory and the technical assistance of Ms. Allison Brudney is gratefully
acknowledged. These studies have been supported by National Institutes of
Health (NIH) grants HD29964 and HD42280.
References
1. Paria, B. C., Reese, J., Das, S. K., and Dey S. K. (2002) Deciphering the cross-talk
of implantation: advances and challenges. Science 296, 2185–2188.
2. Jaeger, L. A., Johnson, G. A., Ka, H., et al. (2001) Functional analysis of autocrine
and paracrine signaling at the uterine-conceptus interface in pigs. Reprod. Suppl.
58, 191–207.
3. Spencer, T. E. and Bazer, F. W. (2002) Biology of progesterone action during
pregnancy recognition and maintenance of pregnancy. Front. Biosci. 7, d1879–
d1898.
4. Fazleabas, A. T. and Strakova, Z. (2002) Endometrial function: cell specific
changes in the uterine environment. Mol. Cell. Endocrinol. 186, 143–147.
5. Rao, C. V. (2001) An overview of the past, present, and future of nongonadal LH/
hCG actions in reproductive biology and medicine. Semin. Reprod. Med. 19, 7–17.
6. Licht, P., von Wolff, M., Berkholz, A., and Wildt, L. (2003) Evidence for cycle-
dependent expression of full-length human chorionic gonadotropin/luteinizing
hormone receptor mRNA in human endometrium and decidua. Fertil. Steril. 79
(Suppl 1), 718–723.
7. Licht, P., Russu, V., Lehmeyer, S., and Wildt, L. (2001) Molecular aspects of
direct LH/hCG effects on human endometrium-lessons from intrauterine
microdialysis in the human female in vivo. Reprod. Biol. 1, 10–19.
8. Licht, P., Russu, V., and Wildt, L. (2001) On the role of human chorionic gona-
dotropin (hCG) in the embryo–endometrial microenvironment: implications for
differentiation and implantation. Semin. Reprod. Med. 19, 37–47.
9. Fazleabas, A. T., Donnelly, K. M., Srinivasan, S., Fortman, J. D., and Miller, J. B.
(1999). Modulation of the baboon (Papio anubis) uterine endometrium by chori-
onic gonadotrophin during the period of uterine receptivity. Proc. Natl. Acad. Sci.
USA 96, 2453–2458.
10. Banaszak, S., Donnelly, K. M., Brudney, A., Chai, D., Chwalisz, K., and
Fazleabas, A. T. (2000) Modulation of the action of chorionic gonadotrophin on
10
The Common Marmoset Monkey as a Model
for Implantation and Early Pregnancy Research
Summary
This chapter describes methods used to investigate implantation in the common marmoset
monkey, Callithrix jacchus. A reverse-transcriptase polymerase chain reaction-strategy with
which to detect transcripts for steroid receptors and enzymes involved in estradiol biosynthesis
is described, and an immunohistochemistry approach for detecting proteins within the implan-
tation site is presented.
Key Words: Marmoset; early pregnancy; steroid receptors; aromatase; 17β-hydroxysteroid
dehydrogenase type 1 and type 7.
1. Introduction
The marmoset monkey (Callithrix jacchus) belongs to the New World Mon-
keys and is widely used as a primate model for reproductive medicine (1,2).
One of its advantages is its small size, which is the reason why it is easy to
handle and breed. The marmoset has a high fecundity and has no restricted
breeding season like other laboratory primates such as rhesus monkeys. Because
its cycle can be controlled by prostaglandin F2α (PGF2α) application (3), exact
prognosis of ovulation and therefore status of pregnancy is possible. The mar-
moset monkey has a placenta hemochorialis like humans and, consequently, it
shows similarities to the human situation in morphology and function (4). Our
knowledge about factors concerning the implantation process in humans is lim-
ited as a result of ethical constraints, which make primate models necessary.
Furthermore, there is an urgent need for more information on mechanisms of
implantation. This is easily understandable in view of current progress in repro-
ductive medicine, especially because pregnancy rates in assisted reproduction
are still unsatisfactory (5).
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
111
2. Materials
2.1. Reproductive Staging of Animals
1. Adult (>18 mo old) marmoset monkeys. Characteristics: 8- to 10-d follicular phase
and 18- to 20d luteal phase (1); no visible menstruation, nonseasonal primate.
2. PGF2α (0.8 µg per animal, Estrumate, Pitman-Moore, Germany) application can
induce luteolysis when applied between days 10–15 of luteal phase (6).
3. Immunoassays (6):
a. Progesterone for monitoring cycle stage (detection range: 0.1–50 ng/mL).
b. Relaxin (RLX) for early pregnancy detection (detection range: 0.02–5 ng/mL).
4. Ultrasound examination with 7.5-, 10-, and 15-MHz probes (Logiq 400 Pro CL,
General Electrics, Solingen, Germany) of ovarian and uterine activity throughout
the cycle and pregnancy.
2.3. Immunohistochemistry
1. Fixation solution: 4% paraformaldehyde, pH 7.3.
2. Buffer for microwave pretreatment: 10 mM citrate buffer, pH 6.0 at 120°C.
3. Tris-buffered saline (TBS), poly-L-lysine (0.01%).
4. ABC-method (DAKO Diagnostika, Hamburg, Germany; Vectastain, Vector
Laboratories, Burlingame, CA, USA).
5. Primary antibodies:
a. Monoclonal mouse antibody against estradiol α receptor (ERα; cat. no. B10,
Euromedex; Souffel Weyersheim, France).
113
Transcript GenBank Forward primer Reverse primer (°C) (bp)
Aromatase (ARO) AY034779 5' ACA ACT CGG CCC 5' AGG AGC TGC AAT CAG 60 498
CTC TTT AT 3' CAT TT 3'
Estrogen receptor α (ERα) X03635 (human) 5' ATG ACC ATG ACC 5' CGG AGA CAC GCT GTT 58 315
CTC CAC AC 3' GAG T 3'
Progesterone receptor (PR) Z86038 5' GTA TTC CAA ATG 5' AAC CAA TTG CCT TGA 60 591
AAA GCC AAG C 3' TGA GC 3'
17-β-Hydroxysteroid AF272013 5' GGC CTG CAC TTG 5' GGC CTG CAG CAT CCG 60 330
Marmoset Implantation/Early Pregnancy Model
8/29/05, 11:18 AM
gene (18S) CCA CAT CCA A 3' GCA TCG A 3'
113
114 Einspanier et al.
3. Methods
3.1. Reproductive Staging of Animals (see Notes 1–4)
The analysis of progesterone and RLX content in the peripheral blood (~2
times per week) provide a method of cycle (~28 d) and pregnancy (~144 d)
classification. Application of PGF2α during mid luteal phase (10–15 d luteal
phase) induces luteolysis of the formed corpora lutea and the initiation of new
growing follicles. Eight to ten days later, ovulation of preovulatory follicles
(1–4) occurs, followed by an increase of blood progesterone concentrations
above 10 ng/mL (1). For exact tissue collection, cycle staging is further con-
firmed by transabdominal ultrasound examination (8). This examination is car-
ried out on unshaved as well as unsedated marmoset monkeys for a time period
of approx 10 min. By ultrasound examination, identification of the number of
follicles and corpora lutea is possible, as well as the status of the uterus (preg-
nant vs nonpregnant, see Note 4). RLX content analysis in peripheral blood
allows early pregnancy detection at day 15 of luteal phase (6).
3.2. Tissue Samples
Uteri are collected by hysterectomy from cyclic and pregnant common mar-
moset monkeys (German Primate Centre, Germany) and immediately fixed in
4% buffered formaldehyde up to 8 h or in liquid nitrogen. The following cycle
stages are routinely collected: mid luteal phase (days 8–12 of luteal phase;
nonpregnant) and pregnancy stages (days 17–135 of pregnancy). Samples from
4. For steroid receptor detection, the paraffin sections are subjected to an antigen
retrieval protocol, incubating them for 10 min in 10 mM citrate buffer, pH 6.0 at
120°C, then allowing them to cool over 30 min.
4. Notes
1. Experiments with marmosets should be approved by the local ethics commission
on animal welfare.
2. In the wild, marmoset monkeys live in social groups of 8–15 members; therefore,
a social structure is recommended for their welfare in captivity. Moreover, single
housing of females often results in variation of cycle length. For reproductive
studies, at least pair housing (one adult male and female) is required. Also, if
collection of tissue from nonpregnant females is necessary, it is recommended
that females be kept with vasectomized or castrated males.
3. The use of trained monkey is advantageous for blood collection, injections, or
ultrasound examination. Training greatly decreases stress. Stress can induce ir-
regular cycles or abortion, resulting from high glucocorticoid levels.
4. Collection of appropriately staged tissue is dependent on accurate endocrinologi-
cal monitoring. The marmoset is a particularly good model as compared with Old
World monkeys because of its sensitivity to PGF2α. PGF2α induces luteolysis and
the initiation of a new wave of follicle development. As ovulation approaches,
more frequent blood collections facilitate accurate determination of ovulation
and the initiation of pregnancy. This requires the availability of rapid assay sys-
tems within 24 h. Hormonal profiles for progesterone, estradiol, chorionic gona-
dotropin, and RLX provide the requisite information for determining cycle and
pregnancy staging. Further confirmation of cycle or pregnancy stage is given by
using ultrasound examination. Detection of pregnancy is possible on day 15 of
luteal phase by the appearance of a double endometrial echo indicating fluid ac-
cumulation in the uterus (8). Taken together, all of these methods enable exact
staging and therefore collection of the marmoset tissues for experimentation on
early pregnancy.
5. Marmoset organs and implantation sites in general are of limited size. Accurate
manual dissection under the stereomicroscope is a prerequisite for valid results.
When drawing conclusions from RT-PCR experiments, the origin of RNA
extracted must be clearly defined. Possible “contamination” by surrounding
tissue with a presumed differing gene expression profile has to be considered. It
is recommended to verify RT-PCR data by immunohistochemistry and vice versa.
6. Two alternative methods for disrupting and homogenizing frozen tissues in RLT
buffer containing β-mercaptoethanol (β-Me/RLT) are used. For tissues smaller
than 4 mm3, a conventional rotor-stator homogenizer is used to lyse the tissue
directly in the appropriate volume of buffer β-Me/RLT. For larger tissue samples
or whole organs, a cryostat microtome is used for sectioning into 30-µm slides at
–30°C, which are then lysed in buffer β-Me/RLT. Quick lysis of cryopreserved
samples is important during homogenization in order to inactivate endogenous
RNases.
7. Be cautious of contamination with RNase in processes like RNA isolation and
reverse transcription. The risk of contamination can be decreased by using a dedi-
cated set of automatic pipettors or by using disposal tips with aerosol barrier
filters certified to be free of RNase. Preparation of all solutions and buffers with
RNase-free glassware, diethylpyrocarbonate (DEPC)-treated water, and chemi-
cals reserved for work with RNA is also recommended. Additionally, use
microfuge tubes certified to be free of RNase.
8. Homologous Callithrix sequences are not available; therefore the HUSAR pro-
gram package has been used to deduce PCR primers with heterologous cDNAs
(e.g., from human or mouse). Annealing temperature as well as cycle number is
crucial for the reliability of the resulting transcript determination. More than 40
cycles should never been used to estimate relative mRNA concentrations. All
PCR products generated with heterologous primer pairs should be sequenced.
Newly generated sequences should then be used to design new homologous
primer pairs. For quantification purposes real-time RT-PCR is recommended.
9. The literature describing immunohistological detection of steroid receptors is not
entirely consistent. One contributing factor is the use of different fixation times.
We have analyzed different fixation periods for steroid receptor detection in mar-
moset tissues. We observe a massive decline in steroid receptor expression in
tissues 1 cm3 with fixation durations longer than 24 h, optimal fixation duration
is up to 8 h. Shrinkage or distortions of these fragile tissues can also occur with
too long fixation period.
10. An example of our use of these techniques is described as follows. First, the
temporal dynamics of steroid receptor expression has been analyzed in total RNA
preparations from whole marmoset uteri throughout nonconceptive as well as
conceptive cycles. Transcripts for both, ERα and PR, are expressed throughout
mid luteal phase and entire pregnancy (Fig. 1). Secondly, using immunohistol-
ogy, ERα is strongly expressed within both maternal and fetal compartments
during early pregnancy (day 25), whereas PR is mainly expressed within the
maternal compartment. The expression levels (gene and protein) for both steroid
receptors are relatively constant as pregnancy progresses (Figs. 1 and 2). Examina-
tion of local expression and distribution patterns provides insights into embryo–
uterine interactions during implantation. The absence of apparent differences in
steroid receptor expression between nonconceptive and conceptive uteri suggest
other regulatory factors are involved.
Similarly to ERα, the three enzymes catalyzing the last steps in the synthesis
of estradiol (ARO, 17HSD1, and 17HSD7) are detectable in nonconceptive and
conceptive marmoset uteri by RT-PCR (Fig. 1). 17HSD1 mRNA expression is
minimal and restricted to early pregnancy, whereas 17HSD7 is expressed later in
pregnancy (day 95) and is undetectable by the end of pregnancy. ARO is detected
at day 35 of pregnancy and through the remainder of pregnancy. Transcripts for
enzymes involved in estradiol synthesis are present in the uterus at the time of
implantation (7). Again, the immunohistochemical results for ARO, 17HSD1,
and 17HSD7 are consistent with the RT-PCR analyses (see Fig. 1). There is a
local up regulation of 17HSD1 and 17HSD7 mainly in the fetal compartments
with weak expression in the maternal compartment during early pregnancy (day
25 of pregnancy). ARO is weakly expressed in the fetal as well as in the maternal
compartments during early pregnancy (Fig. 2).
These complementary approaches for monitoring gene and protein expression
provide insights into the etiology of pregnancy failure and potential therapeutic
strategies. The common marmoset is an excellent model to obtain appropriately
staged tissues, which closely mimics important features of human reproduction (4).
11. Fetal vs maternal compartments can be distinguished using vimentin and
cytokeratin immunostaining. Trophoblasts are visualized by using cytokeratin
staining, whereas decidual cells and stromal cells at the implantation side show
positive staining for vimentin.
Acknowledgments
The authors would like to thank the German Primate Centre for the marmo-
set tissue. This work was supported by grants from the Deutsche
Forschungsgemeinschaft [DFG Ei 333/6-3 and Ei333/11-4 (A.E.) and Ei 296/
10-2 (R.E.)].
References
1. Hearn, J. P. (1983) The common marmoset (Callithrix jacchus), in Reproduction
in New World Primates. New Models in Medical Science (Hearn, J. P., ed.). MTP,
Lancaster, UK: pp. 181–215.
2. Einspanier, A. and Gore, M. (2005) Definition of primate model: female fertility,
in The Laboratory Primate: Reproduction Part 1 (Wolfe-Coate, S., ed.). Elsevier.
3. Summers, P. M., Wennink, C. J., and Hodges, J. K. (1985) Cloprostenol-induced
luteolysis in the marmoset monkey (Callithrix jacchus). J. Reprod. Fertil. 73,
133–138.
III
IN VITRO TROPHOBLAST AND PLACENTAL MODEL
SYSTEMS
124 Quinn, Kunath, and Rossant
Mouse Trophoblasts 125
11
Mouse Trophoblast Stem Cells
Summary
The trophectoderm is one of the earliest cell types to differentiate in the forming mamma-
lian embryo. It is responsible for the initial implantation and the formation of the trophoblast
components of the placenta, an organ essential for nutrient and waste exchange between the
fetus and its mother. The trophoblast can be modeled in vitro using trophoblast stem cells.
Trophoblast stem cells require fibroblast growth factor (FGF)4, heparin, and contact with
embryonic fibroblasts, or fibroblast-conditioned medium. They grow as tight epithelial colo-
nies, which express markers of the early trophectoderm and have been shown to contribute to
all of the components of the placenta through chimera studies. These cells can be passaged
indefinitely and can be differentiated by removal of FGF4 and fibroblasts and will express
genetic markers of later placental cell types. This chapter will discuss the initial derivation of
trophoblast stem cells from the blastocyst stage, maintenance, differentiation, flow cytometry
and transfection techniques that can be used with these cells.
Key Words: Trophoblast stem cell; trophectoderm; derivation; culture maintenance; differ-
entiation; flow cytometry; transfection.
1. Introduction
In a mouse blastocyst at embryonic day (E) 3.5, the specification of the
trophectoderm and the inner cell mass is the first differentiation to occur. By
E 4.5, there are three cell types: the primitive endoderm, which will form the
visceral and parietal endoderm; the primitive ectoderm, which will form the
embryo proper; and the trophectoderm, which will produce all the trophoblast
tissues (1,2). The trophoblast is essential for survival of the mammalian con-
ceptus because it mediates implantation and ultimately creates the placenta,
which allows nutrient and waste exchange between the fetus and its mother (3).
The outer cells of the blastocyst—the trophectoderm—can be divided into
two distinct components: polar and mural (4). The mural trophectoderm is com-
prised of the cells that are most distal to the inner cell mass. These cells will
differentiate into primary trophoblast giant cells. Giant cells undergo
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
125
126 Quinn, Kunath, and Rossant
endoreduplication, which results in large polyploid cells (5). The mural tro-
phectoderm and its resulting primary giant cells are important for the initial
implantation of the blastocyst (3). This differentiation continues laterally toward
the border of the inner cell mass (4). The polar trophectoderm is located in direct
contact with the inner cell mass (4). These cells remain diploid and continue to
divide, giving rise to the trophoblast lineage. This includes the extra-embry-
onic ectoderm and the ectoplacental cone and, eventually, the components of
the mature chorioallantoic placenta—the spongiotrophoblast, labyrinth and
giant cell layer (3). This chapter discusses trophoblast stem cells as an in vitro
model of the trophoblast cell lineage.
Embryonic stem cells from the primitive ectoderm are a well-established in
vitro model (6–8). These cells can be genetically manipulated and have pro-
vided insight into the development of the embryo and essential genes involved
in this process (9). Trophoblast stem (TS) cells may be used in a similar fash-
ion to elucidate the mechanism of differentiation and the role of genes and cell
types in the development of the placenta.
Trophoblast stem cells are diploid, permanent, and self-renewing when they
are maintained in stem cell conditions. They express markers of the trophecto-
derm, extra-embryonic ectoderm, and ectoplacental cone (see Note 1). TS cells
can be derived from E 3.5 blastocysts, the extra-embryonic ectoderm from E 6.5
conceptuses, and the chorionic ectoderm from E 7.5 to E 10 embryos (10–12).
Specific mutant TS cell lines can be developed if the gene in question is not
required for stem cell initiation or maintenance. TS cells require: fibroblast
growth factor (FGF)4 , heparin, and embryonic fibroblasts (EMFIs) or embry-
onic fibroblast-conditioned medium (FCM) to maintain their stem cell mor-
phology of tight adherent epithelial colonies. These cells have been shown to
contribute to all trophectoderm derivates through chimera experiments and can
be maintained in culture indefinitely (10).
When stem cell factors are removed, TS cells differentiate and show an in-
crease in expression of genetic markers for the spongiotrophoblast, labyrinth,
and giant cells and decrease in expression of genes from the blastocyst, extra-
embryonic ectoderm, and ectoplacental cone. Ultimately, these cells become ter-
minally differentiated giant cells with large cytoplasm and high ploidy (10).
This chapter describes methods for TS cell derivation from blastocysts,
maintenance, differentiation, fluorescence-activated cell sorting (FACS), and
transfection.
2. Materials
2.1. Embryonic Fibroblasts
1. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, St. Louis, MO, Cat. Nos.
D2650 and D5879).
Mouse Trophoblasts 127
2.6. Transfection
1. LipofectAMINE PLUS (Gibco-BRL, Gaithersburg, MD, cat. no. 10964-013):
RPMI culture medium, PLUS reagent, lipofectamine.
Mouse Trophoblasts 129
3. Methods
The methods detailed in this chapter describe the (1) the isolation and cul-
turing of mouse embryo fibroblasts, (2) derivation of TS cells from blastocysts,
(3) maintenance, and (4) differentiation of TS cells, as well as (5) protocols to
perform flow cytometry to sort cells and analyze DNA content and (6) to per-
form DNA transfection.
Fig. 1. Fully expanded blastocyst collected by uterine flushing which can be used to
derive trophoblast stem cells. The polar trophectoderm (PT) overlying the inner cell
mass (ICM) will give rise to most of the trophectoderm derivatives and the mural
trophectoderm (MT) will mediate implantation and give rise to primary giant cells.
Phase contrast, scale bar 50 µm.
Fig. 3. Early trophoblast stem (TS) cell colony surrounded by endoderm-like cells.
These round refractile cells are present in TS cell cultures if the blastocyst outgrowth
is allowed to get too large prior to disaggregation. The endoderm-like cells are diffi-
cult to alleviate since they do not require fibroblast-conditioned medium or fibroblast
growth factor-4 to continue to grow. Phase contrast, scale bar 250 µm.
3. This process needs to be repeated every 48 h until the TS cells reach approxi-
mately 50% confluent and require passaging.
Fig. 4. Trophoblast stem (TS) cell colony grown on plastic tissue culture dish in TS
cell conditions. The stem cell colony has tight epithelial borders. Giant cells (GC) can
be seen at the edge of the colony. Phase contrast, scale bar 250 µm.
3.3.4. Freezing/Thawing
TS cells can be frozen for indefinite periods and then later thawed for use.
This allows a certain level of security, because it is not necessary to derive new
TS cells every time one wants a new plate, and it is possible to expand and
store lines of interest for extended periods of time.
3.3.4.1. FREEZING
1. Obtain cells in suspension (1 mL) using the protocol outlined under Subhead-
ing 3.3.2.
2. Add 1 vol of 2X Freezing medium cooled to 4°C.
3. Place 1 mL in a freezing vial.
4. If freezing several lines, keep those in the 2X freezing medium on ice until all
can go in the freezer.
5. Place tubes in an isopropanol slow-freeze container.
6. Slowly freeze in –70°C Freezer for at least 48 h.
7. Transfer to liquid nitrogen.
Mouse Trophoblasts 139
3.3.4.2. THAWING
To recover a cell line from a frozen vial, culture and passage freshly thawed
cells at least twice to ensure they have recovered sufficiently before beginning
any experiments. After the initial thaw, it is normal to have a large number of
floating cells, some giant cells and some stem cell colonies (see Note 12). If
the plate appears confluent (it may require aspirating the media and rinsing
with PBS to see through the floaters), the cells require a passage. Otherwise,
continue to feed the cells until they reach 80% confluency then follow the pro-
tocol for passaging. Cells often recover more rapidly when they are thawed
onto EMFIs.
1. Remove the vial of cells from liquid nitrogen or –70°C freezer.
2. Warm in 37°C water bath until just thawed, approx 3 min.
3. Use a 1 mL pipet to transfer contents of vial to a 14-mL Falcon tube containing
1 mL TS medium.
4. Spin at 200g for 3 min.
5. Aspirate to remove DMSO contained in the freezing medium.
6. Resuspend into an appropriate medium depending on if they are on EMFIs or on
plastic.
7. Plate all cells from vial onto a surface area, which is smaller than the original
plate.
8. 24 h after initial plating aspirate medium (expect many floaters).
9. Rinse with PBS.
10. Add appropriate fresh medium.
3.4. TS Cell Differentiation
As TS cells differentiate in vitro, markers of later cell types of the trophec-
toderm lineage show an increased expression whereas markers of the blasto-
cyst, extra-embryonic ectoderm, and ectoplacental cone show a decrease (see
Note 1). The changes in gene expression are associated with changes in cell
morphology and DNA content. These changes are indicative of cells that are
changing from tight epithelial TS cell colonies to intermediate cell types and
finally to terminal giant cells with expansive cytoplasm and large polyploid
nuclei. Most TS cell cultures will differentiate to predominantly giant cells by
the sixth day of differentiation although some genotypes require a longer pe-
riod of time (Fig. 5).
The rate of differentiation is influenced by several factors, including geno-
type and cell density. It is important to select a consistent density to work with.
TS cell plated at a very low density differentiate very rapidly to giant cells and
do not show an increased expression of intermediate markers of
spongiotrophoblast and labyrinth cells. Typically, a density that results in the
culture becoming nearly confluent by day 6 of differentiation is used (~2 × 105/
60-mm plate). A protocol for the induction of differentiation is presented.
140 Quinn, Kunath, and Rossant
Fig. 5. Differentiated trophoblast stem cells grown on plastic dishes. These cells
have been without fibroblast-conditioned medium + F4H for 6 d and show a character-
istic giant cell morphology with a large cytoplasm and nucleus. Phase contrast, scale
bar 250 µm.
4. Notes
1. TS cells show regulation of different genetic markers throughout differentiation.
Table 1 outlines a few of these genes as well as some genes that can be used to
screen colonies for endoderm-like cells, which might be contaminating a culture.
2. The protocols outlined in this chapter are for deriving TS cells from E 3.5 blasto-
cysts. Embryos flushed at E 2.5 and cultured overnight can be used to plate on
EMFIs for TS cell derivation.
3. TS cells have not been successfully derived from C57BL6 mice. Naturally mated
ICR mice can carry 8–15 embryos.
4. Mouth pipets or finger pipets are required to manipulate blastocysts. Pulling pi-
pets is a delicate process—practice first! To control the blastocysts within the
pipet, ensure that there are at least three air bubbles before attempting to pick up.
In order to keep the mouthpiece clean, place the cap of a 14-mL Falcon tube
(with a hole in it) on the tubing below the mouthpiece. The mouth pipet can be
covered when not in use with a cap of a 14-mL Falcon and the main body of the
Falcon tube. Further directions for mouth pipetting can be found on page 177 in
ref. 9.
5. When flushing blastocysts, the uterine horn will bulge when KSOM is added and
a slightly cloudy liquid will emerge from opposite end. It is important not to
squeeze or puncture uterine horn.
6. EMFIs can “condition” the medium for approx 10 d; after that point 70% FCM is
required.
7. If adding new medium before the blastocysts have fully attached, be careful not
to dislodge or aspirate them.
8. Ensure that each blastocyst and subsequent TS line are kept separate from all
others to avoid contamination.
9. Primitive endoderm-like cells are round and highly refractile. They can be found
in TS cultures if the blastocyst outgrowth becomes too large before the initial
dissociation. These cells grow well in TS medium with or without F4H and are
very difficult to remove.
Mouse Trophoblasts 145
Table 1
Genes Used to Characterize TS Cell Cultures Throughout Differentiation
TS cell expression
Gene Name Expression profile Reference
10. Table 2 provides guidelines for the appropriate dish, amount of medium, and
passage requirements for various stages throughout the derivation of TS cell lines.
11. Table 3 provides the area and requirements of commonly used tissue culture
dishes in the maintenance and differentiation of TS cells.
12. Cultures that have a high rate of floating cells should be rinsed thoroughly.
Aspirate the media, rinse with room temperature PBS, and aspirate. Add PBS
for 5 min, aspirate, and feed cells with fresh medium.
13. Cells cannot be differentiated on EMFIs, because they will condition the media
and will inhibit differentiation.
14. Giant cells are very adherent and are difficult to trypsinize. If after 5 min of
trypsinization the cells remain attached, try to dislodge cells by pipetting up and
down in trypsin only before stopping the reaction with TS medium. If the cells
still remain attached, use a cell scraper to dislodge the rest of the cells.
15. Ensure that all cells are in suspension, especially giant cells, which adhere very
strongly to the plate.
146 Quinn, Kunath, and Rossant
Table 2
A Guideline for Passaging at Different Stages Throughout Derivation
Amount Amount
Stage Size of dish of medium to passage
Table 3
Tissue Culture Dishes and Conditions
Well Area cm2 Volume to feed Volume of trypsin
16. Cells used in FACS sorting must be in a single cell suspension or they will be lost
in the filtration step.
17. Transient transfections in TS cell occurs with a success rate of approx 1%.
18. In transient transfections, the ratio of reporter plasmid to gene of interest must be
optimized; often, higher concentrations of DNA are helpful.
19. Approximately 50% cell death is expected with optimal transfection efficiency
when PBS is used.
20. On a 10-cm plate, there are often approx 100 drug-resistant colonies present after
12 d.
References
1. Gardner, R. L. (1982) Investigation of cell lineage and differentiation in the ex-
traembryonic endoderm of the mouse embryo. J. Embryol. Exp. Morphol. 68, 175–
198.
2. Snell, G. D. and Stevens, L. C. (1966) Early embryology, in Biology of the Labo-
ratory Mouse (Green, E. L., ed.). McGraw-Hill, New York: pp. 205–245.
Mouse Trophoblasts 147
21. Cross, J. C. (2000) Genetic insights into trophoblast differentiation and placental
morphogenesis. Semin. Cell. Dev. Biol. 11, 105–113.
22. Parr, B. A., Cornish, V. A., Cybulsky, M. I., and McMahon, A. P. (2001) Wnt7b
regulates placental development in mice. Dev. Biol. 237, 324–332.
23. Lescisin, K.R., Varmuza, S., and Rossant, J. (1988) Isolation and characterization
of a novel trophoblast-specific cDNA in the mouse. Genes. Dev. 2, 1639–1646.
24. Deussing, J., Kouadio, M., Rehman, S., Werber, I., Schwinde, A., and Peters, C.
(2002) Identification and characterization of a dense cluster of placenta-specific
cysteine peptidase genes and related genes on mouse chromosome 13. Genomics
79, 225–240.
25. Ma, G. T., Soloveva, V., Tzeng, S. J., et al. (2001) Nodal regulates trophoblast
differentiation and placental development. Dev. Biol. 236, 124–135.
26. Basyuk, E., Cross, J. C., Corbin, J., et al. (1999) Murine Gcm1 gene is expressed
in a subset of placental trophoblast cells. Dev. Dyn. 214, 303–311.
27. Anson-Cartwright, L., Dawson, K., Holmyard, D., et al. (2000) The glial cells
missing-1 protein is essential for branching morphogenesis in the chorioallantoic
placenta. Nat. Genet. 25, 311–314.
28. Yu, C., Shen, K., Lin, M., et al. (2002) GCMa regulates the syncytin-mediated
trophoblastic fusion. J. Biol. Chem. 277, 50,062–50,068.
29. Stecca, B., Nait-Oumesmar, B., Kelley, K. A., Voss, A. K., Thomas, T., and
Lazzarini, R. A. (2002) Gcm1 expression defines three stages of chorio-allantoic
interaction during placental development. Mech. Dev. 115, 27–34.
30. Colosi, P., Swiergiel, J. J., Wilder, E. L., Oviedo, A., and Linzer, D. I. (1988)
Characterization of proliferin-related protein. Mol. Endocrinol. 2, 579–586.
31. Faria, T. N., Deb, S., Kwok, S. C., Talamantes, F., and Soares, M. J. (1990) On-
togeny of placental lactogen-I and placental lactogen-II expression in the devel-
oping rat placenta. Dev. Biol. 141, 279–291.
32. Shida, M. M., Jackson-Grusby, L. L., Ross, S. R., and Linzer, D. I. (1992) Placen-
tal-specific expression from the mouse placental lactogen II gene promoter. Proc.
Natl. Acad. Sci. USA 89, 3864–3868.
33. Campbell, W. J., Deb, S., Kwok, S. C., Joslin, J. A., and Soares, M. J. (1989)
Differential expression of placental lactogen-II and prolactin-like protein-A in
the rat chorioallantoic placenta. Endocrinology 125, 1565–1574.
34. Hamlin, G. P., Lu, X. J., Roby, K. F., and Soares, M. J. (1994) Recapitulation of
the pathway for trophoblast giant cell differentiation in vitro: stage-specific ex-
pression of members of the prolactin gene family. Endocrinology 134, 2390–2396.
35. Becker, S., Wang, Z. J., Massey, H., et al. (1997) A role for Indian hedgehog in
extraembryonic endoderm differentiation in F9 cells and the early mouse embryo.
Dev. Biol. 187, 298–310.
36. Dziadek, M. and Adamson, E. (1978) Localization and synthesis of
alphafoetoprotein in post-implantation mouse embryos. J. Embryol. Exp.
Morphol. 43, 289–313.
Connexins and Trophoblast Cell Lineage 149
12
Connexins and Trophoblast Cell Lineage Development
Summary
The mouse is a valuable model for studying basic mechanisms of gene regulation in tropho-
blast cell lineage differentiation. Elements of placental development are conserved across spe-
cies, including trophoblast proliferation, differentiation, migration, and vessel invasion. Among
the regulatory processes, direct intercellular communication between trophoblast cells via gap
junction channels seems to play a crucial role in placental development and physiology. Here
we describe in detail the generation of trophoblast stem (TS) cell lines from connexin-deficient
mice. The design of differentiation and proliferation assays are specified including marker gene
sets which are important for analyzing and comparing the differentiation capacity of the
connexin-deficient TS cell lines. Furthermore, we show that TS cells are capable of forming
tumors after subcutaneous injection into nude mice, providing the opportunity to investigate
trophoblast invasion into host vessels in vivo.
Key Words: Connexins; gap junction; placenta; trophoblast stem cells; trophoblast stem
cell tumors.
1. Introduction
Despite the critical role of the placenta in governing the outcome of preg-
nancy, there is limited information available about the molecules involved in
the differentiation of this organ. Failure of appropriate placental development,
especially in the first trimester, is associated with significant complications in
pregnancy, including miscarriage, preeclampsia, and intrauterine growth
restriction (1). The human placenta is difficult to study for several reasons.
Besides the ethical problems of abortion and the availability of sufficient tis-
sues for research, one major point is that the most important steps of tropho-
blast differentiation occur within the first weeks of gestation. The mouse model
is valuable and helpful for studying basic mechanisms of gene regulation in
trophoblast cell lineage differentiation. Placentation in the mouse and human
involves similar cell biological events, including trophoblast proliferation,
differentiation, migration, and vessel invasion. Among the genes regulating these
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
149
processes, the direct intercellular communication between trophoblast cells via gap
junction channels plays a crucial role in placental development and physiology.
Gap junctions are clusters of intercellular membrane channels that connect
the cytoplasm of two neighboring cells. Each cell contributes one half of the
channel (connexon), which is comprised of six connexin (Cx) subunits. The
hydrophilic central pore allows the transfer of ions and small molecules up to 1 kD
including nucleotides (cAMP, cGMP), inositol 1,4,5-trisphosphate (IP3), Ca2+, and
metabolites. The Cx gene family consists of 19 members in the mouse and 20
members in the human genome (2). Connexins show tissue-specific expression
and are temporally regulated during embryonic and placental development.
Gap junctions play an obligate role in cellular and tissue function that has been
proven by generating knockout mice. Recently, evidence is accumulating for a
dual role of gap junctions in signal transduction mediated not only by their
channel properties but, in addition, by the carboxy-terminus (C-terminus) of
the connexin protein (3). It has been shown that the C-terminus is able to inter-
act with other cellular components and that these protein–protein interactions
mediate intracellular signalling (4). Of interest are connexins that lead to a
placental phenotype if deleted such as Cx26, Cx31, and Cx45, providing a
strong rationale for examining the role of gap junctions in placental develop-
ment (5–7).
Cx26 knockout mice die in utero at day 9.5 post conception (pc) when the
chorioallantoic placenta starts to function. The main reason for this early death
in utero is impaired glucose uptake into the fetal compartment (5). Cx26 chan-
nels connect the two layers of syncytiotrophoblast in the labyrinth layer (5).
No obvious changes in trophoblast differentiation could be detected. Thus, the
Cx26 channel seems to serve as a channel for the diffusion of molecules
across the placental barrier but is not involved in trophoblast cell lineage
development.
Cx31 is expressed in the trophectoderm and, subsequently, in early tropho-
blast derivatives (extra-embryonic ectoderm, ectoplacental cone, chorionic ecto-
derm; see ref. 8). In the mature placenta, Cx31 stays expressed in the
spongiotrophoblast throughout pregnancy. If this channel is deleted, a loss of
more than 60% of the embryos between day 10.5 and 13 pc is observed (6).
The placental phenotype revealed a dramatically reduced size of the placenta
on day 9.5 pc with nearly no labyrinth and spongiotrophoblast but an abun-
dance of trophoblast giant cells. Clearly, the missing channel leads to an im-
balance along the trophoblast cell lineage differentiation in favor of enhanced
differentiation to giant cells. This phenomenon is accompanied by an acceler-
ated decline of proliferating trophoblast stem cells in the placenta. Forty per-
cent of the embryos survive because of a placental rescue starting around day
12 pc (6). Reasons for this partial rescue of Cx31-deficient placentas could be
the induction of the Cx43 channel in the spongiotrophoblast at day 10 pc, which
could serve the function of the Cx31 channel (6). The induction of additional
Cx43 channels accompanies the differentiation of spongiotrophoblast cells to
trophoblast giant cells. The terminally differentiated giant cells only express
Cx43 (6). Thus, the different trophoblast populations of the mouse placenta are
defined by a specific connexin expression pattern with Cx26 in the syncytiotro-
phoblast, Cx31 in the spongiotrophoblast, followed by a coexpression with
Cx43, whereas the terminal differentiated giant cells produce Cx43 exclusively.
Of particular interest is the Cx31 channel, because it is expressed in the early
trophoblast cell lineage and its loss is associated with a failure in trophoblast
differentiation (6).
In the past, it was difficult to study molecular mechanisms in early tropho-
blast development because of a lack of appropriate in vitro systems and the
technical problems of isolating trophoblast tissues without contamination with
maternal and embryonic tissues. Progress in investigating signal cascades re-
sponsible for appropriate placental development has been achieved by generat-
ing trophoblast stem (TS) cells. Rossant and her colleagues have established
permanently growing TS cell lines from the mouse blastocyst or the extra-
embryonic ectoderm in the presence of fibroblast growth factor (FGF)4 (see
ref. 9 and Chapter 11 of this volume). Upon removal of FGF4 or addition of
retinoic acid, TS cells are capable of differentiation (9,10). TS cells also effec-
tively develop into all trophoblast cell lineages in vivo, as shown by aggrega-
tion experiments and by blastocyst injection (9,10). Furthermore, this approach
gives the opportunity to establish TS cells from gene-deficient animals, such
as the Cx mutants, to get more insight in the associated sets of signaling mol-
ecules that are in charge of controlling placental differentiation. In comparison
with placental tissue, TS cells provide an easier tool with which to solve cell
and molecular mechanisms, especially for the application of genomic and
proteomic approaches.
To study the effect of a specific connexin in trophoblast differentiation, we
have established Cx26, Cx31, and Cx43 gene-deficient TS cell lines from blas-
tocysts of the corresponding knockout mice. Using these TS cell lines, the in-
fluence of a specific Cx inactivation on differentiation, proliferation, and
invasion capacity of the TS cell lines was investigated. TS cells provide a tool
with which to understand the null phenotype and a means of distinguishing
specific roles in trophoblast lineage development vs mature trophoblast cell
function. For example, Cx31 is implicated in trophoblast cell lineage develop-
ment, whereas Cx26 regulates transplacental transport (11).
In this chapter, the generation of TS cell lines from Cx knockout mice will be
described in detail, because generation of trophoblast stem cells from knockout
blastocysts seems to be accompanied by more methodological problems, espe-
cially if the genes that are deleted alter the differentiation pathway. Here we
describe marker gene sets, which are important to analyze the differentiation
capacity of Cx-deficient TS cell lines. Furthermore, we show that TS cells are
capable of forming tumors after subcutaneous injection into nude mice. These
tumors, unlike embryonic stem (ES) cell tumors, are only transient because of
the rapid differentiation into the invasive pathway (11). This differentiation
results in the formation of trophoblast giant cells that are not proliferative but
normally invade the decidual compartment and the maternal vessels. The dif-
ferentiation of TS cells into invasive giant cells provides the opportunity to
investigate trophoblast invasion into host vessels using the nude mouse model.
2. Materials
1. TS cell medium: RPMI 1640 (Gibco, Karlsruhe, Germany) supplemented with
20% heat-inactivated fetal bovine serum (Biochrom, Berlin, Germany), 1 mM
sodium pyruvate (Gibco, Karlsruhe, Germany), 100 µM β-mercaptoethanol
(Sigma, Munich, Germany), 2 mM L-glutamine (Gibco, Karlsruhe, Germany),
100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, Karlsruhe, Germany).
2. Mouse embryonic fibroblast (EMFI)-conditioned TS cell medium (EMFI-CM).
3. C-TS cell medium: 75% EMFI-CM, 25% TS cell medium, 25 ng/mL human
recombinant FGF4 (R&D-systems, Wiesbaden, Germany) and 1 µg/mL heparin
(Sigma, Munich, Germany).
4. TS cell freezing medium: C-TS cell medium containing 10% dimethylsulfoxide
(Merck, Darmstadt, Germany).
5. Cell dissociation medium: 1X trypsin-ethylenediamine tetraacetic acid (EDTA)
solution (Gibco, Karlsruhe, Germany) containing 0.25% trypsin and 1 mM
EDTA.
6. Standard protocol for RNA isolation from cell cultures and Northern blotting
equipment.
7. cDNA probes for Northern blotting. GenBank accession numbers are indicated
in parentheses: Cx26 (BC013634), Cx31 (X63099), Cx31.1 (M91236), Cx43
(NM010288), β-actin (X03672), Mash-2 (NM008553), Pl-1(M35662) and Tpbpa
(NM009411).
8. Male athymic nude mice (Han: NMRI nu/nu), 8–12 wk old (Animal Facility of
the University Hospital Essen, Essen, Germany).
3. Methods
3.1. Preparation of Fibroblast-Conditioned TS Medium
1. For preparation of EMFI-CM, 10.5 mL TS cell medium is incubated on confluent
10-cm plates of mitomycin C arrested EMFI for 72 h.
2. The conditioned medium is centrifuged (4000g, 15 min, room temperature), fil-
tered (0.2 µm) and stored at –20°C (see Note 1).
3. The plates of mitomycin C arrested EMFI can be reused for two more rounds of
EMFI-CM preparation.
4. Notes
1. We routinely use 75% of EMFI-CM to prepare the C-TS medium for growing
and maintaining TS cells in an undifferentiated state. Others report using 80%
(12), 70% (9,10) or 50% of EMFI-CM (13) to keep TS cells undifferentiated. The
optimal percentage should be empirically determined. In our experience, the FBS
and the FGF4 used are the most critical factors for a successful culturing of TS
cells. Several distributors and much FBS should be tested on established TS cell
lines, because some sera lead to very poor proliferation or enhanced spontaneous
differentiation of the cells. In our hands, FGF4 shows the best results on promot-
ing TS cell proliferation. Established TS cell lines may also be cultured using
FGF1 (14,15) or FGF2 (15), but when problems in TS cell cultures arise, we
recommend using FGF4. We did not observe an influence of the plastic ware
from different companies on cell viability.
Acknowledgments
The authors would like to thank Dr. J. Rossant for providing the cDNA for
Mash-2, Pl-1, and Tpbpa. We also thank Natalie Knipp, Gabriele Sehn, and
Georgia Rauter for excellent technical assistance in developing these methods.
This work was supported by grants from the National Institutes of Health (NIH)
(1R01 HD42558-01), the Deutsche Forschungsgemeinschaft (DFG Wi 774/
10-3), and the Deutscher Akademischer Austauschdienst (DAAD).
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NY: pp. 267–287.
13
Rcho-1 Trophoblast Stem Cells
A Model System for Studying Trophoblast Cell Differentiation
Summary
The biology of trophoblast cell development can be investigated using in vitro model sys-
tems. The Rcho-1 trophoblast stem cell line was derived from a rat choriocarcinoma and is an
effective tool for elucidating regulatory mechanisms controlling trophoblast cell differentia-
tion. In this chapter, we describe methods used in the maintenance and manipulation of the
Rcho-1 trophoblast cell line.
Key Words: Trophoblast differentiation; rat placenta; trophoblast giant cells; Rcho-1 tro-
phoblast stem cells; choriocarcinoma.
1. Introduction
Trophoblast cells possess specialized phenotypes and arise from a common
stem cell population directed along a multi-lineage differentiation pathway (1).
Trophoblast stem cells develop from the blastocyst and are maintained by sig-
nals emanating from the inner cell mass (2,3). In the rat, trophoblast stem cells
can be directed toward at least five recognizable differentiated trophoblast cell
phenotypes: trophoblast giant cells, spongiotrophoblast cells, invasive tropho-
blast cells, glycogen cells, and syncytial trophoblast (Fig. 1) (4,5). Differenti-
ated trophoblast cell populations can be distinguished on the basis of
morphology, location, and patterns of gene expression. These cell types are
arranged into two distinct zones of the chorioallantoic placenta—the junctional
zone and the labyrinth zone—and contribute to a complex uteroplacental struc-
ture prominent during the last week of gestation, the metrial gland (Fig. 1).
Each differentiated cell lineage specializes in activities supportive of preg-
nancy, some of which are well established whereas others are the source of
both speculation and ongoing investigation. Some specific trophoblast func-
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
159
13_Sahgal_159_178_F
160
Fig. 1. A schematic representation of rat trophoblast cell lineages and their location within the mature uteroplacental compart-
8/29/05, 11:19 AM
ment. In the rat, trophoblast stem cells can be directed toward at least five recognizable differentiated trophoblast cell phenotypes:
trophoblast giant cells, spongiotrophoblast cells, invasive trophoblast cells, glycogen cells, and syncytial trophoblast. These cell
types are arranged into two distinct zones of the chorioallantoic placenta, the junctional zone and the labyrinth zone; and contrib-
ute to a complex uteroplacental structure prominent during the last week of gestation: the metrial gland.
Sahgal et al.
Studying Trophoblast Cell Differentiation 161
Table 1
Rcho-1 Trophoblast Stem Cell Line Applications for Studying Trophoblast
Cell Biology
Trophoblast cellular process References
types is possible, but is not optimal using classic monolayer culture practices
(Canham, L. N. and Soares, M. J., unpublished results).
Cancer cells, such as those represented by the RCHO and Rcho-1 tropho-
blast stem cell lines, are caricatures of normal development and represent
potentially important models for dissecting molecular mechanisms control-
ling differentiation (78). The key is in identifying and appreciating which regu-
latory pathways are characteristic of normal development and which are
associated with the transformed phenotype. Thus, it is imperative to perform
complementary experimentation using primary cultures of trophoblast cells and
in vivo models.
In this chapter, we describe methods developed in our laboratory for using
the Rcho-1 trophoblast stem cell model to study various aspects of trophoblast
cell biology.
2. Materials
1. Culture media:
a. Standard Growth Medium: RPMI-1640 culture medium (Mediatech Cellgro,
Herdon, VA) containing 50 µM 2-mercaptoethanol (Bio-Rad Laboratories,
Hercules, CA), 1 mM sodium pyruvate (Sigma Chemical Co., St. Louis,
MO), 100 µg/mL penicillin, and 100 U/mL streptomycin (Mediatech
Cellgro), and 20% heat-inactivated fetal bovine serum (FBS, Altanta
Biologicals, Norcross, GA).
b. Standard Differentiation Medium-Type I: NCTC-135 culture medium
(Sigma) containing 50 µM 2-mercaptoethanol (Bio-Rad), 1 mM sodium pyru-
vate (Sigma), 100 µg/mL penicillin and 100 units/mL streptomycin
(Mediatech Cellgro), and 1–10% heat-inactivated donor horse serum (HS;
Atlanta Biologicals).
3. Methods
3.1. Routine Maintenance and Expansion of Rcho-1 Trophoblast Stem
Cells
1. Rcho-1 trophoblast cells are routinely maintained in 75-cm2 flasks in Growth
Medium, in an atmosphere of 5% CO2/95% air at 37°C in a humidified incuba-
tor. Cells are grown under subconfluent conditions. Initially, cells are plated at
1–2 × 106 cells per flask and fed at two day intervals (see Notes 1–3).
Table 2
Genes Expressed in Proliferating Rcho-1 Trophoblast Stem Cells
Gene Functional group GenBank accession no. References
Cdx2 Transcription AJ278466 unpublisheda
Eomes Transcription AY457971 unpublisheda
Id-1 Transcription L23148 17 and unpublisheda,b
Mash2 Transcription X53724 17 and unpublisheda
SOCS 3 Signal transduction AF075383 32 and unpublisheda
Cyclin D3 Cell cycle D16309 14 and unpublishedb
Abbreviations: Eomes, Eomesodermin; Id-1, Inhibitor of DNA binding 1; Mash, mammalian
achaete schute; SOCS3, suppressor of cytokine signaling 3.
aSahgal, N., Canham, L. N., and Soares, M. J., unpublished results.
bCanham, L. N., Sahgal, N., and Soares, M. J., unpublished results.
Table 3
Trophoblast Giant Cell-Associated Genes Expressed in Differentiating Rcho-1
Trophoblast Cellsa
GenBank Antibodies: source
Gene accession no. (cat. no.) References
PRL family
PL-I D21103 Chemicon International, 9,13,26,38,44
Temecula, CA (AB1288)
PL-II M13749 Chemicon (AB1289) 9,13,26,38,44
PLP-A NM_017036 Chemicon (AB1290) 9,13,44
PLP-Fα NM_022530 None currently available 42,44
PLP-M NM_053791 None currently available 44
Steroidogenic regulators
P450scc J05156 Chemicon (AB1244, AB1294) 35,36
3β-HSD L17138 None currently available Unpublishedb
P450c17 NM_012753 See references 37
Others
PSG36 M32474 None currently available Unpublishedb
HAND1 NM_021592 Santa Cruz Biotechnology, 17 and unpublishedc
Santa Cruz, CA (sc-9413)
Abbreviations: PRL, prolactin; PL, placental lactogen, PLP, prolactin-like protein; P450scc, side
chain cleavage; P450c17, 17α hydroxylase; 3βHSD, 3β hydroxysteroid dehydrogenase; PSG, preg-
nancy specific glycoprotein.
aThis list of genes reflects the trophoblast giant cell phenotype of the differentiating Rcho-1 tro-
4. After a maximum of seven days, the wells are rinsed with PBS, and stained with
Crystal Violet Solution (300 µL/well) for 10 min with agitation.
5. Cell cultures are then washed repeatedly in tap water, and allowed to dry.
6. Crystal violet dye is then eluted with ethylene glycol.
7. Cell density can be quantified by measuring absorbance of each eluate at 600 nm.
In this assay, cell number is directly correlated with absorbance of the cellular
eluates.
4. Notes
1. We routinely use RPMI-1640 culture medium as a base growth medium. Rcho-1
trophoblast stem cells grow vigorously in RPMI-1640 culture medium but some-
times at the cost of poor pH regulation. We compensate for the lack of pH control
by changing the culture medium more frequently (daily) and/or by supplement-
ing the cultures with HEPES (10–20 mM). High humidity is essential for optimal
Rcho-1 trophoblast stem cell growth. A serum-free system has not been defined
for propagating the Rcho-1 trophoblast stem cells. At this juncture the inclusion
of FBS is essential. We routinely use high concentrations (20%) of FBS, which
the cells appear to prefer. The high FBS concentration may also minimize some
of the variabilities associated with different lots of serum.
2. Cell density is a key for the appropriate maintenance and expansion of the Rcho-
1 trophoblast stem cell line. The most common problem in working with Rcho-1
trophoblast stem cells is the desire to grow them to confluence. Confluence and
proliferation are not compatible. As the cells become more dense, they begin to
spontaneously differentiate or die. The differentiating cells have a more flattened
appearance and will ultimately develop into trophoblast giant cells, whereas the
dead cells lift from the surface of the culture plate. In order to prevent spontane-
ous cell death or differentiation, the Rcho-1 trophoblast stem cells must be pas-
saged as recommended.
3. Rcho-1 trophoblast stem cell cultures are heterogeneous. Both proliferative and
differentiated cells can be observed in expanding cultures. Manipulating various
aspects of the culture procedure can influence the cellular composition of the cell
line. Cell composition can influence growth rates and features of differentiation.
Maintaining the cells at higher densities or any type of significant stress (humid-
ity, pH, CO2 deprivation, and so on) can lead to differentiation (giant cell forma-
tion) or cell death, both of which result in an irreversible termination of the
culture. Harvesting the Rcho-1 trophoblast cells following brief treatment with
throughout the cultures (Fig. 2). As these cultures are maintained in Standard
Growth Medium, colonies of stem cells will also begin to appear. Cells in these
colonies are tightly packed and rise above the surface of the plate. If needed, the
stem cell colonies can be removed by brief trypsinization without detachment of
the differentiated trophoblast giant cells. In both protocols, mitogen withdrawal
is the key. In the absence of FBS, some cells differentiate, others die, and some
stem cells apparently become dormant. The enhanced trophoblast giant cell for-
mation following re-introduction of Standard Growth Medium suggests that
endoreduplication is stimulated by factors present in FBS.
8. Under our culture conditions, Rcho-1 trophoblast stem cell differentiation is most
prominently directed toward the trophoblast giant cell lineage. Giant cell forma-
tion proceeds over time and may be accelerated by re-introduction of FBS con-
taining medium. Evidence for differentiation along other trophoblast cell lineages
(Fig. 1; spongiotrophoblast cells, glycogen cells, syncytial trophoblast, and the
specialized invasive trophoblast cells of the metrial gland) is apparent but gener-
ally modest to minimal. This restricted differentiation to trophoblast giant cells is
likely, at least in part, a reflection of culture conditions rather than developmen-
tal capabilities of the Rcho-1 trophoblast stem cells. We may be able to learn
from differentiation strategies developed for studying embryonic stem cells (79).
Other cell lineages can be detected by monitoring the expression of genes or gene
products specific for spongiotrophoblast cells, syncytial trophoblast, and the spe-
cialized invasive trophoblast cells of the metrial gland (Table 4). Glycogen cells
are generally identified by their accumulation of glycogen. Exposure of differen-
tiating cells to dimethylsulfoxide can inhibit trophoblast giant cell differentiation
and reactivate part of the trophoblast stem cell phenotype (Sahgal, N., Canham,
L., and Soares, M. J., unpublished results).
9. Balzarini and colleagues use alkaline phosphatase enzyme activity as a measure
of differentiation of RCHO trophoblast stem cells (22,25). The assay is simple
and can readily be adapted to a multi-well format. We have not utilized the assay
mainly because alkaline phosphatase is known to be expressed in many cell types
and thus does not reflect a specific measure of trophoblast cells.
10. We have utilized an assortment of different housekeeping genes to monitor RNA
integrity and loading efficiency. These have included β-actin, glyceraldehyde-3'-
phosphate dehydrogenase (G3PDH), β-tubulin, and 28S ribosomal RNA. Some
of these, including G3PDH and β-tubulin are sometimes problematic in that their
expression is affected by cell differentiation or the treatments employed.
11. Aspects of the invasive phenotype can also be monitored by determining the ex-
pression of gelatinase B and/or α1 integrin and through the analysis of gelatinase
B activity in conditioned medium by substrate gel electrophoresis (zymography;
see ref. 75).
12. Rcho-1 trophoblast stem cells can be maintained in vivo by transplantation into
various host tissues. We have routinely used the kidney capsule but these cells
have also been successfully transplanted to other sites, including the liver, cere-
bral ventricles, lungs, testes, and uteri of rats (7,10,11,85–92). In vivo transplan-
Table 4
Other Trophoblast Cell Lineage-Specific Gene Markers
Trophoblast Cell lineage Gene name GenBank accession no. References
Spongiotrophoblast PLP-B M31155 80,81
PLP-Fβ AY741310 Unpublisheda
SSP NM_172073 82
Syncytial trophoblast GCM-1 NM_017186 Unpublishedb
Invasive trophoblast PLP-L NM_138527 5,83
PLP-N NM_153738 84
tation of the Rcho-1 trophoblast cells has been effectively used to elevate circu-
lating levels of lactogenic hormones. The predominant lactogen expressed by the
transplants appears to be PL-I. Lactogenic and luteotrophic actions on the mam-
mary glands and ovary, respectively, represent effective indicators of systemic
action of the products of the transplants. Please be aware that Rcho-1 trophoblast
cells are potentially capable of producing other peptide and steroid hormones;
thus the physiological consequences of trophoblast stem cell transplantation may
be complex.
Acknowledgments
We would like to thank past and current members of our laboratory for their
efforts in developing and characterizing the methods described in this chapter.
This work was supported by a National Institutes of Health (NIH) KO8 award
to NS (HD42171) and grants from the NIH (HD20676, HD39878, HD48861)
and the Hall Family Foundation.
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(1993) A factor(s) from a rat trophoblast cell line inhibits prolactin secretion in
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140, 2159–2166.
14
Bovine Trophoblast Cell Culture Systems
A Technique to Culture Bovine Trophoblast Cells
Without Feeder Cells
Summary
Bovine trophoblastic cells are the first cells to differentiate during embryogenesis and play
pivotal role in morphological and physiological development of the placenta. We have devel-
oped culture systems for bovine trophoblast stem cells isolated from in vitro fertilized blasto-
cysts in the absence of feeder cells. These cells continuously proliferate in Dulbecco’s modified
Eagle’s/F12 culture medium supplemented with bovine endometrial fibroblast-conditioned
medium. The cells possess epithelial morphology, express cytokeratin, and form dome-like
structures (vesicles). Methods for the maintenance, subculture, storage, and measurement of
bovine trophoblast stem cell growth are described. The cells exhibit characteristics of bovine
trophoblastic stem cells and possess the ability to differentiate into binucleate cells and express
placental lactogen, prolactin-related protein-1, pregnancy-associated glycoprotein-1, and inter-
feron τ.
Key Words: Trophoblastic cell line; BT-1; binucleate cells; trophoblast differentiation; pla-
cental lactogen; collagen gel; microarray; gene expression; bovine.
1. Introduction
Trophectoderm is the first cell type to differentiate from the embryo at the
blastocyst stage and its cell lineage contributes to placental formation. Factors
controlling early decisions in the development of inner cell mass (ICM) and
trophectoderm cell lineages are not completely understood. Embryonic stem
cells derived the ICM are pluripotent, whereas trophoblast stem cells have a
more restricted developmental capacity (1). Some trophoblast cell lines have
been developed in various species and have been used for cell differentiation
studies (2–4). Fibroblast growth factor (FGF)4 has a critical role in maintain-
ing mouse trophoblast stem cells in an undifferentiated status.
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
179
2. Materials
1. Culture medium: Dulbecco’s modified Eagle’s/F-12 medium (DMEM/F-12;
Sigma, St. Louis, MO, USA) containing 100 international units (IU)/mL penicil-
lin and 100 mg/mL streptomycin (Sigma), and 10% heat-inactivated fetal bovine
serum (FBS; Sigma).
2. Cell substratum: acid-soluble porcine type I collagen solution (3 mg/mL) and gel
reconstitution solution (0.05 N NaOH solution containing 2.2% NaHCO3 and
200 mM HEPES).
3. Tenfold-concentrated physiological salt solution consisting of 1.52 M NaCl, 54 mM
KCl, 10 mM CaCl2, 8 mM MgCl2, 56 mM glucose, and 100 mM HEPES, pH 7.4.
4. Cell extraction medium: 0.1 M crystal violet (Wako Chemical, Osaka, Japan),
0.1% citric acid (Wako chemical), and 0.1% Triton X-100 (Sigma).
5. Collagen solution: acid-extracted collagen, 3 mg/mL (Cell Matrix, Nitta gelatin,
Osaka, Japan).
6. Freezing reagent: CellBanker (Zenyaku kogyo Co, Tokyo, Japan).
7. 5-Bromo 2'-deoxyuridine 5'-triphosphate (BrdU) Labeling and Detection Kit II
(Roche Diagnostics, Mannheim, Germany).
9. Hoechst 33342 (Molecular Probes, OR, USA).
10. Paraformaldehyde (Wako Chemical).
11. Bovine serum albumin (BSA, Sigma).
12. Transfer pipet (Becton Dickinson Labware, Franklin Lake, NJ, cat. no. 357575).
13. Mouse monoclonal and rabbit polyclonal anti-bovine PL (5).
14. Alexa 546-conjugated goat anti-mouse immmunoglobulin (Ig)G antibody (Molecu-
lar Probes).
15. Alexa 488-conjugated goat anti-rabbit IgG antibody (Molecular Probes).
16. 24-well culture plates and 25-cm2 culture flasks (Becton Dickinson).
17. Cell freezing vessel: BICELL (Nihon Freezer Co., Ltd, Tokyo, Japan).
18. Screen Cup with a 80 mesh screen (pore size 180 µm, Sigma).
19. Sterile plastic transfer pipets and 35-mm plastic culture dishes (Becton Dickinson).
3. Methods
3.1. Establishiment of Bovine Trophoblast Stem Cell Cultures
Bovine trophoblast stem cells can be established from in vitro cultured blas-
tocysts.
1. In vitro matured (IVM)/in vitro fertilized (IVF) bovine blastocysts are obtained
as described previously (6,9).
2. Blastocysts are individually plated into 24-well culture dishes that are coated
with acid-extracted collagen and cultured in DMEM/F-12 supplemented with
10% FBS, 50% fibroblast-conditioned medium, and 50 µM beta-mercaptoethanol
(see Note 1).
3. Following attachment cells outgrow from blastocysts in a week. These cells are
collected and maintained as follows under Subheading 3.1.1.
Fig. 1. Bovine trophoblast (BT)-1 cell features. (A) Small cell explants just after
plating in a new culture dish; (B) BT-1 cells 1 d after plating; (C) 2 d after plating; (D) 7
d after plating; (E) freely floating BT-1 vesicles; (F) BT-1 vesicles 1 day after plating.
Note that vesicles attach and exhibit cellular outgrowth. Scale bar = 500 µm
Fig. 2. Phase contrast (A), Hoechst 33342 (B), and (C) placental lactogen (PL)
staining (with monoclonal anti-PL antibody) images of bovine trophoblast (BT)-1 cells
cultured on collagen gels for 17 ds. Some binucleate cells are indicated by arrows.
These binucleate cells have an intense Hoechst fluorescence in their nuclei and express
PL. Scale bar =100 µm.
2. After three washes with phosphate-buffered saline (PBS), the cells are blocked
and permeabilized with PBS containing 10% normal goat serum and 0.5% Triton
X-100 for 30 min at room temperature.
3. Incubation with the mouse monoclonal (diluted 1:1000) or the rabbit polyclonal
(1:8,000) anti-PL antibody is carried out in PBS containing 1% BSA, 0.05%
NaN3, and 0.3% Triton X-100 for 2 h at room temperature.
4. After three washes with PBS, Alexa 546-conjugated goat anti-mouse IgG anti-
body (1:400) or Alexa 488-conjugated goat anti-rabbit IgG antibody (1:200) in
PBS containing 1% BSA, 0.05% NaN3, and 0.3% Triton X-100 is applied for 1 h
at room temperature.
5. Hoechst 33342 (5 µg/mL) is added into the secondary antibody solution to stain
nuclei.
6. After three washes with PBS, PL and Hoechst 33342 signals can be viewed using
an inverted epifluorescence microscope with appropriate filters.
4. Notes
1. Fibroblast-conditioned medium. We generally use DMEM/F12 containing 10%
FBS culture medium for maintenance of bovine trophoblast stem cells. As previ-
ously described, the BT-1 cells were established from blastocysts using fibro-
blast-conditioned medium (6). However, conditioned medium is not necessary
for the maintenance of cell growth following the establishment of the cell line.
2. Collagen-coated culture dishes. Acid-extracted collagen suspension is diluted
10-fold with distilled water and poured into dishes. After a 60-min incubation
at room temperature, they were rinsed with culture medium and used for culture.
3. Cell concentration. Adequate density of bovine trophoblast stem cells is impor-
tant for sustaining cell growth. When cells are plated at low density, cells tend to
become flatten and stop proliferating. Passaging the cells at a 1:2 ratio is optimal
for subculture.
4. Freezing for storage and thawing for re-culture. After cyropreservation, bovine
trophoblast stem cell viability is low. To successfully establish a culture from a
frozen vial, seed cells from a cryovial into a small culture dish (35 mm or smaller).
Supplementation of the cultures with BT-1 trophoblast stem cell conditioned
medium may facilitate recovery and growth of previously frozen bovine tropho-
blast stem cells.
5. A custom-designed cDNA microarray using uteroplacental cDNAs has been suc-
cessfully utilized for analyzing transcriptome in BT-1 cells as well as in the pla-
centa and uterus (7). Results from DNA microarray studies should be confirmed
by other appropriate procedures (e.g., RT or real-time PCR). Table 1 provides an
overview of the gene expression profile in BT-1 cells. The BT-1 cells express an
array of transcripts including placental prolactin family proteins, pregnancy-
associated glycoproteins and IFN-τ, and other genes involved in steroidogen-
esis and cytokine signaling. In this experiment, BT-1 cells were maintained with
DMEM/F12 supplemented with 20% FBS. The intensity of expression is shown
as a relative value compared to that of the glyseraldehyde-3-phosphate dehydro-
genase (GAPDH) as internal reference. Figure 3 shows the expression profiles
of selected genes under different culture conditions. The cells are routinely grown
in a medium supplemented with 10 % FBS. In this experiment, differences in
gene expression pattern under different culture conditions with 20% FBS, 2%
FBS or 10 % horse serum (HS)-supplemented medium were determined. BT-1
cells express an assortment of trophoblast marker genes (PL, PRP-I, PAG, and
IFN-τ) when maintained in 20% FBS-supplemented medium. However, the cells
cease or decrease expression of these genes when the serum supplementation is
switched to 10% HS. This is in marked contrast to the Rcho-1 rat trophoblast
cell line (11). BT-1 cells represent both proliferative and endocrine phenotypes
depending on the presence of FBS. In contrast, both bone morphogenetic protein
(BMP)4 and Oct 3/4 are stably expressed regardless of sera. Both eomesodermin
and Oct 3/4 are thought to be markers representing the undifferentiating status
in mouse trophoblast stem cell (1). It has been reported that BMP4 triggers
Acknowledgments
The authors thank Dr. K. Imai for in vitro fertilization and culture of
bovine embryo. We also thank Drs. H. Ishiwata and K. Kizaki (N.I.A.S.),
and G. Tsujimoto (Kyoto University) for fabricating and analyzing bovine
utero-placental cDNA microarray.
References
1. Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., and Rossant, J. (1998)
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of a feeder-dependent, porcine trophectoderm cell line obtained from a 9-day blas-
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4. Miyazaki, H., Imai, M., Hirayama, T., et al. (2002) Establishment of feeder-inde-
pendent cloned caprine trophoblast cell line which expresses placental lactogen
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5. Nakano, H., Takahashi, T., Imai, K., and Hashizume, K. (2001) Expression of
placental lactogen and cytokeratin in bovine placental binucleate cells in culture.
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6. Shimada, A., Nakano, H., Takahashi, T., Imai, K., and Hashizume, K. (2001)
Isolation and characterization of a bovine blastocyst-derived trophoblastic cell
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centa 22, 652–662.
7. Ishiwata, H., Katsuma, S., Kizaki, K., et al. (2003) Characterization of gene ex-
pression profiles in early bovine pregnancy using a custom cDNA microarray.
Mol. Reprod. Dev. 65, 9–18.
8. Nakano, H., Shimada, A., Imai, K., Takezawa, T., Takahashi, T., and Hashizume,
K. (2002) Bovine trphoblastic cell differentiation on collagen substrata: formation
of binucleate cells expressing placental lactogen. Cell Tissue Res. 307, 225–235.
9. Konishi, M., Aoyagi, Y., Takedomi, T., Itakura, H., Itoh, T., and Yazawa, S.
(1996) Production and transfer of IVF embryos from individual inhibin-immu-
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Sci. 58, 893–896.
10. Quackenbush, J. (2002) Microarray data normalization and transformation. Nat.
Genet. 32(Suppl.), 496–501.
11. Faria, T. N. and Soares, M. J. (1991) Trophoblast cell differentiation: establish-
ment, characterization, and modulation of a rat trophoblast cell line expressing
members of the placental prolactin family. Endocrinology 129, 2895–2906.
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15
In Vitro Induction of Trophoblast from Human Embryonic
Stem Cells
Ren-He Xu
Summary
Human embryonic stem (ES) cells can proliferate without a known limit and can form advanced
derivatives of all three embryonic germ layers. What is less widely appreciated is that human ES
cells can also form the extra-embryonic tissues that differentiate from the embryo before gas-
trulation. The use of human ES cells to derive early human trophoblast is particularly valuable,
because it is difficult to obtain from other sources and is significantly different from mouse
trophoblast. Here we describe a method by using bone morphogenetic protein (BMP)4 , a mem-
ber of the transforming growth factor (TGF)-β superfamily, to induce the differentiation of
human ES cells to trophoblast. Immunoassays (as well as DNA microarray and reverse-tran-
scription polymerase chain reaction analyses—data not shown) demonstrate that the differenti-
ated cells express a range of trophoblast markers and secrete placental hormones. When plated
at low density, the BMP4-treated cells form syncytia that express chorionic gonadotrophin
(CG). This technique underscores fundamental differences between human and mouse ES cells,
which differentiate poorly, if at all, to trophoblast. Human ES cells thus provide a tool for
studying the differentiation and function of early human trophoblast and could provide a new
understanding of some of the earliest differentiation events of human postimplantation devel-
opment.
Key Words: Human embryonic stem cells; trophoblast; bone morphogenetic protein.
1. Introduction
The trophectoderm is the first differentiated cell type in the mammalian
embryo, and it forms the outer epithelium of the blastocyst and later contrib-
utes (as the trophoblast) to the outer layers of the placenta. The trophectoderm
is crucial for implantation and maintenance of pregnancy. When formed into
chimeras with intact preimplantation embryos, mouse embryonic stem (ES)
cells rarely contribute to the trophoblast, and the manipulation of external cul-
ture conditions has, to date, failed to direct mouse ES cells to differentiate to
trophoblast (1). Mixed populations of spontaneously differentiated rhesus mon-
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
189
2. Materials
1. Human ES cells. Human ES cell lines H1, H7, H9, and H14 were used for the
trophoblast induction by BMPs. Their National Institutes of Health (NIH) regis-
try numbers are WA01, WA07, WA09, and WA14, respectively (http://
stemcells.nih.gov/research/registry/index.asp#warf). These cell lines are avail-
able at the WiCell Research Institute upon application and licensing (http://
www.wicell.org/forresearchers/index.jsp?catid=4).
2. Mouse embryonic fibroblast (MEF) (see below for details).
3. Human uterine fibroblast (HUF) cell line (6).
4. Four-well and six-well culture plates, 50-, 75-, and 90-mm Filter Units (Nalge
Nunc International, Rochester, NY).
5. Six-well Transwell plate (Corning Incorporation, Corning, NY).
6. 0.22-mM Filter Unit (Millipore, Bedford, MA).
7. 40 mM mesh (BD Labware, Bedford, MA).
8. Falcon (35/2054) 5 mL polystyrene round bottom tube (BD Labware).
9. Knockout Dulbecco’s modified Eagle’s/F12 medium (DMEM/F12), knockout
serum replacement (SR), 100X MEM nonessential amino acid solution, L-glutamine,
and Trypsin/ethylenediamine tetraacetic acid (EDTA) solution (Invitrogen,
Carlsbad, CA).
10. Human basic fibroblast growth factor (bFGF) (Invitrogen). Dissolve 10 µg bFGF
in 1 mL of 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)
(without Ca2+ and Mg2+). Aliquot and store at –80°C.
11. Collagenase type IV (Invitrogen). Dissolve it at 1 mg/mL in DMEM/F12 and
sterilize with a 0.2-µm cellulose acetate filter. Store at 4°C. Use within 2 wk.
3. Methods
The methods described below outline (1) cell culture, (2) trophoblast induc-
tion, and (3) biological assays of the induced trophoblast.
Fig. 1. Mouse embryonic fibroblast (MEF) cells. MEF cells plated at 2.12 × 105
cells/mL in a T75 flask serve as feeder cells to produce conditioned medium.
2. Filter to sterilize.
3. Store at 4°C and use within 2 wk.
Fig. 2. Trophoblast induction. Human embryonic stem cell line H1 cells were
treated with (C,D) or without (A,B) 100 ng/mL bone morphogenetic protein-4 for 7 d.
The cells were photographed at 5× (A,C) and 20× (B,D).
j. Return the plate to incubator. Move the plate back-and-forth and side-to-side
several times to evenly disperse the cells across the surface of the wells.
k. Incubate cells overnight without touch to ensure that colonies attach.
l. Add fresh conditioned media daily.
Fig. 3. Syncytial cell. The left panel shows a syncytial cell formed after 2 wk of
treatment of individualized embryonic stem cells by bone morphogenetic protein-4.
The right panel shows immunofluorescence for CG-β and Hoechst 33342 fluorescence
for the nuclei in the cell.
nuclei. The observed highest number of nuclei in one single syncytial cell was
100 (Fig. 3). These differentiated phenotypes remain for a long time without
obvious changes (see ref. 5 [http://genome-www.stanford.edu/es_cell/supple
ment.shtml]).
2. Passage human ES cells into the inner chamber, and culture them in a total of 3 mL
conditioned medium added to the inner chamber and the outer chamber of the
Transwell plate (both chambers link through openings on the side wall of the
inner chamber).
3. Two days post passage, add 100 ng/mL BMP4 to the culture for 7 d to induce
trophoblast.
4. HUF cells are maintained and expanded in RPMI culture medium supplemented
with antibiotics, sodium pyruvate, and FBS.
5. Seed 2 × 104/well HUF cells in MEF medium in a new six-well plate, and culture
them for 2 d.
6. Remove the spent media, and wash with PBS once.
7. Aspirate the spent media from the Transwell, transfer each of the inner chambers
that contains BMP4-induced trophoblast to the well that contains HUF cells.
8. Add 3 mL human ES cell medium to the inner and outer chambers.
9. Co-culture the cells for 7 d.
10. Collect spent media daily to test for the production of placental hormones.
6. Add 1 mL Trypsin/EDTA solution to the well, and incubate at 37°C for at least
5 min, break up the cell colonies by pipetting up and down several times, and
then add 1 mL ES cell medium to neutralize the Trypsin/EDTA solution.
7. Scrape the cells from the plate with a glass pipet, and transfer the cells to a 15-mL
tube.
8. Break up the cell colonies by pipetting up and down several times, and add human
ES cell medium to a final volume of 10 mL.
9. Pellet the cells by spinning at 200g for 5 min.
10. Remove the supernatant and re-suspend the pellet in FACS buffer and count the
cells.
11. Pellet the cells by spinning at 200g for 5 min, remove the supernatant (leave
about 0.1 mL supernatant), and briefly mix to re-suspend the cells in the residual
supernatant.
12. Add 1 mL 2% paraformaldehyde to the tube, mix well, and incubate at room
temperature for 10 min.
13. Pellet the cells by spinning at 200g for 5 min, and remove the supernatant.
14. Add FACS buffer plus 0.1% Triton X100 to resuspend and permeabilize the cells,
and also achieve a cell concentration of 5 × 106/mL.
15. Add 100 µL of the cell suspension containing 5 × 105 cells per tube to both a test
tube and a control tube (using a Falcon 5-mL polystyrene round-bottomed tube).
16. Add 1 µL mouse anti-human CG-β antibody (5 mg/mL) to the test tube, and 5 µL
of mouse IgG (1 mg/mL) to the control tube.
17. Briefly vortex the tubes to mix and incubate the tubes at 4°C overnight.
18. Add 1 µL fluorescein-labeled rabbit anti-mouse IgG antibody to both tubes, and
incubate for 30 min on ice.
19. Wash the cells twice with 1-mL FACS buffer plus 0.1% Triton X100 by centrifu-
gation at 200g for 5 min for each wash.
20. Re-suspend the cells in 0.3 mL of FACS buffer.
21. Analyze the samples on a FACSCalibur flow cytometer using the Cellquest
acquisition and analysis software (see Note 5).
4. Notes
1. For synchronous differentiation of human ES cells to trophoblast, ES cells should
be passaged as small colonies (about 200 µm in size), and BMP4 added when the
cells are about 30% confluent. Big colonies often end up with the cells in the
middle of the colonies remaining undifferentiated.
2. We have observed that the potency of BMP4 added to ES cell cultures twice on
alternative days is equivalent to that of BMP4 added daily for 7 d, as evaluated
by morphology and CG secretion.
3. According to microarray and reverse transcription-polymerase chain reaction as-
says (5), the expression levels of ES cell- and trophoblast-related genes change
dynamically in the ES cells during BMP4 treatment. From 3 h through 7 d of
BMP4 treatment, the expression of the following trophoblast-related genes are
elevated: TFAP2A, TFAP2C, MSX2, GATA2, GATA3, SSI3, HEY1, FZD,
PlGF, CGB, CGA, LHB, GCM1, INSL4, PAEP, PAPPE, DEPP, MET, and HLA-
G1 (see ref. 5 [http://genome-www.stanford.edu/es_cell/supplement.shtml]). At
day 7, ES cell marker genes OCT4 and TERT are downregulated.
4. For detection of CG-β expression in trophoblast by immunocytochemistry or flow
cytometry, it is essential to enhance the signal by pretreating the cells with the
Golgi blocker brefeldin A for 4 h, permeabilizing the fixed cells with Triton X-
100, and incubating the cells with anti-CG-β antibody at 4°C overnight.
5. A total of 10,000 events are required. Analysis is restricted to live events based
on light scatter properties. The fluorescein signal is collected through a 530/30
Fig. 6. Flow cytometry analysis for chorionic gonadotrophin (CG)-β positive cells
in human embryonic stem cells cultured in conditioned medium (CM) with or without
bone morphogenetic protein-4 treatment for 7 d. HCG stands for human CG.
band pass filter, and the mean fluorescence for both the IgG control and the test
samples are determined. All data are normalized via division of the test mean by
the control mean (Fig. 6).
Acknowledgments
I thank Dr. James Thomson, his laboratory and the WiCell Research Institute
for contributions to this work. It was supported by WiCell Research Institute, a
non-profit subsidiary of the Wisconsin Alumni Research Foundation.
References
1. Beddington, R. S. P. and Robertson, E. J. (1989) An assessment of the develop-
mental potential of embryonic stem cells in the midgestation mouse embryo.
Development 105, 733–737.
2. Thomson, J. A., Kalishman, J., Golos, T. G., et al. (1995) Isolation of a primate
embryonic stem cell line. Proc. Natl. Acad. Sci. USA 92, 7844–7848.
3. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998) Embryonic stem
cell lines derived from human blastocysts. Science 282, 1145–1147.
4. Andrews, P. W., Oosterhuis, J., and Damjanov, I. (1987) Cell lines from human
germ cell tumors, in Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach (Robertson, E., ed.). IRL, Oxford: pp. 207–246.
5. Xu, R. H., Chen, X., Li, D. S., et al. (2002) BMP4 initiates human embryonic stem
cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264.
16
Isolation and Culture of Term Human Trophoblast Cells
Summary
Experimentation with most human cell types is restricted to the use of cell lines, and this
limits our ability to extrapolate interpretations to the in vivo condition. However, in studying
human trophoblast cells, we have a unique opportunity to obtain large quantities of readily
available human tissue. In this chapter, we outline the methodology for purification of human
trophoblast cells from term placentas. The procedures are based on enzymatic dissociation of
villous placental tissue, followed by gradient centrifugation and immunomagnetic bead purifi-
cation. Purity may be assessed by immunocytochemistry or flow cytometry using a number of
markers to identify both cytotrophoblast cells and cellular contaminants. The resulting cytotro-
phoblast cell populations have excellent viability and purity, and may be subjected to long-
term culture.
Key Words: Human; placenta; cytotrophoblast cell; cell culture.
1. Introduction
The ability to establish primary cell cultures from human organs is a rare
opportunity. Even when it is possible, the investigator must often rely on lim-
ited quantities of tissues obtained from clinical biopsies. Furthermore, it is
implicit that tissues could be diseased, particularly when samples are obtained
from nonelective surgery. In contrast, those who study the trophoblast cell ben-
efit from the large size of a readily available source material: the term placenta.
An individual can easily process up to 50 g of villous placental tissue, harvest-
ing upwards of 250 million cells. A further benefit is that there is little ethical
controversy surrounding the use of human placenta for biomedical research
because it is almost invariably discarded following delivery.
In this chapter, we describe the isolation and purification of villous cy-
totrophoblast cells from the term placenta. In situ, these cells serve as precur-
sor cells for the continually regenerating syncytiotrophoblast, and are located
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
203
Fig. 2. Immunomagnetically purified from the term placenta. (A) Cells bound to the immunomagnetic column, represent-
ing nontrophoblastic cells. (B) Cells not bound to the column, representing trophoblastic cells in a state of early differentia-
tion; (C) Cells not bound to the column and treated with 10 ng/mL epidermal growth factor (EGF); these cells have undergone
extensive syncytialization. Cells were plated in 60 mm Primaria dishes at a density of approx 5 × 106 per dish (A) or 6 × 106
(B,C) in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum. After allowing adherence for 4
h, the nonadherent cells were removed by gentle washing. Thereafter, media were removed and replaced every 48 h 6 d.
8/29/05, 11:20 AM
205
206 Petroff et al.
Table 1
Summary of Immunostaining After Purification
Protocol (Reference) Steps Markers Staining
Kliman et al. (10) Percoll gradient hCG (sTB) 1–5%
hPL (sTB) Absent
SP1 (sTB) Absent
Vimentin (non-TB) Absent
Chymotrypsin (non-TB) Absent
Douglas & King (11) Percoll gradient and
Immunopurification
(Anti-HLA class I/II) Cytokeratin-18 (CTB) 100%
Vimentin (non-TB) Absent
Guilbert et al. (12) Immunopurification PLAP (sTB) 4–46%
(Anti-CD9 and
Anti-MHC class I/II)
Current protocol Percoll gradent and β-hCG (sTB) <1%
immunopurification Cytokeratin-7 (sTB, CTB) ~100%
(Anti-HLA class I) CD14 (Macrophage) <1%
CD9 (non-CTB) <3%
Abbreviations: CTB, cytotrophoblast; hCG, human chorionic gonadotropin; hPL, human placental
lactogen; PI, propidium iodide; PLAP, placental alkaline phosphatase; PS, phosphatidyl serine; SP1,
pregnancy-specific β1-glycoprotein; sTB, syncytiotrophoblast; TB, trophoblast; FACS, fluorescence-
activated cell sorting.
The methods described in this chapter outline the enzymatic dispersion, den-
sity gradient centrifugation, and HLA class I-depletion of trophoblast cells
(Fig. 3). The latter step exploits the fact that virtually every placental cell type
other than the villous cytotrophoblast cell expresses classical HLA class Ia
molecules, so that these contaminating cell types are depleted. We routinely
harvest >97% pure cytotrophoblast cells based on immunocytochemical stain-
ing for cytokeratin-7 and the absence of CD14-positive cells.
2. Materials
2.1. Collection of Semipurified Trophoblast Cells
1. Sterile field sheets (Baxter, Deerfield, IL).
2. Sterile 4 in. × 4 in. gauze pads (Baxter).
3. Two pairs of small straight blade, sharp point scissors (Fine Science Tools, North
Vancouver, BC).
4. Two pairs of fine-toothed forceps (Fine Science Tools).
5. Sterile 250-mL beaker for tissue collection, preweighed.
6. Sterile 1-L beaker for liquid waste.
7. Sterile 500-mL Erlenmeyer flask for tissue dissociation.
8. Sterile 150-mm Petri dish for tissue mincing.
9. Cell dissociation sieve, fitted with 40 mesh screen (Sigma-Aldrich, St. Louis,
MO; cat. no. CD-1).
10. Benchtop water-bath shaker (for example, LabLine model SHKE7000; Barnstead
Intl., Dubuque, IA).
11. Shandon cytospin centrifuge (Shandon, Pittsburgh, PA).
12. 15-mL sterile polypropylene or polystyrene centrifuge tubes.
13. 100-µm Falcon nylon mesh cell strainers (Becton Dickinson, Franklin Lakes, NJ,
cat. no. 2360).
14. Biohazard bags.
15. Bleach.
16. Falcon Opticul 50-mL sterile polypropylene centrifuge tubes (Becton Dickinson).
17. Cryogenic cell storage vials.
18. Cell-freezing chamber.
19. 1 L 0.9% NaCl: dissolve 9 g NaCl in deionized H2O to yield a 1 L solution.
Filter-sterilize (0.2 µm).
20. 10X Hank’s balanced salt solution (10X HBSS), 1 L: 5.36 mM KCl (4 g), 4.4 mM
KH2PO4 (0.6 g), 1.37 M NaCl (80 g), 3.37 mM Na2HPO4 ( 0.4788 g), 55.5 mM
D-glucose (10 g). Dissolve each of the constituents in deionized H2O to yield 1 L
solution. Filter-sterilize (0.2 µm).
21. 1X Ca/Mg-free HBSS (CMF-Hank’s): Combine 100 mL 10X HBSS with 25 mL
1-M HEPES (Sigma, cat. no. H-0887) with deionized H2O to yield 800 mL H2O.
Adjust pH to 7.4 and bring volume to 1 L. Filter sterilize.
22. Enzyme digestion buffer: combine 35 mL 10X HBSS with 1.65 mL 7.5% Na
Bicarbonate (Sigma, cat. no. S-8761), 8.75 mL 1-M HEPES, and 266.1 mL deion-
ized H2O; filter-sterilize and distribute into three bottles containing 133.5 mL,
89 mL, and 66.8 mL. Store at 4°C.
23. 2.5% Trypsin: 10X concentrate (Invitrogen, cat. no. 15090-046). Thaw a 100-mL
bottle of trypsin and distribute 33 mL over three sterile tubes. Store at –20°C, and
thaw just prior to use.
24. DNase I: (Sigma, cat. no. D-5025, 150,000 U, approx 50 mg solid, ~3000 U/mg).
Just before use, add 5 mL sterile 0.9% NaCl to a 150,000-U vial of DNase. Filter-
sterilize and keep on ice until use or store at –20°C for later use.
Table 2
Dilution Scheme for Preparation of Percoll Gradients
90% Percoll (mL) CMF-Hanks (mL) Final concentration
15.6 4.4 70%
14.4 5.6 65%
13.3 6.7 60%
12.2 7.8 55%
11.1 8.9 50%
10.0 10.0 45%
8.9 11.1 40%
7.8 12.2 35%
6.7 13.3 30%
5.6 14.4 25%
4.4 15.6 20%
3.3 16.7 15%
2.2 17.8 10%
1.1 18.9 5%
3. Methods
3.1. Collection of Semipurified Trophoblast Cells (see Note 3)
1. Obtain a human placenta and process as soon as possible after delivery (see Note
4). If variation due to potential effects of labor is a concern, placentas from Cae-
sarian section deliveries may be used. Always use caution when handling human
biological material.
2. Place the placenta on a sterile field, with the maternal side (basal plate) facing up.
Prepare histological, RNA or protein samples if desired (see Note 5). Using sharp,
fine-point scissors and blunt forceps, dissect one cotyledon at a time. Remove the
overlying basal plate tissue, about 3 mm from the surface. Avoiding the chori-
onic plate, collect 40–50 g of villous tissue into the preweighed 250-mL beaker.
3. Rinse tissue several times with 0.9% NaCl by swirling with forceps, using the
1-L beaker for liquid waste. Transfer all of the tissue to a 150-mm Petri dish
and mince finely with scissors. Transfer half the tissue to the cell dissociation
sieve and rinse with 0.9% NaCl extensively until the eluate becomes clear. Trans-
fer the minced tissue to a 500-mL sterile Erlenmeyer flask, and repeat with the
second half of the tissue.
4. The dissociation is performed in three stages. During the dissociation of the sec-
ond and third batches, the cell suspensions from the first and second batches are
centrifuged and resuspended in turn. To prewarmed enzyme dilution buffer (labeled
batch I), add DNase and trypsin as indicated above. Add the mixture to the Erlen-
meyer flask containing the tissue, and incubate for 15–20 min at 37°C in a rotat-
ing water-bath shaker (150 rpm). During this incubation, add 1.5 mL FBS to
22 15-mL centrifuge tubes.
5. After the first 15-min dissociation, set the digestion flask at a tilt until tissue
settles. Remove about 13.5 mL of the supernatant, taking care not to collect undis-
sociated tissue. Slowly layer the suspension over the 1.5 mL serum in 15-mL coni-
cal centrifuge tubes. Repeat for seven additional tubes or until most of the
digestion supernatant is transferred. Centrifuge the tubes at 1000g for 15 min at
25°C at room temperature.
6. While batch I is in the centrifuge, add 100 mL warm enzyme digestion solution
containing enzymes (labeled batch II) and repeat digestion. Again collect super-
natant and layer over FBS as for batch I. Repeat digestion a third (final) time.
7. For each batch, following centrifugation, aspirate the supernatant without dis-
turbing the pellet. The trophoblast cells are predominantly in the white portion of
the pellet, overlying the red blood cells.
8. Resuspend the cell pellet in each tube in 1 mL culture medium, and combine the
resuspended pellets for each digestion group. Hold cells at room temperature.
9. After collecting the cells from all three digestion stages, filter the suspension
using a 100-µm nylon cell strainer inserted in the top of a sterile 50-mL conical
centrifuge tube. If the filtration of the cell suspension slows, lift upward on the
filter to draw a vacuum within the tube. Centrifuge at 1000g for 10 min, and
resuspend in 6 mL CMF-Hank’s. The total volume of the resuspended cells
should be approx 8 mL.
10. Carefully layer half of the cells onto each of two preformed Percoll gradients.
Centrifuge the gradients at 1200g for 20 min at room temperature in a swinging
bucket rotor without a brake.
11. Aspirate upper diffuse “band” (Fig. 4; usually down to approx 25–30 mL mark on
tube), and use a Pasteur pipet fitted with a bulb to manually collect the cells that
fractionate near center of tube into each of two sterile 50-mL tubes. The band con-
taining the cytotrophoblast cells is diffuse and usually between the 30-mL and 12-
to 15-mL graduations on tube. If using a separate reference tube containing density
marker beads, the cells will correspond to the location between the 1.048 and 1.060
marker beads. Dilute the cell fractions fourfold with culture medium and centrifuge
at 1000g for 5 min. Resuspend cells in each tube in 10 mL culture medium, com-
bine, and determine yield by trypan blue dye exclusion. Typical yields are between
1.5 and 3 × 108 cells per approx 40 g tissue at greater than 95% viability.
12. To assess cell characteristics by immunocytochemistry, cytocentrifuge slides can
be prepared. For one spot, use 50,000–100,000 cells in a 200-µL vol containing
at least 50% serum. Centrifuge in the Shandon cytospin unit at 700 rpm for 3
min. Remove funnel, turn slide and filter paper end-for-end in holder, replace
funnel and repeat. Place slides on bench to air dry, and store slides at –20°C.
10. Rinse the tube that contained the cells with 500 µL ice-cold CSB and apply to
column. Wash the column two to three times with 500 µL ice-cold CSB, each
time collecting in same effluent tube. If using more than one column, combine
effluents from the columns.
11. If desired, recover the bound cells (i.e., noncytotrophoblastic cells that remain
trapped within the column). Remove column from separator by pulling the col-
umn horizontally out of the magnet. Remove flow resistor from bottom of col-
umn, and place column above a fresh collection tube. Add 1 mL ice-cold CSB
onto the column. Using the plunger supplied with the column, flush cells from
column.
12. Centrifuge the cells at 400g and resuspend in cytotrophoblast culture medium.
Prepare a dilution in trypan blue and determine yield and viability. Typical yields
are 60–70% of the starting population. For characterization, prepare cytospin
preparations of each cell type (see Note 7).
13. Plate the purified cells in culture medium at a minimum of 2 × 105 cells/cm2 in
appropriate culture vessels (see Note 8). Allow cells to attach to the surface of
the plate for 4–24 h, after which rinse twice with prewarmed culture medium to
remove the nonattached cells. Refresh medium every 24–48 h.
4. Notes
1. This procedure allows for preparation of six tubes of stepwise 5–70% Percoll
gradients. Each cytotrophoblast preparation from 40 g placental tissues requires
two of these gradients; therefore, six are sufficient for three cytotrophoblast cell
preparations. All six gradients can be prepared at one time, but for best results,
use the gradients within 24 h of preparation. The diluted Percoll solutions may be
stored for later preparation of gradients.
2. Preparation of Percoll gradients is time-consuming. Some laboratories utilize
peristaltic pumps to reduce the workload. We have found that pipetting time can
be greatly reduced by resting the tip of the 5-mL pipet on the side of the tube just
above the liquid level and gently swinging the tip side-to-side against the tube to
induce layering of a broad stream of liquid. The result is that liquid can be pipetted
at a faster rate because the broader stream will reduce the pressure per unit of
surface area of the liquid.
3. We usually reserve 2 or 3 d for isolation of semi-purified cytotrophoblast cells.
The first day is devoted to preparation of reagents and Percoll gradients, and the
second day to the purification procedure itself. At the conclusion of the second
day, one may either cryopreserve the semipurified cytotrophoblast cells, or pro-
ceed directly to the final step of trophoblast isolation, immunomagnetic purifica-
tion (see Subheading 3.2.).
4. In the United States, term placentas are regarded as discarded tissues and can
therefore be obtained without patient consent. Obtaining additional information
about the medical background of the patient will be subject to regulation by the
Health Insurance Portability and Accountability Act (HIPAA), and investigators
must go through the appropriate channels to ensure privacy and consent of the
patient.
5. It is often desirable to obtain histological specimens that include basal plate pla-
centa as well as villous tissue, because the basal plate tissue contains extravillous
trophoblast cells. To do this, score a 1-mm × 3-mm rectangular portion of the
surface of the basal plate with sharp scissors. Make the deeper cuts, blotting away
unclotted blood if necessary, and use blunt, flat-tipped forceps to gently lift the
tissue upwards. Trim the tissue to the desired size and fix or freeze as necessary.
To obtain amniochorion histological samples, lay a section of amniochorion
membrane on a sterile field with the amnion face downward. Trim a 1-mm × 5-mm
slice, and either fix in desired fixative or, for frozen histology, use the scissors
and forceps to roll the membrane. Place the roll on its side for embedding; upon
microscopy, it will be possible to simultaneously observe the amnion, chorion,
and decidua.
For extraction of RNA and/or protein, we usually obtain specimens from the
villous portion of the placenta after removal of the basal plate connective tissue.
This can therefore be done concomitantly with collection of the tissue to be dis-
sociated. We collect and pool these samples from several cotyledons to avoid
potential intercotyledonary differences. To snap-freeze, samples can be dropped
directly into liquid nitrogen in an RNase-free container and subsequently trans-
ferred into sterile, precooled polypropylene tubes for storage at –80°C.
6. If the MACS columns run slowly, there are several steps that may be taken to
improve flow. First, a micropipet fitted with a yellow tip can be used to disrupt
any cells that may have settled on the top of the column. Without introducing
bubbles into the cell suspension over the column, pipet up and down to dislodge
settled cells. If this fails to speed up the column, use the plunger supplied with
the column to introduce light pressure. Place the plunger over the head of the
column and gently press down. Applying too much pressure by forcing the
plunger into the column will shear and detach the nontrophoblast, antibody/bead-
bound cells from the magnet, thus contaminating the eluted cell population. If
application of pressure fails to hasten the elution, we have found that the problem
often resides within the needle, which can get clogged with cells. Thus, the needle
can simply be removed and replaced with a new, sterile needle. If the problem is
indeed due to the needle, the flow will immediately hasten upon removal of the
clogged needle; thus, a new needle must be in place as quickly as the old one is
removed. As a last resort, the column itself can be replaced, and the unfiltered
cells transferred to a new column. However, this will result in the sacrifice of any
cells that may be trapped within the original column.
7. At this point, the cells can be analyzed by flow cytometry (19) or immunocy-
tochemistry on cytospin preparations. By the latter method, we have used anti-
cytokeratin-7 (Dako, Carpinteria, CA; clone OVTL 12/30), anti-CD14 (Zymed,
San Francisco, CA; clone RPA-M1), and anti-hCGβ (Neomarkers, Freemont, CA;
clone CG05) (4,20).
8. Cytotrophoblast cells may be cultured on Permanox Chamber Slides (Nalge Nunc).
Although adherence to this type of plastic is better than glass chamber slides, it is still
relatively poor. Thus, we seed at a very high density to guarantee that sufficient num-
bers of cells attach to the slide (e.g., seed 300,000 cells per well in eight-well slides).
Acknowledgments
The authors thank Mr. Stanton Fernald and Ms. Erin Skorina for their assis-
tance in preparation of graphics. This work was supported by grants R01
HD045611 (M.G.P), P01 HD39878 Project III (J.S.H.), U54 HD33994 (J.S.H)
Project III, and R01 HD24212 (J.S.H) from the National Institutes of Health.
References
1. Benirschke, K. and Kaufmann, P. (2000) Pathology of the Human Placenta.
Springer-Verlag, New York: pp. 55–56.
2. Morrish, D. W., Dakour, J., Li, H., et al. (1997) In vitro cultured human term
cytotrophoblast: a model for normal primary epithelial cells demonstrating a spon-
taneous differentiation program that requires EGF for extensive development of
syncytium. Placenta 18, 577–585.
3. Kamat, A., Alcorn, J. L., Kunczt, C., and Mendelson, C. R. (1998) Characteriza-
tion of the regulatory regions of the human aromatase (P450arom) gene involved
in placenta-specific expression. Mol. Endocrinol. 12, 1764–1777.
4. Petroff, M. G., Chen, L., Phillips, T. A., Azzola, D., Sedlmayr, P., and Hunt, J. S.
(2003) B7 family molecules are favorably positioned at the human maternal-fetal
interface. Biol. Reprod. 68, 1496–1504.
5. Morales, P. J., Pace, J. L., Platt, J. S., et al. (2003) Placental cell expression of
HLA-G2 isoforms is limited to the invasive trophoblast phenotype. J. Immunol.
171, 6215–6224.
6. Huppertz, B., Frank, H. G., Reister, F., Kingdom, J., Korr, H., and Kaufmann, P.
(1999) Apoptosis cascade progresses during turnover of human trophoblast: analy-
sis of villous cytotrophoblast and syncytial fragments in vitro. Lab. Invest. 79,
1687–1702.
7. Ka, H. and Hunt, J. S. (2003) Temporal and spatial patterns of expression of in-
hibitors of apoptosis in human placentas. Am. J. Pathol. 163, 413–422.
8. Abbasi, M., Kowalewska-Grochowska, K., Bahar, M. A., Kilani, R.T., Winkler-
Lowen, B., and Guilbert, L. J. (2003) Infection of placental trophoblast by Toxo-
plasma gondii. J. Infect. Dis. 188, 608–616.
9. Li, H., Dakour J., Kaufman S., Guilbert, L. J., Winkler-Lowen, B., and Morrish,
D.W. (2003) Adrenomedullin is decreased in preeclampsia because of failed
response to epidermal growth factor and impaired syncytialization. Hyperten-
sion 42, 895–900.
10. Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., and Strauss, J. F. (1986)
Purification, characterization, and in vitro differentiation of cytotrophoblasts from
human term placentae. Endocrinology 118, 1567–1582.
11. Douglas, G. C., and King, B. F. (1989) Isolation of pure villous cytotrophoblast
from term human placenta using immunomagnetic microspheres. J. Immunol.
Methods 119, 259–268.
12. Guilbert, L. J., Winkler-Lowen, B., Sherburne, R., Rote, N. S., Li, H., and Morrish,
17
Production of Human Trophoblast Cell Lines
Guy St J. Whitley
Summary
Our understanding of important biological phenomena such as invasion, migration, and
apoptosis has advanced greatly through the prudent use of in vitro models based on isolated
cells in culture. Established cell lines are readily manipulated using simple molecular biologi-
cal techniques and their abundance as homogenous populations allows the rapid accumulation
of statistically significant data. The study of human trophoblast function in vitro has been ham-
pered by the difficulties associated with obtaining and culturing primary human trophoblasts
including the paucity of material and contamination with other cell types. This chapter describes
a cheap and simple method for the production of human trophoblast cell lines using poly-L-orni-
thine. It details the production, isolation and initial characterization of these cells and provides
simple tips on how to store and maintain a bank of cells for future needs.
Key Words: Cytotrophoblasts, transfection, cell line, poly-L-ornithine, SV40.
1. Introduction
The power of interventional over purely observational studies is immense
and our understanding of important biological functions such as cell division,
apoptosis and migration has advanced significantly following studies using
cells grown in culture. The two options for these experiments are either to use
primary cells isolated from fresh tissue or to use established cell lines. It is the
aim of this chapter to describe the methods used to generate cell lines. Cell
lines may be obtained spontaneously as out-growths from malignant tissue or
from normal primary cultures that have been manipulated to express viral
oncogenes which overcome the cells normal growth controls.
The study of human trophoblast function in vitro has been hampered by the
difficulties associated with obtaining and culturing trophoblasts. Although pure
preparations of the different subpopulations of trophoblast can be obtained by
fluorescence-activated cell sorting (1) yields can be low and the procedures
require specialized equipment. Pure extravillous trophoblasts may be obtained
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
219
following out-growth from chorionic explant cultures (2), although this again
results in relatively low cell yields. Normal human cells cultured in vitro have
a limited proliferative lifespan, dividing a given number of times before they
undergo a permanent growth arrest, known as replicative senescence. This
seems particularly true of trophoblasts cells that grow poorly in culture. Other
problems associated with using primary cells are heterogeneity between prepa-
rations, phenotypic instability of the isolated cells and, in some circumstances,
availability of tissue. Established cell lines would therefore seem to offer a
number of advantages since they are homogenous populations that grow readily
in culture providing a plentiful supply of cells with a stable phenotype over
many passages. Conversely, the methods employed to extend lifespan (trans-
fection or spontaneous immortalization/transformation) may alter regulation
of cell division that could impact on differentiated functions and gene expres-
sion. However, it should be emphasised that no matter which route is taken
(either primary culture or established cell line), no in vitro model is perfect.
Therefore, one of the most important considerations when using in vitro mod-
els is that we recognize their limitations and design the experiments and inter-
pret the results accordingly.
A number of human trophoblast cell lines already exist; some of the more
widely used lines are listed in Table 1. Early trophoblast lines were derived
from choriocarcinomas and these include BeWo, JEG, and JAR (Table 1).
These lines have proved useful in many studies, but may have characteristics
related to their malignant origins. More recently, a number of lines that have
retained phenotypic function have been reported that arose spontaneously from
primary cultures of normal first trimester chorionic villous explants or chori-
onic villous samples; these lines include the HTR-8 (3) and the ED series of
trophoblast cell lines (4,5). Unfortunately, caution should now be taken when us-
ing ED27 as they have at some stage become contaminated with the nontrophoblast
cell line HeLa (6).
Transfection of cells with viral oncogenes from Simian virus-40 (SV40),
adenovirus, or human papilloma virus (HPV) have been used to establish lines.
The most common of these used in the production of trophoblast cell lines is
the early region of SV40 containing the small and large T-antigen. The large
T-antigen binds to and inactivates the protein products of p53 and retinoblas-
toma (RB), two tumor suppressor genes. Expression of the T-antigen increases
the proliferation of the cells in culture and delays the onset of cellular senes-
cence. As increased growth can lead to a reduction in differentiated function,
constructs containing conditional mutants including the temperature sensitive
variants of SV40 have been used to generate these lines. At the permissive
temperature the large T-antigen is expressed and functional and the cells pro-
liferate however at the nonpermissive temperature the stability of the mutant
Table 1
Commonly Used Human Trophoblast-Derived Cell Lines
Cell line Source Means of production Reference
BeWo Choriocarcinoma Spontaneous 24
ED27 First trimester Spontaneous 5
ED31 5
ED77 4
HP-A1 Term placenta Infection Adenovirus(ori)SV40tsA209 9
HP-A2 Infection Adenovirus(ori)SV40tsA209
HP-W1 Infection Adenovirus(ori)SV40wt
HT Term placenta Spontaneous 25
HT-116 First trimester Spontaneous 26
HTR-8 First trimester Spontaneous 3
HTR-8/SVneo HTR-8 Electroporation with pSV3neo 3
JAR Choriocarcinoma Spontaneous 27
JEG Choriocarcinoma Spontaneous 28
IST-1 First trimester villous HPV16 E6/E7 11
explant
NHT First trimester Spontaneous 29
NPC First trimester Spontaneous 30
RSVT-2, 2/C HTR-8 Electroporation with pRSVT 30
SGHPL-4 First trimester Poly-L-ornithine with pSV3neo 31
SPA-26 First trimester Infection SV40tsa255 8
TCL-1 Term choriodecidua pZipSV40 32
TL Term Spontaneous 33
gene product is compromised and division of the cells falls. The theory is that
at the nonpermissive temperature, the resulting cell will re-establish lost func-
tions. This type of construct has been used to produce both first and third tri-
mester trophoblast cell lines (7–9).
Although the expression of the T-antigen does extend the lifespan of the
cultures, it seldom produces immortal human cell lines. Eventually, cells
undergo a delayed phase of growth arrest and enter what is termed SV40-
crisis. This is associated with a reduction of telomere length (10). Recent work
has suggested that the expression of human telomerase catalytic component
could overcome this, although further work is required to determine if this has
adverse effects on the phenotype of the resultant cell lines.
Another commonly used construct for the production of cell lines is the
early region of HPV-16. This contains two gene products known as E6 and E7,
expression of which is also known to overcome cellular senescence in human
cells. Although E6 and E7 alone have been used to derive lines in some cell
2. Materials
1. Culture medium:
a. Standard growth medium will depend on the method of primary cell isola-
tion. We use Ham’s-F10 containing 10% (v/v) heat-inactivated fetal calf
serum (FCS), 2 mM glutamine and penicillin (100 U/mL), and streptomycin
(0.1 mg/mL).
b. Transfection medium Dulbecco’s modified Eagle’s medium (DMEM; Sigma,
Dorset UK) containing 2 mM glutamine, 100 U/mL penicillin and 0.1 mg/mL
streptomycin, and 5% (v/v) FCS.
c. Selection medium is Ham’s-F10 plus 0.3 mg/mL geneticin (G418) and 10%
(v/v) FCS.
d. Cell freezing and storage medium: standard growth medium containing 10%
(v/v) dimethylsulfoxide (DMSO; MERCK, Poole, Dorset, UK).
2. Poly-L-ornithine (Sigma Chemicals, cat. no. P4538) is prepared as a 5 mg/mL
stock in water, filter-sterilized through a 0.2-µm syringe and stored as 50-µL
aliquots at –20°C (see Note 1).
3. Chloroquine diphosphate 100 mM stock solutions (60 mg/mL in H2O) filter-ster-
ilized and stored in foil wrapped tubes at –20°C.
4. Geneticin is prepared fresh in medium and filter sterilized through a 0.2-µm sy-
ringe filter.
5. The recombinant plasmid used for transfection, pSV3-neo, contains both the large
and small T-antigens of the early region of SV40 and the bacterial neomycin
3. Methods
3.1. Transfection
The first and, possibly, most important step in the process of developing a
cell line is to obtain a primary culture rich in the cells of interest. The methods
for isolating and purifying different populations of trophoblast are presented
elsewhere in this volume, and so will not be considered here. Suffice it to say a
pure population of cells is not essential, but the greater the purity the greater
the chance of successfully obtaining useful transfectants. DNA introduced into
mammalian cells can be either retained in the cytoplasm and transiently expressed
or transported to the nucleus and integrated into the genome of the cell. Tran-
sient expression is appropriate for the production of proteins in relatively high
concentrations over short periods of less than 10 d but will not result in the
production of stable lines. Stable integration is a more rare event—perhaps as
few as 0.1% of the transfected cells will result in stable integration. It is there-
fore important to optimize the efficiency of transfection.
1. Seed cells into 9-cm dishes 24 h prior to transfection in order to achieve approx
80% confluence on the day of transfection (see Note 2).
2. For each 9-cm dish, place 3 mL of DMEM into a flat bottomed Bijou con-
tainer and add 10 µg of plasmid DNA and 5 µg/mL of poly- L-ornithine (see
Note 1). Mix gently and allow standing for 30 min at room temperature. Remove
the medium from the cells and add the DNA solution. Return the cells to the
incubator and gently rock the plate every 45 min (see Note 4).
3. After 6 h, aspirate the medium and replace with 2 mL DMEM 5% (v/v) FCS
containing 30% (v/v) DMSO (see Note 5). After 2 min, remove the supernatant
and wash gently with Ham’s-F10 plus 10% (v/v) FCS. Aspirate and replace with
a further 10 mL of Ham’s-10 plus 10% (v/v) FCS and maintain the cells in a
humidified incubator at 37°C in 5 % CO2.
4. Notes
1. Poly-L-ornithine is available from Sigma in a number of different molecular weights,
a molecular weight of 11,000 is desirable (cat. no. P4538); with a molecular weight
in the range of 5000–15,000 is recommended for this purpose.
2. There has been some controversy over antisera that should be used in the detec-
tion of HLA-G. At the 14th Rochester Trophoblast Conference (12) it was sug-
gested that the two antibodies of choice were 87G (21), and G233 (22). As the
Acknowledgments
I wish to thank Judith Cartwright and Alan Johnstone for helpful discus-
sions while preparing this chapter.
References
1. King, A., Jokhi, P. P., Smith, S. K., Sharkey, A. M., and Loke, Y. W. (1995)
Screening for cytokine mRNA in human villous and extravillous trophoblasts
using the reverse-transcriptase polymerase chain reaction (RT-PCR). Cytokine 7,
364–371.
2. Aplin, J. D., Haigh, T., Jones, C. J., Church, H. J., and Vicovac, L. (1999) Devel-
opment of cytotrophoblast columns from explanted first-trimester human pla-
cental villi: role of fibronectin and integrin alpha5beta1. Biol. Reprod. 60,
828–838.
3. Graham, C. H., Hawley, T. S., Hawley, R. G., et al. (1993) Establishment and
characterization of first trimester human trophoblast cells with extended lifespan.
Exp. Cell Res. 206, 204–211.
4. Diss, E. M., Gabbe, S. G., Moore, J. W., and Kniss, D. A. (1992) Study of throm-
boxane and prostacyclin metabolism in an in vitro model of first-trimester human
trophoblast. Am. J. Obstet. Gynecol. 167, 1046–1052.
5. Morgan, M., Kniss, D., and McDonnell, S. (1998) Expression of
metalloproteinases and their inhibitors in human trophoblast continuous cell lines.
Exp. Cell Res. 242, 18–26.
6. Kniss, D. A., Xie, Y., Li, Y., et al. (2002) ED(27) trophoblast-like cells isolated
from first-trimester chorionic villi are genetically identical to HeLa cells yet ex-
hibit a distinct phenotype. Placenta 23, 32–43.
7. Chou, J. Y. (1978) Human placental cells transformed by tsA mutants of simian
virus 40: a model system for the study of placental functions. Proc. Natl. Acad.
Sci. USA 75, 1409–1413.
24. Pattillo, R. A. and Gey, G. O. (1968) The establishment of a cell line of human
hormone-synthesizing trophoblastic cells in vitro. Cancer Res. 28, 1231–1236.
25. Ho, C. K., Li, S. Y., Yu, K. J., Wang, C. C., Chiang, H., and Wang, S. Y. (1994)
Characterization of a human tumorigenic, poorly differentiated trophoblast cell
line. In Vitro Cell Dev. Biol. Anim. 30A, 415–417.
26. Zdravkovic, M., Aboagye-Mathiesen, G., Guimond, M. J., Hager, H., Ebbesen,
P., and Lala, P. K. (1999) Susceptibility of MHC class I expressing extravillous
trophoblast cell lines to killing by natural killer cells. Placenta 20, 431–440.
27. Hussa, R. O., Story, M. T., and Pattillo, R. A. (1975) Regulation of human chori-
onic gonadotropin (hCG) secretion by serum and dibutyryl cyclic AMP in malig-
nant trophoblast cells in vitro. J. Clin. Endocrinol. Metab. 40, 401–405.
28. Kohler, P. O. and Bridson, W. E. (1971) Isolation of hormone-producing clonal
lines of human choriocarcinoma. J. Clin. Endocrinol. Metab. 32, 683–687.
29. Rong-Hao, L., Luo, S., and Zhuang, L. Z. (1996) Establishment and characteriza-
tion of a cytotrophoblast cell line from normal placenta of human origin. Hum.
Reprod. 11, 1328–1333.
30. Khoo, N. K., Bechberger, J. F., Shepherd, T., et al. (1998) SV40 Tag transforma-
tion of the normal invasive trophoblast results in a premalignant phenotype. I.
Mechanisms responsible for hyperinvasiveness and resistance to anti-invasive
action of TGFbeta. Int. J. Cancer. 77, 429–439.
31. Choy, M. Y. and Manyonda, I. T. (1998) The phagocytic activity of human first
trimester extravillous trophoblast. Hum. Reprod. 13, 2941–2949.
32. Lewis, M. P., Clements, M., Takeda, S., et al. (1996) Partial characterization of
an immortalized human trophoblast cell-line, TCL-1, which possesses a CSF-1
autocrine loop. Placenta 17, 137–146.
33. Ho, C. K., Chiang, H., Li, S. Y., Yuan, C. C., and Ng, H. T. (1987) Establishment
and characterization of a tumorigenic trophoblast-like cell line from a human
placenta. Cancer Res. 47, 3220–3224.
18
Culture and Transfection of Human Choriocarcinoma
Cells
Michael W. Wolfe
Summary
In vitro models for human trophoblasts were initially established more than three decades
ago from isolated choriocarcinomas. They have proven to be extremely valuable for the study
of the cellular, molecular, and endocrine aspects of human trophoblasts. This chapter describes
basic methods for culture and maintenance of the Jeg-3, Jar, and BeWo human choriocarci-
noma cell lines as well as an effective paradigm for introducing DNA into the cells.
Key Words: Trophoblast cells; Jeg-3 cell; Jar cell; BeWo cell; medium; transfection;
lipofectamine.
1. Introduction
In vitro models for studying human trophoblast function were initially estab-
lished more than three decades ago. Dr. Roy Hertz (1) isolated choriocarcinomas
from affected women and transferred them to the cheek-pouch of hamsters.
The tumors were maintained over a number of years by serial transfer to addi-
tional animals. Three different strains of tumors were developed. Interestingly,
animals harboring the tumors exhibited endocrine changes consistent with the
presence of human chorionic gonadotropin (hCG), a known endocrine hor-
mone secreted by human trophoblasts and choriocarcinomas. One of these
strains was subsequently used to develop a number of the choriocarcinoma cell
lines that are extant today.
Pattillo and coworkers obtained choriocarcinoma tissue from Dr. Hertz at
serial transfer 304 and placed it in a co-culture with human decidual explants
(2,3). From this co-culture, they established the BeWo choriocarcinoma cell
line. This cell line maintains many of the morphological characteristics of
human trophoblasts including the ability to form syncytia. BeWo cells, like
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
229
2. Materials
2.1. JEG-3 and Jar Cells
1. The JEG-3 and Jar cell lines are available through American Type Culture Col-
lection (ATCC, Manassas, VA; Cat. Nos. JEG-3 - HTB-36; Jar - HTB-144).
2. Cell culture medium: Dulbecco’s modified Eagle’s medium (DMEM) (with
L -glutamine and 4500 mg glucose/L; Invitrogen, Carlsbad, CA, cat. no.
11965-084), fetal bovine serum (FBS) (Invitrogen, cat. no. 16000-044), and peni-
cillin/streptomycin (10,000 U/mL; Invitrogen, cat. no. 15140-122).
3. Trypsinization solution: Trypsin-ethylenediamine tetraacetic acid (EDTA)
(0.25% trypsin, 1 mM EDTA, 4 Na), 1X liquid (Invitrogen, cat. no. 25200-056).
4. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
and 2 mM KH2PO4. Adjust pH to 7.4 and filter-sterilize or autoclave.
5. Freezing medium: cell culture medium containing 5% dimethylsulfoxide
(DMSO) (Sigma, St. Louis, MO; cat. no. D-2650), filter-sterilized.
6. Freezing container: Nalgene Cryo 1°C Freezing Container (Nalgene Nunc Inter-
national, Rochester, NY, cat. no. 5100-0001).
7. Tissue culture plates: 100 cm2 (Greiner Bio-One, Longwood, FL).
2.3. Transfections
1. Trypsinization solution: Trypsin-EDTA (0.25% trypsin, 1 mM EDTA, 4 Na), 1X
liquid (Invitrogen, cat. no. 25200-056).
2. PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4. Adjust
pH to 7.4 and filter-sterilize or autoclave.
3. Culture medium.
4. Hemacytometer (Fisher, cat. no. 02-671-6).
5. Sterile polypropylene snap-cap culture tubes (Greiner, from ISC Bioexpress, cat.
no. T-2840-5).
6. Opti-MEM I reduced serum media (Invitrogen, cat. no. 31985-070).
7. LipofectAmine transfection reagent (Invitrogen, cat. no. 18324-012).
8. Plus reagent (Invitrogen, cat. no. 11514-015).
9. TK-, CMV-, or SV40-driven Renilla expression plasmid (Promega, Madison, WI).
10. Tissue culture plates: 12-well (Greiner Bio-One).
11. Passive lysis buffer (Promega, cat. no. E1941).
12. Dual-Luciferase reporter assay system (Promega, cat. no. E1960).
3. Methods
3.1. Culture Medium
3.1.1. JEG-3 and Jar Cells
1. In a laminar flow hood or biosafety cabinet (see Note 2), add the following to a
sterile, 500-mL bottle: 445 mL of DMEM, 50 mL of FBS, and 5 mL of penicillin/
streptomycin (see Note 3).
2. The medium can be filtered through a 0.22-µm disposable filter if desired.
3. Store at 4°C in the dark.
3.2. Cell Removal From Liquid Nitrogen: JEG-3, Jar, and BeWo Cells
1. Warm the cell culture medium to 37°C.
2. Remove a vial of frozen cells from a liquid nitrogen storage tank and thaw the
cells in a 37°C water-bath. Record vial removal in the liquid nitrogen storage log.
3. Immediately after the cells are thawed, transfer the cell suspension to a sterile,
conical 50-mL tube containing 30 mL of culture medium, and mix the cell sus-
pension gently (see Note 4).
4. Place 10 mL of the cell suspension into each of three 100-cm2 tissue culture
plates and label the plates with the cell line name, passage number, and date.
5. Grow cells in a 37°C incubator supplied with 5% CO2 and 95% air.
6. Replace the cell culture medium every 2–3 d.
3.4. Liquid Nitrogen Storage of Cells: JEG-3, Jar, and BeWo Cells
1. Harvest the cells from the plates by trypsinization as described in Subheading
3.3., steps 1–6.
2. Centrifuge to pellet the cells at 216g for 10 min at room temperature.
3. Aspirate the supernatant media and gently resuspend the cell pellet in 1 mL of
freezing medium per plate of harvested cells.
4. Label 1.0- or 2.0-mL cryo vials: cell line, passage number (one greater than the
previous passage), date, and your initials. Use vials that are appropriate for liquid
nitrogen storage.
5. Transfer 1.0 mL of cell suspension into each vial.
6. Transfer the vials to the freezing container and place in a –80°C freezer for 6 h to
overnight. Transfer the vials to a liquid nitrogen storage container for long-term
storage (see Note 8). Update the liquid nitrogen storage log with these entries.
Fig. 1. JEG-3 and Jar cell lines were transiently transfected with either a human
α-subunit promoter (–845/+48) linked to luciferase or a promoterless control (0.5 µg
per well) along with a Renilla expression plasmid as an internal control (0.1 µg per
well) as described under Subheadings 3.5.1. and 3.5.2. (see Note 12). Approximately
6 h after the transfection medium was removed, cells were washed two times with
phosphate-buffered saline (PBS) and lysed using 100 µL of Passive lysis buffer. Lysates
(30 µL) were assayed for luciferase and renilla activity using the Dual-Luciferase assay
system. Luciferase activity (relative light units [RLU]) of the human α-subunit pro-
moter was adjusted to that of Renilla and presented as the mean + SE (n = 3). Activity
of the promoterless luciferase vector was 293 ± 50 (LipofectAmine only) and 442 ± 58
(LipofectAmine and Plus) RLUs in JEG-3 cells and 1129 ± 175 (LipofectAmine only)
and 610 ± 49 (LipofectAmine and Plus) RLUs in Jar cells.
3. Seed the appropriate number of 12-well culture plates with cells at a density of
30,000 cells per mL, adding 1 mL per well.
4. Place the plates into the 37°C incubator (supplied with 5% CO2 and 95% relative
humidity) and allow the cells to attach overnight.
5. Day 2. The following morning, warm the Opti-I MEM solution to 37°C.
6. Determine the concentration of all DNAs to be transfected using a spectropho-
tometer (see Note 10).
7. For each DNA to be transfected, add 0.75 mL of Opti-I MEM to separate snap-
cap tubes (see Note 2). This represents the appropriate volume for transfections
performed in triplicate (0.25 mL per well).
8. Add 1.5 µg of DNA to the appropriate tubes (e.g., human α-subunit promoter-
luciferase construct or RSV β-galactosidase expression vector) (0.5 µg per well).
9. Add 0.3 µg of a Renilla expression vector (TK-, CMV-, or SV40-driven vector)
to each tube as an internal control (0.1 µg per well).
10. Add 9 µL of Plus reagent to each tube and swirl to mix. Incubate at room tem-
perature for 15 min (3 µL per well).
11. In a sterile conical tube, prepare a solution containing 6 µL of LipofectAmine
and 0.75 mL of Opti-I MEM per DNA to be transfected.
12. Add 0.75 mL of the LipofectAmine/Opti-I solution to each tube prepared in steps
7–10. Swirl to mix and incubate at room temperature for 15 min (2 µL
LipofectAmine and 0.25 mL Opti-I MEM per well).
13. Place in the hood the 12-well plates containing the Jar cells seeded the previous
day. At the end of the 15 min incubation, aspirate the medium from one to two
plates at a time and add 0.5 mL of the DNA/Plus/LipofectAmine/Opti-I solution
to each well. Carefully label the plates and record the DNA transfected into each
well.
14. Place the plates back into the 37°C incubator for 5 h.
15. At the end of the 5 h incubation, add 0.5 mL of Jar culture medium (prewarmed)
to each well (see Note 13).
16. Day 3. In the morning, warm the Jar culture medium to 37°C.
17. Aspirate the DNA/Plus/LipofectAmine/Opti-I solution from each well and re-
place with 0.5 mL of Jar culture medium (see Note 11). Return the plates to the
37°C incubator.
18. Cultures can be harvested 6–72 h later and assayed for reporter activity (Fig. 1;
time is experiment dependent; method depends on assay used to detect reporter
activity) (see Note 12).
4. Notes
1. A clonal variant of the BeWo cell line exists (b30) that has characteristics that
differ slightly from the parental line (forms a tight monolayer) and is cultured
under slightly different conditions (see Volume 2, Chapter 11).
2. All procedures should be performed in a laminar flow hood or biosafety cabinet
to maintain sterile conditions.
3. Alternative growth media suggested by ATCC is as follows: JEG-3 cells: 90%
MEM with 2 mM L-glutamine and Earles’ balanced salt solution to contain 1.5 g/
L sodium bicarbonate, 0.1 mM nonessential amino acids, and 1.0 mM sodium
pyruvate; 10% FBS; Jar cells: 90% RPMI-1640 medium with 2 mM L-glutamine
adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES,
and 1.0 mM sodium pyruvate; 10% FBS; BeWo cells: 90% - Ham’s F12K medium
with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate; 10% FBS.
4. We typically do not remove the cells from the freezing medium. The 1:30 dilu-
tion is adequate to remove any inhibitory effects that the DMSO may cause on
cell growth and viability. Nonetheless, the cells can be pelleted (216g for 10 min)
at this step to remove the DMSO-containing medium and subsequently resus-
pend the cell pellet in 30 mL of fresh culture medium.
5. The BeWo cells grow much more slowly than do the JEG-3 and Jar cells; there-
fore, it will take longer for plates to reach confluency. In addition, because the
cells are on the plate for a longer period of time, they tend to attach more tightly.
Longer incubations with trypsin/EDTA may be required in order to effectively
detach the cells from the plate.
6. At this point, the cells can be washed two times with 5 mL of PBS to speed up the
subsequent trypsinization.
7. Only trypsinize a maximum of five plates at a time to prevent overtrypsinization.
If there are more than five, tryspinize them in groups with staggered additions.
8. The cells can remain in the –80°C freezer for longer periods of time (a few days),
but extended time at –80°C reduces cell viability when thawed.
9. With the coverslip in place and using a Pasteur pipet, transfer a small amount of
the undiluted cell suspension (invert tube three to five times to ensure that cells
are resuspended) to both chambers of the hemacytometer (allow the chambers to
fill by capillary action; do not overfill). Using an inverted microscope, count all
of the viable cells in four of the 1-mm squares (a 1-mm square is composed of a
4 × 4 grid of smaller squares) in one chamber. Repeat this with the other cham-
ber. Determine the cell density using the following equation: Cells per mL =
(total cell count/eight squares) × dilution (1 in this case) × 104. The total number
of cells harvested would be: Cells per mL × volume of cell suspension.
10. We have determined that more consistent results are obtained when all of the
DNAs to be transfected in a single experiment are quantified at the same time and
near the time (within a few days) of performing the transfection. It is also impor-
tant to use super-coiled DNA that is mycoplasma-free.
11. Alternatively, treatments can be administered at this point.
12. The LipofectAmine protocol is adequate for experiments where a high level of
transfection efficiency is not required. Transfection efficiency in JEG-3 cells can
be boosted two- to threefold (Figs. 1 and 2) by following a modification of the
approach described under Subheading 3.5.2., which uses a combination of
LipofectAmine and Plus reagent (3 µL of LipofectAmine and 3 µL of Plus
reagent per well). Conversely, transfection of the Jar cells can be simplified
(although at the cost of lower transfection efficiency) by modifying the approach
under Subheading 3.5.1. (3 µL of LipofectAmine). Finally, although a specific
protocol for the transfection of BeWo cells is not provided, we have had success
using LipofectAmine in combination with the Plus reagent as described for JEG-
3 cells.
13. Alternatively, add 0.5 mL of serum-free Jar culture medium.
Fig. 2 (see companion CD for color version). JEG-3 and Jar cell lines were tran-
siently transfected with a construct containing the Rous sarcoma virus (RSV) pro-
moter linked to a β-galactosidase reporter gene or a promoterless control
β-galactosidase vector (0.5 µg per well) as described under Subheadingss 3.5.1. and
3.5.2. (LipofectAmine alone or LipofectAmine in combination with Plus reagent; see
Note 12). Approximately 6 h after the transfection medium was removed, cells were
stained for β-galactosidase activity (12–14).
Acknowledgments
This work was supported by National Institute of Child Health and Human
Development (NICHD) (HD39695).
References
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3. Pattillo, R. A., Gey, G. O., Delfs, E., and Mattingly, R. F. (1968) Human hormone
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thesis. Ann. NY Acad. Sci. 172, 288–298.
6. Pattillo, R. A., Hussa, R. O., Huang, W. Y., Delfs, E., and Mattingly, R. F. (1972)
Estrogen production by trophoblastic tumors in tissue culture. J. Clin. Endocrinol.
Metab. 34, 59–61.
7. Kohler, P. O. and Bridson, W. E. (1971) Isolation of hormone-producing clonal
lines of human choriocarcinoma. J. Clin. Endocrinol. Metab. 32, 683–687.
8. Kohler, P. O., Bridson, W. E., Hammond, J. M., Weintraub, B., Kirschner, M. A.,
and Van Thiel, D. H. (1971) Clonal lines of human choriocarcinoma cells in cul-
ture. Acta Endocrinol. Suppl. 153, 137–153.
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equine glycoprotein hormone α subunit expression in trophoblast cells. Biol.
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ment of clonally related cells in the chicken optic tectum: lineage analysis with
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6, 1745–1755.
19
In Vitro Methods for Studying Vascularization
of the Murine Allantois and Allantoic Union
with the Chorion
Karen M. Downs
Summary
Despite the importance of the definitive chorio-allantoic placenta in fetal survival, fetal
development, and long-term health of the adult, little is known about how the placenta’s indi-
vidual components, the allantois and the chorion, proliferate and develop. In this chapter, two
techniques will be described: (1) explanting murine allantoises for culture in isolation, and (2)
grafting murine allantoises into living whole mouse embryos. Together, these will enable study
of differentiation of allantoic mesoderm into the umbilical vasculature, and the mechanism(s)
by which the allantois unites with the chorion to form the chorio-allantoic placenta.
Key Words: Allantois; chorion; chorio-allantoic fusion; explants; placenta; vasculogenesis.
1. Introduction
The definitive chorio-allantoic placenta of eutherian mammals is a compos-
ite of two tissues, the umbilical cord and the chorionic disk. The umbilical
vasculature carries fetal blood to and from the chorionic disk for exchange of
nutrients, wastes, and gases with the mother. Despite their importance in fetal
survival and development, little is known concerning how the two major tis-
sues of the placenta develop, with an especial paucity of information on devel-
opment of the umbilicus.
Although the details of chorio-allantoic placentation vary between species,
humans and rodents share major features (1). First, both possess a hemochorial
placenta, in which maternal blood directly bathes trophoblastic cells of the
chorionic disk. Second, fetal/maternal exchange is carried out within the chori-
onic labyrinth. Third, the umbilicus and future chorionic disk are initially well
separated in the conceptus, uniting to become the chorio-allantoic placenta.
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
241
Given the similarities between rodent and human placentae, that genetic muta-
tions are readily available in the mouse, and that microsurgery and other experi-
mental manipulations can be carried out in living mouse embryos during
placentation, the mouse is thus a sound choice for establishing fundamental
paradigms of early placental ontogeny.
The focus of this review will be on the umbilical component of the chorio-
allantoic placenta. In the mouse, the umbilical cord is derived from a wholly
mesodermal organ, the allantois, which grows as an extension of the antero-
posterior axis, the primitive streak (reviewed in ref. 2). Until recently, little
was known about the allantois other than what it looked like and where it came
from in the conceptus (3,4) (Fig. 1).
As a result of studies carried out over the past decade, the timing of forma-
tion and whereabouts of three allantoic cell types have been elucidated.
Mesothelium ensheathes the allantois as soon as the allantoic bud appears
in the exocoelom (early neural plate stage, approx 7.25 d post conception [dpc])
(5). Angioblasts, the precursor cells of endothelium (6), are apparent in the
distal allantoic core slightly later as a small population of Flk-1-containing
cells (late neural plate stage, approx 7.5 dpc) (5,7,8). Chorio-adhesive cells are
a subset of distal mesothelium; they mediate allantoic union with the chorion
(12), and are recognized by their content of vascular cell adhesion molecule
(VCAM)-1 (headfold stage, approx 7.75 dpc), which is required for chorio-
allantoic union (9,10). Recently, thanks in large part to the classical methods of
embryology described in this chapter, a model of differentiation of allantoic
mesoderm into mesothelium, angioblasts/endothelium, and chorio-adhesive
cells has been put forth (5).
Thus, allantoic mesoderm differentiates into the umbilical vasculature,
which is then secured onto the chorionic disk. Detailed methods through which
to study allantoic vascularization and fusion with the chorion are discussed
under Subheadings 3.6. and 3.7. General protocols for addressing these major
biological questions are described under Subheadings 3.1.–3.5., and include
directives for animal husbandry (see Subheading 3.1.), obtaining rat serum
(see Subheading 3.2.), preparation of dissection and culture media (see Sub-
heading 3.3.), sharpening forceps (see Subheading 3.4.), and dissecting and
culturing whole embryos (see Subheading 3.5.).
Please note that, strictly speaking, the correct terminology for the product of
the fertilized egg, which contains both the embryonic and extraembryonic com-
ponents, is “conceptus”; however, because use of the word “embryo” is more
common throughout modern literature, “embryo” will be used in place of “con-
ceptus” in the detailed protocols.
Fig. 1 (see companion CD for color version). Morphology of the murine allantois.
A–C, Brightfield photomicrographs of pre-fusion allantoic stages in histological sec-
tions (6 µm) stained with hematoxylin and eosin. D, Histological section (6 µm) of the
chorio-allantoic fusion junction immunostained with anti-Flk-1 (white arrowhead,
brown color on the accompanying compact disk) to demonstrate penetration of Flk-1-
positive allantoic blood vessels into chorionic ectoderm. A, Allantoic bud (al), neural
plate stage/early bud stage (7.25 days post conception [dpc]) (20). The mesodermal
allantoic core is covered in a simple squamous epithelium, called mesothelium (5). B,
Enlargement of the allantois in the exocoelomic cavity (x), three-somite pair stage
(8.25 dpc). C, The allantois is just making contact with the chorion (14), in prepara-
tion for enduring fusion (14), five-somite pair stage (8.5 dpc). D, Chorio-allantoic
fusion and penetration of the allantoic vasculature into the chorion, 12-somite pairs
(9.5 dpc). Other abbreviations: ac, amniotic cavity; am, amnion; ps, primitive streak
region; ys, yolk sac. Scale bars: 50 µm (A,D); 75 µm (B,C).
2. Materials
2.1. Mice
1. F2 generation of embryos from intercrosses of the F1 inbred hybrid strain (C57Bl/
6 × CBA) (B6CBAF1/J; Jackson Laboratories, Bar Harbor, Maine) (see Note 1).
Fig. 4 (see compaion CD for color version). Chimeric allantois. Donor allantois
(dal) is darkly stained (blue on the accompanying compact disk) and has fused with
both the host’s regenerating allantois (hal-r) and chorion (ch). Other abbreviations:
am, amnion; b, embryonic brain; ys, yolk sac. Scale bar, 100 µm.
2. Embryos hemizygous for the Rosa26 lacZ transgene (17), which are produced by
mating an F1 hybrid female (see Subheading 2.1., item 1) with a male of similar
(C57Bl/6 × CBA) genetic background but which bears two copies of the Rosa26
lacZ transgene (18) (see Note 2).
Table 1
Reagents and Suppliers for Dissection and Culture Media for Mouse Embryos
Between 7.25 and 8.5 Days Post Coitum
Dissection medium Culture medium
(DMEM + H + S) (DMEM–S)
Media component (per liter) (per liter)a
DMEM (Invitrogen-Gibco, Carlsbad, CA, 13.5 g 13.5 g
12800-017) with 4500 g/L glucose,
L-glutamine (584 mg/L) and NaPyruvate
(110 mg/L); w/o NaHCO3
HEPES, free acid (Sigma H-3375) 2.385 g –
NaHCO3 (Sigma S-8875) – 3.7 g
NaCl 0.8 g –
Amino acids (Sigma)b:
cysteine-HCl (C-1276) 4.8 mg 4.8 mg
L-alanine (A-7469) 3.6 mg 3.6 mg
L-asparagine (A-0884) 6.0 mg 6.0 mg
L-aspartic acid (A-8949) 5.3 mg 5.3 mg
L-proline (P-5607) 4.6 mg 4.6 mg
L-glutamic acid (G-1626) 5.9 mg 5.9 mg
Penicillin-streptomycinc (Invitrogen-Gibco 15070-063) 2 mL 2 mL
(100 U/mL pen, 100 mg/mL strep)
Heat-inactivated Fetal Calf Serum (FCS)d 7.5% –
(Invitrogen-Gibco 16000-044)
Heat-inactivated rat serum (prepared in house) – 50.0%e
DMEM, Dulbecco’s modified Eagle’s medium.
aBefore rat serum is added, all components are combined and the pH of the culture medium is adjusted
to 7.3 by bubbling in CO2. The solution is then filter-sterilized, aliquoted, and stored indefinitely at –86°C.
bThe amino acids are made as a 100X stock, i.e., 100 times the amounts shown above are combined
in approx 980 mL sterile double-distilled water, the pH is adjusted to 9.0 with 5 N NaOH. Bring the
volume to 1000 mL with double-distilled sterile water. Dispense 10 mL aliquots to 15 mL sterile
polypropylene tubes and store, unfiltered, indefinitely at –86°C.
cPenicillin-streptomycin stock: 5000 U/mL penicillin G sodium and 5000 µg/mL streptomycin sul-
fate in 0.85% (physiological) saline. Upon delivery from the manufacturer, thaw solution in warm water
and dispense in 6 mL aliquots to 15 mL sterile polypropylene tubes via a sterile volumetric disposable
pipette. Store the aliquots indefinitely at –86°C. When making dissection or culture media, completely
thaw a 6-mL aliquot and remove 2 mL; refreeze the remaining 4 mL.
dAs soon as the fetal calf serum (FCS) is delivered to the lab, thaw it in warm water; monitor the
bottle for breakage during the thaw. If the FCS has not been heat-inactivated from the supplier, dispense
37.5 mL aliquots of FCS to 50 mL sterile polypropylene conical tubes and heat-inactivate it at 56°C for
1 h. Store the FCS indefinitely at –86°C.
eHeat-inactivated rat serum is prepared by bleeding isofluorane-anesthetised rats from the descend-
ing dorsal aorta and immediately centrifuging the blood (see Subheading 3.2.2.). The serum is stored at
–86°C. Just before use, the serum is heat-inactivated at 56°C for 30 or 60 min, spun at 1625g for 5 min,
and diluted 1:1 with culture medium. It is then distributed to embryo culture tubes or 24-well plates,
which are gas- and temperature-equilibrated for at least 1 h before embryos or allantoises are added.
3. Methods
3.1. Maintenance of Mouse Strains
The F1 inbred hybrid strain (B6CBA/J) provides not only standard embry-
onic material for most allantoic explant experiments, but host recipient embryos
for allantoic grafting experiments, as well.
1. F1 hybrids are purchased from the Jackson Laboratories when they are 6–8 wk of
(breeding) age, and are maintained on a 12-h light/dark cycle.
2. The lights are turned off at 1300 h so that most experiments can be carried out in
the late morning/early afternoon, rather than late into the night.
3. Estrous females are selected (19) just before the lights are turned off; single
estrous females are then paired with a single stud male.
4. Copulation plugs are checked during the dark cycle at 1630 h on the day of pair-
ing with the aid of a red light.
5. If a plug is detected at 1630 h, pregnant females are dissected 8 d later, at 1100 h,
when the majority of conceptuses are at the headfold stage (20). If no copulation
plug is detected at 1630 h, females are checked again at 0830 h the next morning,
after which embryos are dissected between 1300–1600 h seven days later.
Table 2
Stock Solutions for β-Galactosidase Activity*
Number Chemical FW Concentration Preparation
I K3Fe(CN)6 329.2 200 mM 65.85 mg/mL PBS
II K4Fe(CN)6.3H2O 422.4 200 mM 84.48 mg/mL PBS
III MgCl2 203.3 1M 10.16 g/50 mL H2O
IV X-gal 408.6 1M 40 mg/mL DMSO
(5-bromo-4-chloro-3-indolyl (dimethyl sulfoxide)
β-D-galactopyranoside)
*All reagents are purchased from Sigma. Nonsterile distilled water is used to make solutions I–III,
which are stored indefinitely at 4°C. Aliquot 250 µL of solution IV into sterile Eppendorf tubes, cover
each one with foil, and store indefinitely at –86°C. X-gal can be thawed and re-frozen five times.
Table 3
Working X-Gal Solution*
Amount of working solution
Stock solution 50 mL 10 mL Final concentration
tion in this Table. The phosphate-buffered saline (PBS) is made in nonsterile distilled water; use it at
room temperature to ensure that all components go into solution.
Although most of our experiments are initiated at the headfold stage (approx
7.75–8.0 dpc), a variety of other prefusion stage embryos (neural plate/early bud,
EB, through five to sixsomite pairs (20); approx 7.25–8.5 dpc) can be obtained
by following these guidelines of animal husbandry.
To distinguish allantoic cells from those of the host in allantoic grafting
experiments, use lacZ-labeled allantoises as donor material. Adult Rosa26 lacZ/
lacZ homozygotes are maintained on the same lighting regime as the F1 hybrid
animals, described in the previous section. To obtain hemizygous lacZ/+ embryos,
F1 hybrid females, described above, are selected for estrus (described above), and
mated with individual stud Rosa26 lacZ/lacZ males, at the times described
above. Plugs are checked as described above.
14. At this point, release the forceps and gently grab the end of the plunger with the
same hand; this allows the other hand to switch grip on the syringe barrel and
cradle it from underneath.
15. From this point on, slowly pull the plunger toward you, while maintaining an eye
at all times on the tip of the needle. By this time, about 1–2 mm of the needle tip
will have penetrated the aorta. The blood will look cherry red; it is highly oxy-
genated. However, you might mistakenly enter the vein, in which case the venous
blood is quite dark purple. Serum obtained from venous blood seems not to have
an adverse affect on embryo culture (K. Downs, unpublished). If entry into the
dorsal aorta is too superficial, blood will not enter your syringe. In that case, you
can safely remove the needle, and try again.
16. During exsanguination, and as you gently pull the plunger toward you, the needle
may clog. Stop pulling the plunger, and gently rotate the needle 90° within the
lumen of the aorta, then resume pulling on the plunger. As the rat becomes exsan-
guinated, it will eventually stop breathing. From this point on, only approx
1–2 mL more of blood might be collected, bringing the total to 10–15 mL.
The procedure, from the time of removing the rat from the anesthesia chamber to
complete exsanguination, takes approx 3–4 min.
17. As you withdraw the needle from the aorta, place a Kimwipe over the entry site
to absorb any residual blood that might spurt out.
18. Using the hemostat, disconnect the needle from the syringe, dispose of it in a
sharps container, and gently expel the fresh blood down the side of a sterile
15-mL tube.
19. Immediately euthanize the rat by cutting open its rib cage and piercing its heart.
Immediately thereafter, spin the collected blood in a tabletop centrifuge at 1625g
for at least 10 min. Place the rat in a nearby carcass bag. If only one centrifuge is
available, delay procedure on the next rat. If two centrifuges are available, you
can immediately prepare the next rat. Do not worry if the tubes spin for longer
than 10 min.
interface. This procedure releases the serum and results in collapse of the clot
toward the interface. Take care to minimize contact with red blood cells. Some-
times a clot is not visible; nonetheless, “squeeze” the plasma as if it contained a
clot.
6. Spin the tubes containing the squeezed clots for 10 min at 1625g, and decant the
serum to fresh 15-mL tubes via a sterile 9-in. Pasteur pipet. If the clot reappears,
squeeze it again, and spin it down.
7. It is desirable to mix the serum from rat to rat, so as to partially normalize it.
Thus, serum from several rats can be collected in one tube.
8. To remove residual red blood cells from the serum, spin the tubes containing
pooled serum for 10 min at 1625g.
9. Avoiding the typically tiny pellet of red blood cells at the bottom of the conical
tube, use a sterile 5-mL pipette to aliquot serum to 6-mL labeled cryotubes in
1-, 2-, and 3-mL volumes.
10. These are then frozen at –20°˚C overnight, after which they are placed indefi-
nitely at –86˚°C (see Note 6).
vacuum. Add 37.5 mL of heat-inactivated FCS to each aliquot, bringing the total
volume of each half to 500 mL. Dispense approx 40 mL of the dissection medium
into each of about 25 sterile 50-mL polypropylene conical tubes labeled “DMEM
+ H + S” and the date. Store indefinitely at –86°C.
5. After thawing, store dissection medium at 4°C for no longer than 1 mo.
for 5 min at 1625g at room temperature. With a sterile 9-in. Pasteur pipet, trans-
fer all but the bottom-most serum into a sterile 50-mL polypropylene tube. It is
important that one add the serum to the test tube before adding the culture medium
(DMEM–S) in case some is spilled. Then, add an equal volume of culture medium
(DMEM–S) via a sterile 5-mL disposable volumetric pipet attached to a dedi-
cated automated pipettor. Gently mix.
4. Label embryo culture tubes and distribute 1 mL of complete culture medium to
each one. With caps in the loose position, balance the culture tubes against each
other in the incubator’s roller apparatus, placed at 8° off the horizontal and roll-
ing at 0.5 rpm (25). We never turn off the roller apparatus. Gas- and temperature-
equilibrate the complete culture medium for at least 1 h before placing embryos
into the tubes. When equilibrated, the color of the medium will be fleshy-pink.
Any unused medium can be stored for up to 24 h at 4°C and used the next day; if
you do this, mix it with fresh medium, rather than using it separately, because in this
way, all embryos in that experiment will be exposed to similar culture conditions.
3.4. Protocol for Sharpening Forceps (Modified From ref. 21)
Assess the state of your forceps. Forceps should have the following charac-
teristics:
• Tines are of similar thickness.
• Tips of tines meet in a point when examined in profile. There is no gap between
them.
• When examined one above the other, tines do not substantially overlap.
• When examined from the inside, the tips of the tines are pointed.
With the aid of the dissection microscope, carry out the following steps, but
note that fine forceps will require a much more gentle touch during the sharp-
ening procedure than the robust forceps:
1. Place the Arkansas stone on a horizontal surface and squirt it with distilled water.
Hold the forceps vertically over the wet part of the stone with the tips down and
lightly squeezed together. Move the forceps across the stone, applying minimum
pressure. After grinding, dry the tines with a Kimwipe and check them in a dis-
section microscope with ×25 magnification. Continue grinding until the tips are
the same length.
2. Rotate the forceps 90°, and determine whether and where the tines overlap. With
the tips of the tines apart, position the forceps horizontally so that the edges of
both tines are in contact with the stone along their length, and move the forceps
from side to side, applying more pressure to one or the other prong if it overhangs
when the tips are brought together. The tines may also be slightly angled toward
their tip during this procedure to shave off more metal from the tip.
3. Rotate the forceps 180°, examine them for overlap, and repeat step 2 with the
other side of the tines.
4. Rotate the forceps 90°, and examine the thickness of the tines. A gradual tapering
toward the tip, and tines of similar thickness along this length, are desired. For
this, grind the outer surface of each tine from side to side on the stone until the
thickness is reduced and tapered toward the tip.
5. Turn the forceps 180° and repeat step 4 on the other tine.
6. Check that the tips are still of the same length when just touched together, repeat-
ing step 1 if necessary.
7. With very light strokes, round off the inner edges and tip so that, when apposed,
the tips together form a tapered probe. Very fine sandpaper may be used for this
step and for future periodic touch-ups.
8. Check the inner edges of the tines to make sure that they make a point. If not,
gently stroke each tine near the tip along the stone until a point is made; if the tips
no longer meet, repeat step 6 and/or 7.
Before each dissection, make sure that the forceps are in good condition.
Once sharpened as desired, forceps should need only minor touch-ups, which
can be carried out with very fine sandpaper. When not in use, protect the for-
ceps by sheathing the tips with a cut-off yellow pipet tip. After use, and also
before the next dissection, wash the forceps in soapy water, rinse in tap water
and then distilled water, apply a final rinse in absolute alcohol, and wipe dry
with a Kimwipe. Never flame dissection forceps.
2. Using both pairs of fine forceps, pinch the trophoblast, associated parietal endo-
derm, and its Reichert’s membrane (all three tissues are collectively, though col-
loquially, called “Reichert’s membrane) at the embryonic/extraembryonic
junction.
3. With one of the forceps, reflect Reichert’s membrane toward the embryo’s distal
end, rounding it and allowing Reichert’s membrane to retract toward the extraem-
bryonic region.
4. Trim away enough of this reflected membrane so that the embryos can be
staged (20).
4. If the stretched portion of the tubing is less than 120 µm thick, it should snap
smartly, and break in half. If not, throw the tubing away and try another piece.
5. If the glass breaks, the broken ends should be flush, rather than beveled. Discard
any halves that have beveled ends, because these will leave behind bits of the
base of the allantois during aspiration.
6. Score the base (nonpulled end) of the glass microcapillary with the diamond-tip
glass scorer and break cleanly so that the shaft of the final product is approx 1–2
inches long. This length will allow comfortable manipulation of the allantois in
the dissection microscope.
7. Measure the inner diameter of the microcapillary in an eyepiece reticule. It should
be between 60 and 120 µm.
8. Store the microcapillaries in boxes into which has been inserted a strip of thick
foam scored at regular intervals with a razor blade. Use one box each for 60-µm,
90-µm, and 120-µm microcapillaries and label appropriately.
9. Repeat the above procedure on the shorter length of glass, thereby conserving
this expensive commodity.
4. Replace the dish containing the explanted allantoises with the 24-well plate; in
the dissection microscope, release a single allantois into a single well of the plate.
5. Repeat steps 3 and 4.
6. Culture the allantoic explants for up to 24 h. If you wish to culture the explants
for longer periods, change the complete culture medium every 24 h with fresh
gas- and temperature-equilibrated fresh complete culture medium. Do not rinse
allantoises between changes of medium.
7. Examine the explants in the inverted compound or tissue culture microscope.
3.6.4.2. CULTURING ALLANTOISES ON GLASS COVER SLIPS
You may want to mount immunostained allantoic explants or subregions on
a glass microscope slide for closer analysis. Although chamber slides are avail-
able, we have found that these do not support allantoic growth and develop-
ment as well as culture in 24-well dishes (K. Downs, unpublished data).
1. Thus, at least 1 d before allantoic explantation, insert autoclaved glass cover slips
into each well of a sterile 24-well plate, and coat each cover slip with poly-D-
lysine, as described later. Vascularization on poly-D-lysine-coated glass cover-
slips appears to take place as well as on tissue culture plastic, although with a
delay of 4–5 h (8).
2. Prepare poly-D-lysine by dissolving 50 mg poly-D-lysine in 50 mL sterile double-
distilled water for a final solution of 1 mg/mL. Filter poly-D-lysine solution
through a 0.22-µm filter and store at 4°C.
3. Place autoclaved cover slips into individual wells of a 24-well plate using heat-
flamed nonembryo dissection forceps.
4. Pipet 200 µL poly-D-lysine onto each cover slip.
5. Incubate cover slips for 30 min at room temperature.
6. Aspirate poly-D-lysine via a vacuum apparatus, and rinse wells three times each
with at least 2 mL of sterile double-distilled water per well each time. Do not flip
over the cover slips during this procedure.
7. Cover the 24-well plate, and air dry the poly-D-lysine coated glass inserts over-
night up to 2 wk.
8. Proceed as described above for culturing allantoic explants, and carry out stain-
ing as normal. At the last step of the staining procedure, remove the cover slip
with a forceps, place it explant side up on a glass slide, and overlay the explant
with water-based mounting medium and a standard glass cover slip. View the
explant in a compound microscope.
3.6.4.3. CULTURING ALLANTOISES IN SUSPENSION
One may wish to culture whole allantoises or allantoic subregions in sus-
pension, because cell relations after double-immunostaining are more readily
apparent in sectioned specimens than in plated allantoic explants.
1. Isolated allantoises can be cultured in suspension in embryo culture tubes (see
Subheading 2.5., item 16) containing 0.5 mL medium and inserted into the roller
apparatus.
2. Roll the tubes at approx 1 rpm, instead of 0.5 rpm, to prevent the explants from
sticking to the walls of the test tube.
3. Suspended allantoises can be whole mount stained, squashed beneath a glass
cover slip on a glass microscope slide, or especially, prepared for immunohis-
tochemistry in histological sections (7,18).
7. Aspirate the donor material into the tip of the microcapillary. Directing the
microcapillary into the host’s anterior yolk sac puncture, gently release the donor
material into the host’s exocoelom.
8. Place the operated host embryo and the operated donor embryo from which
you’ve removed the donor allantois (by yolk sac puncture) into an embryo cul-
ture tube containing gas- and temperature-equilibrated culture medium, and note
the time that culture was begun. The advantages of this culture arrangement are
that first, at the end of culture, the numbers of somite pairs are compared—they
should be similar, thus confirming synchronicity of the donor allantois and host
embryo; and second, the donor embryo serves as a positive control for X-gal
staining.
9. Continue in this manner until all desired donor and host embryos have been operated.
10. Place unoperated donor and host embryos into separate culture tubes; these serve
as controls for chorio-allantoic union. Embryos of each pair can be distinguished
by trimming the ectoplacental cone of one member of the pair (see Subheading
3.5.1.5., step 3).
11. Culture embryos for up to 24 h.
12. At the end of the culture period, score the operated embryos for the location of
the donor allantois (sometimes this is not possible until after applying appropri-
ate methods of visualization; see Subheading 3.7.2.), and score both operated
and control embryos by morphological features (27) (see Subheading 3.5.3.).
13. Proceed to Subheading 3.7.3.
4. Notes
1. The F2 inbred hybrid strain (B6CBA/J) was used to compile an extensive histo-
logical atlas (30) as well as fate maps of the mouse gastrula (4). Thus, an exten-
sive foundational literature exists for this strain.
2. All cells of hemizygous Rosa26 embryos stain positively for β-galactosidase
activity between 7.5–9.5 dpc, turning blue, with the exception that cells of the
ectoplacental cone are not as intensely blue as the other cells in the embryo, even
Acknowledgments
The author is deeply grateful to the following scientists who contributed to
the foundational success of the protocols described in this document: Professor
Sir Richard Gardner, Dr. Kirstie Lawson, the late Dr. Rosa Beddington, and
Dr. David Cockroft.
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analysis of X-chromosome inactivation and the origin of the germ line in the
mouse embryo. J. Embryol. Exp. Morph. 52, 141–152.
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9. Gurtner, G. C., Davis, V., Li, H., McCoy, M. J., Sharpe, A., and Cybulsky, M. I.
(1995) Targeted disruption of the murine VCAM1 gene: essential role of VCAM-
1 in chorioallantoic fusion and placentation. Genes Dev. 9, 1–14.
10. Kwee, L., Baldwin, H. S., Shen, H. M., et al. (1995) Defective development of the
embryonic and extraembryonic circulatory systems in vascular cell adhesion mol-
ecule (VCAM-1) deficient mice. Development 121, 489–503.
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ment and lethality in embryos lacking a single VEGF allele. Nature 380, 435–439.
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ontogeny: allantoic attachment to the chorion is selective and developmentally
regulated. Development 121, 407–416.
13. Yang, J. T., Rayburn, H., and Hynes, R. O. (1995) Cell adhesion events mediated
by α4 integrins are essential in placental and cardiac development. Development
121, 549–560.
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IV
PHENOTYPIC ANALYSIS OF THE PLACENTA
20
Phenotypic Analysis of the Mouse Placenta
Summary
Placental development is a dynamic and complex process and much of our current under-
standing of the underlying molecular processes comes from analysis of targeted gene mutations
in mice. There are more than 50 strains of mutant mice that have placental defects, and it has
become widely appreciated that placental defects should be suspected in cases where embry-
onic lethality is observed. The degree to which these phenotypes are investigated is highly
variable, owing to a general lack of expertise in the field. However, there has been considerable
progress in developing techniques and reagents for analyzing placental phenotypes that are
relatively simple to apply and that should be accessible to all investigators. This chapter pro-
vides a basic outline of the strategies for the general identification and then the subsequent
detailed investigation of placental phenotypes.
Key Words: Transgenic mice; knockout mice; histology; gene.
1. Introduction
The mouse has become a dominant model system for studying the function
of genes in mammals because of the ability to alter gene functions through
transgenic and gene knockout approaches. The vast majority of embryonic
lethal phenotypes that are due to loss-of-function mutations are associated
with placental defects and, as such, we now know the molecular basis of many
aspects of placental development and function (1–5). In most cases in which
investigators have made knockout mice, a placental phenotype was completely
unsuspected. Indeed, in some cases, phenotypes were initially attributed to
other defects but have been more recently re-interpreted as being due to the
placenta. These problems have highlighted the fact that the placenta, until
recently, has been underappreciated in the broad biomedical research field.
Considerable progress has been made in the last decade, however, in our under-
standing of how the mouse placenta develops and in development of approaches
and methods for studying it in detail. Given this progress, it is now possible to
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
275
that bring maternal blood to the implantation site, and these cells are detectable
soon after implantation. A subtype of spongiotrophoblast cell, called glycogen tro-
phoblast cell, invades interstitially into the uterus but only starting after E 12.5.
In analyzing placental development in mouse mutants, we take a systematic
approach that first involves assessing the stage(s) that may be affected. In gen-
eral, this is done by defining if there is a specific period during gestation when
placental function is abnormal and by noting whether the major developmental
milestones have been achieved. This is usually best accomplished by doing
gross dissection of tissues. This is then followed by histological and marker
analysis. It is important to take this two-stepped approach, and a common mis-
take is to immediately dive into histological analysis, which is very time-con-
suming if done properly. In this chapter we outline the general approaches and
refer to the specific techniques that we use for detailed analysis. Many of the
detailed protocols have been published elsewhere and as such will not be
repeated here.
2. Materials
1. Dissecting scissors.
2. Dissecting stereomicroscope.
3. Watchmaker’s forceps: Dumont #55 (Fine Science Tools Inc., Foster City, CA).
4. 60-mm and 100-mm Petri dishes for dissections.
5. Ice-cold phosphate-buffered saline (PBS).
6. Ice-cold 4% paraformaldehyde (PFA) in PBS, prepared fresh. Made from a 20%
stock solution that can be stored at –20°C.
7. Proteinase K lysis buffer: 50 mM KCl; 10 mM Tris-HCl, pH 8.3; 2.0 mM MgCl2;
0.1 mg/mL gelatin; 0.45% Tween-20; 0.45% Nonidet P-40; 0.1 mg/mL proteinase K.
8. NT solution: 0.15 M NaCl; 0.1 M Tris, pH 7.5.
9. NTMT solution: 0.1 M NaCl; 0.1 M Tris, pH 9.5; 0.05 M MgCl2; 0.1% Tween-
20. Prepare fresh daily.
10. Alkaline phosphatase substrate: 5-bromo,4-chloro,3-indolylphosphate (BCIP)/
nitroblue tetrazolium (NBT) substrate kit (Vector Laboratories, Burlington, ON,
Canada, Cat. No. SK-5400).
11. Nuclear Fast Red (Vector Laboratories, Cat. No. H-3404).
12. Peroxidase-conjugated Isolectin BS1 (Sigma-Aldrich, St Louis, MO, cat no.
L-5391).
13. Rabbit polyclonal anti-laminin antibody (Sigma-Aldrich, cat. no. L-9393).
3. Methods
3.2. Gross Dissections to Survey Stages of Placental Development
and Function
The most critical first step in determining whether you have a placental phe-
notype is to look for signs of placental dysfunction. Mild placental dysfunction
Fig. 2. Anatomy and dissection of the pregnant mouse uterus to remove the decidual
swellings. Briefly, individual implantation sites are separated. The uterine muscle and
membranes are removed by using forceps to carefully tear them away along the
antimesometrial side of the implantation site. The implantation site can then be
removed from the uterus on the mesometrial side by using one set of forceps to
hold the uterus while using a second set to gently shell the implantation site away.
ing motion with the forceps can lift the decidua away from the mesometrial
side. This same general approach is used to remove implantation sites at all
stages of gestation.
3.2. Analysis of the Placenta Between E 5.5 and 8.5
3.2.1. Examination of the Placental Structures
During the first few days after implantation, the conceptus is surrounded by
a relatively thick layer of decidual tissue. The conceptus is positioned within
the tissue in a stereotypical pattern. The conceptus is located centrally in the
radial axis. The ectoplacental cone sits at roughly the mid-point along the
mesometrial to antimesometrial axis and one can usually see an accumula-
tion of maternal blood in a “red band” at this position (Fig. 3). As the concep-
tus grows, the decidual tissue thins around the radial diameter and therefore the
outermost layer of the conceptus (trophoblast giant cells of the parietal yolk
sac), are located closer to the outside. Using these general rules and landmarks
we use one of two approaches to remove the conceptus from the decidua
between E 5.5 and 8.5. In order to visualize the conceptus intact we split the
deciduas along the mesometrial to antimesometrial axis (Fig. 3, longitudinal
bisection). For conceptuses at E 8.5, we position forceps at about the 30:70
position and slowly squeeze the points together in a scissor-like cut. This will
Fig. 3. Alternative approaches for opening the decidual swellings. Implantation sites can be bisected either transversely
or longitudinally with respect to the mesometrial–antimesometrial axis to facilitate the separation of the embryo and pla-
centa into separate compartments of the implantation site or to allow observation of the intact embryo and placenta respec-
tively. To bisect the implantation site, place it gently between fine forceps and carefully close them along the axis you wish
cut. Using a scalpel blade, or one side of a second set of forceps as a blade, make a cut along the line of the first set of forceps
8/29/05, 11:21 AM
to bisect the implantation site as shown.
281
282 Natale, Starovic, and Cross
Table 1
Molecular Markers of Placental Development From Early Postimplantation to
Embryonic Day (E) 8.5
Gene Site
of expression Technique Comments Reference
Pem ExE ISH, IHC E 5.5 in extraembryonic tissues; 19
later in EPC, Ch, SGC
Eomes ExE ISH E 5.5 in extraembryonic tissues; 20
later in EPC, Ch and embryo;
required for trophoblast devel-
opment following blastocyst
formation
Estrrb ExE ISH E 5.5—in extraembryonic 21
ectoderm; E 7.5—specific to
chorion; diminishes following
chorioallantoic attachment
Limk TGC ISH Expressed exclusively in giant 22
cells from E 4.5 onwards
Mash2 EPC, Ch ISH 23
Pl1 TGC ISH Detectable in primary GCs 24
lining the embryonic cavity
Plf TGC ISH Detectable in primary and 25
secondary GCs
make a glancing cut through the parietal yolk sac but leave the ectoplacental
cone and embryo intact. The ectoplacental cone is recognizable because it is
red as a result of the presence of maternal blood. The smaller piece of decidua
will contain part of the parietal yolk sac and can be used to examine tropho-
blast giant cells. For conceptuses younger than E 8.5, the forceps should be
positioned closer to the midline since the conceptuses are much smaller.
The ectoplacental cone and extraembryonic ectoderm/chorion are best stud-
ied in detail using histological and marker analysis (Fig. 1; Table 1). Most
current markers that are available are mRNAs and therefore must be detected using
RNA in situ hybridization. Given that both ectoplacental cone and extraembry-
onic ectoderm/chorion cells proliferate, if the relative size of these tissues is
reduced, it is useful to assess the cell proliferation index by using standard
approaches such as BrdU incorporation, or Ki67 or proliferating cell nuclear
antigen (PCNA) immunostaining which all identify cells undergoing or that
Table 2
Molecular Markers of Placental Development at the Time of Chorioallantoic
Attachment
Gene Site of expression Technique Comments Reference
IHC, immunohistochemistry.
visceral yolk sac creating a window into the extraembryonic cavity. The cut
can then be extended around the circumference thus removing the cup-shaped
decidua/yolk sac tissue, leaving the amnion-covered fetus attached to the pla-
centa by the umbilical cord. A second and even simpler technique can be used
to separate the fetus from placenta, once you are assured that chorioallantoic
attachment is normal based on dissection of other litters—simply place forceps
across the full thickness of the decidua, just below and parallel to the base of
the placenta, slowly squeeze shut and make a cut along the edge of the forceps.
This separates the placenta from the fetus/yolk sac/decidual tissue in one rapid
step (Fig. 3, transverse bisection).
For routine purposes, we fix tissues in 4% PFA, embed in paraffin, and then
make serial sagittal sections through the entire block, mounting two sections
per slide. We then stain every 10th slide with hematoxylin/eosin (H&E). Slides
are examined in order to find the midpoint of the placenta (site of umbilical
attachment), which is used as the major reference point for comparisons
between mutants and wild-type littermates. The major structures to note
are the number and size of trophoblast giant cells, and the thickness of the
spongiotrophoblast and labyrinth layers. These layers have distinct morpho-
logical features that are clear even from H&E-stained materials, though a num-
ber of molecular markers are helpful in defining the layers at a gross level (see
Table 3, Fig. 4, and Note 5). A systematic approach is required in order to
evaluate the development of both the trophoblast and vascular compartments
of the placenta, and a common mistake is to only describe the presence or
absence of fetal capillaries (see Note 6).
3.4.2. Alkaline Phosphatase Histochemical Staining for Trophoblast
Cells Lining the Maternal Blood Sinuses
Trophoblast cells that line the maternal blood spaces express endogenous
alkaline phosphatase activity that can be detected by histochemical staining of
histological sections (Fig. 4).
1. Briefly, following de-waxing and rehydration of tissue sections, slides are washed
in NT solution for 20 min at room temperature followed by washing in NTMT
solution for 10 min at room temperature.
2. To visualize enzymatic activity, the color is developed using a standard alkaline
phosphatase substrate (e.g., BCIP/NBT).
3. Sections are then stained with Nuclear Fast Red and mounted following dehydra-
tion and clearing through a graded series of ethanol and xylene washes.
20_Natale_273_294_F
Table 3
Molecular Markers of Placental Development Associated With Placental Labyrinth Development
Gene Site of expression Technique Comments Reference
Gcm1 Ch, Lab ISH— E 8.5—expressed in subsets of trophoblast 18,27
286
wholemount on chorionic plate; E 9.0 demarcates
chorioallantoic branching and then
expressed in syncytiotroph. in labyrinth
layer; required for chorioallantoic
branching and labyrinth formation
Eomes Lab ISH Marker of trophoblast stem cells 20
Tpbpα EPC, Sp ISH 28
286
Mash2 EPC, Sp ISH Mutant results in loss of spongiotrophoblast
layer and compact labyrinth 23
Hand1 EPC, TGC ISH Expression overlaps with Mash2 in EPC;
required for TGC differentiation 11,29,30
Pl1 TGC ISH, IHC PL1 protein detectable E 9–E 10, not after E 10 24
Pl2 TGC ISH, IHC PL2 protein detectable beginning E 10 31
Plf TGC ISH Expressed strongly E 8–E 10 25
ISH, in situ hybridization; IHC, immunohistochemistry; EPC, ectoplacental cone; TGC, trophoblast giant cell; Ch, chorion; Lab,
8/29/05, 11:21 AM
labyrinth layer; Sp, spongiotrophoblast
Natale, Starovic, and Cross
Phenotypic Analysis of Mouse Placenta 287
Fig. 4. Low and high magnification micrographs of sagittal sections from a pla-
centa at embryonic day (E) 11.5 stained for various markers. Alkaline phosphatase
activity and laminin immunoreactivity are used to identify maternal and fetal-derived
blood spaces in the labyrinth layer, respectively. Tpbpα and proliferin (Plf) mRNAs
are used to identify the spongiotrophoblast and trophoblast giant cell layers respec-
tively. F, fetal capillary; M, maternal blood space; Sp, spongiotrophoblast; TGC, tro-
phoblast giant cell.
Fig. 5. Anatomical organization of the mature placenta and the maternal spiral
arteries. The arrows highlight the orientation of tissue sectioning for sagittal and
transverse sections. Sagittal tissue sections are used for the routine analysis of mor-
phology and marker gene expression. Serial transverse sections are used for detailed
assessment of endovascular trophoblast invasion.
recently that the normal patterns of invasion have been described and methods
developed to identify the two distinct invading cell populations (9). Sagittal
sections can be used to generally survey the extent of invasion, but serial cross
sections (Fig. 5) are needed in order to study the course of invasion of the
endovascular trophoblast giant cells because they traffic along spiral shaped
arteries that come in and out of the planes of sagittal sections. The endovascular
giant cells are apparent from the early postimplantation period to term and are
characterized by positive staining for Plf mRNA, but negative for Pl1 and
Tpbpα mRNA and periodic acid-Schiff (PAS) staining. The interstitial invad-
ing glycogen trophoblast cells are negative for Plf and Pl1 mRNA, but positive
for Tpbpα mRNA and PAS staining (9). Because of the close relationship be-
tween endovascular trophoblast giant cells and maternal spiral artery develop-
ment, the information in a survey of these trophoblast cell subtypes in serial
sections is greatly enhanced by doing a comparative analysis with markers for
endothelium (e.g., Factor VIII or CD31/PECAM1), smooth muscle actin and
NK cells (which are implicated in spiral artery dilation) (9).
4. Notes
1. Identification of pups. Tatooing pups should not routinely be done at birth because
many females are stressed by the intrusion and will reject or cannibalize their
young.
2. Verification of an embryonic lethal phenotype. An embryonic-lethal phenotype
can be difficult to prove as pups that are born stillborn, or that die within hours of
birth, are usually cannibalized by the female. For this reason, it is a good idea to
check the cage in the morning after a delivery to look for signs of dead pups,
before putting a lot of effort into assessing intrauterine development. This is
important because observing intrauterine development involves having to sac-
rifice the pregnant female, and such females are usually in short supply when a
phenotyping project first begins.
3. Initial intrauterine investigation of an embryonic lethal phenotype. We begin our
survey of embryonic lethal phenotypes by examining conceptuses at E 10.5. This
time is chosen for three reasons. First, it is half way through gestation and there-
fore it is convenient to address if lethality occurs in the first or second half of
gestation. Second, the vast majority of placental mutants that have been described
to date occur around mid-gestation. Third, if mutant conceptuses were able to
implant (E 4.5) and therefore initiate a decidual response, there should still be
evidence of it within the uterus even if development of the conceptuses stalled
immediately after implantation. By gestation day 10.5, such implantation sites
would show signs of advanced resorption (hemorrhage, necrosis) and usually no
useful tissue for genotyping can be recovered. However, their presence should
be noted and the objective is to see if the frequency of resorptions accounts for
the mutant class. It is important to remember that there is a normal background
rate of resorptions even in wild-type mice, but in outbred strains of mice the
normal resorption rate is usually less than 5%.
4. Fluorescence-activated cell sorting (FACS) and trophoblast giant cells. In our
experience, trophoblast giant cells cannot reliably be analyzed using FACS. Their
large size results in cell fragmentation during sorting and they also tend to clump.
5. Complexity of the labyrinth layer. The labyrinth layer of the placenta is very com-
plex in that, as described in the Introduction, it contains several trophoblast sub-
types as well as stromal and vascular cell types. H&E staining does not provide
sufficient detail to accurately describe the relative numbers of these cell types.
Therefore, detailed analysis is critical and there are now several robust markers
available (Table 3). Morphometric techniques should also be used to quantitate the
relative volumes of each layer, and relative densities of various cell types and struc-
tures particularly within the labyrinth (e.g., maternal blood sinus, syncytiotropho-
blast layers, mononuclear trophoblast, feto-placental capillaries).
6. Phenotypes affecting the labyrinth layer. Of all the parts in the placenta, the laby-
rinth layer is the one that is most commonly misunderstood and poorly described
in descriptions of mutant phenotypes. A very common mistake is that investiga-
tors simply look at whether the labyrinth is vascularized. Because fetal red blood
cells are nucleated, they are certainly easy to spot. However, it is important to
remember that the fetal vascular network cannot develop unless chorioallantoic
morphogenesis and trophoblast differentiation has occurred to create the villi into
which the vessels develop. Therefore, a simple reduction in the thickness of the
labyrinth layer due to reduced formation of villi would have secondary effect that
would superficially look like “reduced vascularization” of the labyrinth. In Gcm1
mutants, for example, the labyrinth layer completely fails to form as a result of a
block in chorioallantoic morphogenesis (18). In many other cases, the labyrinth
defect is less dramatic and morphometric analysis can be very helpful in identify-
ing the true cause of the phenotype. A case in point is the phenotype of Rb
mutants in which trophoblast differentiation is impaired because of a reduction
in the ability of trophoblast stem cells to exit the cell cycle (8). Using morpho-
metric analysis to assess the volume densities of differentiated trophoblast (villi)
and fetal capillaries, significant reductions are observed in both. However, the
reduction in capillary density within the overall labyrinth is less than the decrease
in villous density. Indeed, in areas of the labyrinth in which villi do form, the rela-
tive capillary density is higher (8).
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21
Phenotypic Analysis of the Rat Placenta
Summary
The rat is an important model for studying the biology of trophoblast-uterine development.
This chapter describes methods that are useful for the characterization of the rat uteroplacental
compartment.
Key Words: Rat; chorioallantoic placenta; choriovitelline placenta; junctional zone; laby-
rinth zone; metrial gland; spongiotrophoblast; trophoblast giant cells; trophoblast cell inva-
sion; prolactin gene family.
1. Introduction
In this chapter, we discuss the rat as an experimental model for investiga-
tions directed toward pregnancy and the uteroplacental interface. We present
an overview on the organization of the rat maternal–fetal interface and present
methods for studying the rat placenta and trophoblast cells.
1.1. Merits of the Rat as an Animal Model for Placental Research
Historically, the rat has been a valuable model for studying most aspects of
reproduction and, in many areas, still remains the preferred model system. The
rat is the dominant preclinical model system used by the pharmaceutical and
agro-chemical industries. Genetic approaches for analysis of the rat are rapidly
advancing. The first draft of the rat genome is completed (1) and significant
progress is being made in developing strategies for the genetic manipulation of
the rat, including transgenic (2–7), in vivo mutagenesis (8), and nuclear trans-
fer (9,10). Finally, panels of consomic rat strains have been established, which
will permit a systematic and physiologically relevant dissection of the rat
genome (11).
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
295
The use of animal model systems provides an essential tool for dissecting
molecular mechanisms controlling cellular development. The maternal–fetal
interface is no exception. The premise of employing any animal model system
is that if the process being studied is fundamental it will likely demonstrate
conservation across species. Although there are some differences in the orga-
nization of the rodent vs the primate maternal–fetal interface, there are over-
riding similarities in the functions and lineages of cells comprising the
maternal–fetal interface. Among these are events transpiring during the last
week of gestation in the rat. During this latter phase of pregnancy in the rat,
trophoblast cells exit the chorioallantoic placenta and invade into the uterine
endometrium, where they establish intimate relationships with the uterine vas-
culature (12). These invasive events in the rat are remarkably similar to inva-
sive events occurring during the latter stages of the first trimester and
throughout the second trimester of pregnancy in the human (13).
If we can understand and appreciate biological processes at the maternal–
fetal interface in species that can be experimentally manipulated (e.g., rat,
mouse), then we can more intelligently study the development of the human
maternal–fetal interface and identify pivotal junctures of cellular control, fa-
cilitating diagnosis and therapeutic intervention.
In some instances, cross-species similarities may prevail, whereas in other
cases, the differences may be most compelling. Nonetheless, our appreciation
for the biology of pregnancy increases. Animal models provide us with a means
of studying biological phenomena at levels that are not ethically permissible
with humans. Viviparity is vital to the success of our species. Mechanisms that
underlie viviparity will exhibit some level of conservation.
1.2. Overview of the Organization of the Uteroplacental Compartment
of the Rat
The uteroplacental compartment of the rat is similar to the mouse and pos-
sesses similarities and differences to the organization of the uteroplacental
compartment of other species with hemochorial placentation (14–17). This has
led to the utilization of an assortment of terms to describe components of the
uteroplacental compartment. Schematic representations of the rat uteroplacental
compartment are presented in Figs. 1 and 2.
The site where blood enters the uterus determines the orientation of the
uteroplacental compartment. This region is referred to as the mesometrial com-
partment, and the opposite end is termed the antimesometrial compartment.
The uterine mesometrial compartment is prominently comprised of stromal
cells, blood vessels (endothelial cells, smooth muscle cells), immune/inflam-
matory cells (natural killer cells, macrophages), smooth muscle cells of the
myometrium, and trophoblast cells. Cellular composition of this compartment
Fig. 1 (see companion CD for color version). Hematoxylin and eosin-stained tissue
section of the midgestation rat uteroplacental compartment (left panel, day 11 of ges-
tation) and a corresponding schematic diagram (right panel).
21_Ain_295_314_F
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298
8/29/05, 11:22 AM
Fig. 2 (see companion CD for color version). Hematoxylin and eosin-stained tissue section of the late gestation
rat uteroplacental compartment (left panel, day 18 of gestation) and a corresponding schematic diagram (right
Ain et al.
panel) with highlighted expanded views of the labyrinth and junctional zones (lower panels).
Phenotypic Analysis of Rat Placenta 299
2. Materials
2.1. Mating and Gestational Staging
1. Holtzman rats are obtained from Harlan Sprague-Dawley (Indianapolis, IN).
2. Saline solution (0.9% NaCl).
3. Glass slide with wells.
4. Microscope (×40–100 magnification).
Table 1
cDNAs Used in the Phenotypic Analysis of Cells Within the Uteroplacental Com-
partment
GenBank
Cell type Gene Gestation stage accession no. Reference
Trophoblast
Ectoplacental cone PLF-RP Early NM_053364 37
Trophoblast giant cell PL-I Early to mid D21103 31
PL-II Mid to late M13749 31,38
PLP-Fα Early to mid NM_022530 37,
unpublisheda
P450scc Early to late J05156 39,40
P450c17 Mid to late NM_012753 40,41
3βHSD Early to late L17138 Unpublishedb
Spongiotrophoblast PLP-B Mid to late M31155 42,43
PLP-Fβ Mid to late AY741310 Unpublisheda
SSP Mid to late NM_172073 44
Labyrinthine TGC PL-II Mid to late M13749 42,45
PLP-K Mid to late NM_138861 32
PLF-RP Mid to late NM_053364 37
Syncytial trophoblast FABP3 Mid to late NM_024162 46
Alk Phos Mid to late NM_013059 47
Invasive trophoblast PLP-L Mid to late NM_138527 12
PLP-M Mid to late NM_053791 12
PLP-N Mid to late NM_153738 48
IGF-II Mid to late X17012 49,
unpublishedc
Decidual cells
Mesometrial α2-MG Early to mid NM_012488 50
Antimesometrial dPRP Early to mid NM_022846 51,52
PLP-J Early to mid NM_031316 32
Natural killer cells Osteopontin Mid NM_012881 Unpublishedd
Abbreviations: TGC, Trophoblast giant cell; PLF-RP, proliferin-related protein; PL, placental lacto-
gen, PLP, prolactin-like protein; P450scc, side chain cleavage; P450c17, 17α hydroxylase; 3βHSD, 3β
hydroxysteroid dehydrogenase; SSP, spongiotrophoblast-specific protein; FABP3, fatty acid binding
protein-3; IGF-II, insulin-like growth factor-II; α2-MG, α2-macroglobulin; dPRP, decidual prolactin-
related protein.
aHo-Chen, J., Bustamante, J. J., and Soares, M. J., unpublished results.
bCanham, L. N. and Soares, M. J., unpublished results.
cAin, R. and Soares, M.J., unpublished results.
dLiu, B. and Soares, M.J., unpublished results.
Table 2
Antibodies Used in the Phenotypic Analysis of Cells Within the Uteroplacental
Compartment
Cell type Antigen Gestation stage Source (Cat. No.) Reference
Abbreviations: TGC, trophoblast giant cells; PL, placental lactogen, PLP, prolactin-like protein; P450scc,
side chain cleavage; P450c17, 17α hydroxylase; SSP, spongiotrophoblast-specific protein; FABP3, fatty acid
binding protein-3; β2-MG, α2-macroglobulin; dPRP, decidual prolactin-related protein; Sm, smooth muscle.
aAlam, S. M. K., Konno, T., and Soares, M. J., unpublished results.
bAin, R. and Soares, M. J., unpublished results.
2. Nitrocellulose (Optitran, Schleicher & Schuell Biosciences, Inc., Cat. No. BA-S 85).
3. Antibodies to the protein of interest (Table 2).
2.9.2. Immunocytochemistry
1. Dry-ice-cooled heptane (Fisher).
2. Cryostat (Leica).
3. Antibodies to the antigen(s) of interest (Table 2).
3. Methods
3.1. Mating and Gestational Staging
1. The rats are maintained on a 14 h light:10 h dark lighting schedule with lights on
at 0600 h (see Note 1).
2. Adult males, preferably older than 10 wk of age, are placed one per cage.
3. Adult females, generally 7–10 wk of age, are transferred to a cage with a male
(no more than two females per male). The fur on one of the females is generally
marked with a dye to distinguish it from the other female.
4. Every morning between 0800 and 0900 h, each female cohabiting a cage with a
male, is removed from the cage for the purpose of obtaining a vaginal lavage.
5. A few hundred microliters of saline are delivered with a pipette into the vagina of
the female and recovered with the same pipet.
6. The contents of each saline lavage are transferred to a well within a multi-well
glass plate.
7. After all of the vaginal lavages are collected, they are examined with the aid of a
microscope (×40–100 magnification).
8. The presence of sperm in the lavage is recorded, as is the cellular content of the
lavage (see Notes 2 and 3).
9. The sperm positive females are transferred to separate cages. The presence of
sperm in the vaginal lavage is considered day 0 of pregnancy (see Note 4).
3.2. Detection of Pregnancy During Early Post Implantation Stages (26)
(see Note 5)
1. Pregnancy detection within the first 48 h post implantation requires intravenous
injection of a vital blue dye, such as Chicago Blue B.
2. A volume of 0.25–0.5 mL/100 g body weight of a 1.0% solution of Chicago Blue
B can be injected into the tail vein of the rat.
3. Implantation reactions are identified by the accumulation of blue bands within
the uterus after 15 min.
3.3. Mid-Gestation Placental Dissection (27) (see Note 6)
1. Embryos with their encapsulating decidual tissues (conceptuses) are retrieved
from the uterus from days 10–13 of gestation.
2. Conceptuses are dissected with the aid of a dissecting microscope (×10–20 mag-
nification).
3. Tissues are collected and washed with HBSS.
4. The overlying decidua basalis and decidua capsularis are removed with fine for-
ceps and gentle dissection.
5. A cut through the mural pole of the trophoblast layer is made and the trophoblast
retracted. Be careful not to cut through the yolk sac/amnion.
6. The visceral yolk, amnion, and embryo are separated from the developing chorio-
allantoic placenta by cutting at the insertion site of the allantois with microdis-
section spring scissors.
7. The entire trophoblast component is flattened (allantoic insertion site is up) and
can be further separated into chorioallantoic and choriovitelline layers with the
aid of microdissection spring scissors. The inner dark circle of tissue (more vas-
cular) comprising the chorioallantoic tissue is cut away from the lighter surround-
ing tissue (less vascular) consisting of the choriovitelline tissue.
8. Dissected decidua basalis, decidua capsularis, and trophoblast components can
each be processed as required, and/or stored at –80°C, until further use.
7. Membranes are washed once with 2X SSPE and 0.1% SDS for 30 min at 42°C
and twice with 0.1X SSPE and 0.5% SDS at 60°C for 30 min each.
8. Membranes are then wrapped with plastic wrap and exposed to Kodak Bio-Max
film for 1–4 h and developed.
4. Notes
1. The Holtzman rat is an outbred strain closely related to the Sprague-Dawley rat.
The rats are easy to handle. Under the 14 h light:10 h dark lighting schedule the
female rats tend to have a 5-d estrous cycle. Our approach for mating and the
pregnancy dating system also applies to other strains. Males used for breeding
are obtained at 10 wk of age and usually continue to be effective breeders until
approx 1 yr of age. Although, most vendors provide timed-pregnant female rats,
we have not found their dating of pregnancies to be reliable and prefer to gener-
ate our own timed pregnancies.
2. The process is repeated daily until mating is confirmed by the presence of sperm.
Occasionally, seminal plugs are present in the vagina. Unlike the mouse, seminal
plugs tend to fall out of the rat vagina. Special suspended caging, if permitted,
can be used and dark paper placed in the trays beneath the cages each evening
and checked the following morning for the presence of the whitish-yellow,
opaque seminal plugs. This is generally the procedure used for generating
pseudopregnant rats with vasectomized males.
3. Inspection of the cellular content is helpful. Based on the cellular composition of
the vaginal lavage it is possible to determine whether the animal is cycling. Cells
present in the vaginal lavage are impacted by circulating concentrations of the
ovarian steroid hormones, estrogen and progesterone. Vaginal lavage’s contain-
ing nucleated and/or nucleated with cornified cells characterize the estrous cycle
stage coinciding with behavioral estrus and receptivity. Cornified cells are char-
acteristic at the time of ovulation and lavages containing leukocytes are domi-
nant post-ovulation during the luteal phase. A cycling female cohabiting a cage
with a male would raise the question of the male’s fertility.
4. It is always important to determine the pregnancy dating system used when re-
viewing any experimentation with pregnant rats. For us, classifying the presence
of vaginal sperm as day 0 of pregnancy is historical. Others consider the presence
of sperm in the vagina as day 1 of pregnancy. It would likely be most accurate to
consider 1200 h on the day sperm is found to be day 0.5 of pregnancy. It is criti-
cal to appreciate that trophoblast/placental development can differ markedly
within a day. Thus, regardless, of the pregnancy dating system used, it is impera-
tive to provide the information in any scientific report forthcoming from the re-
search. De Rijk and colleagues have published a useful guide for assessing placental
morphology and pregnancy-dependent maternal hematological indices (35).
5. The detection of early postimplantation pregnancy by intravenous injection of a
vital dye is based on capillary permeability changes. The vital dyes bind to circu-
lating proteins such as albumin. As the capillary permeability increases after im-
plantation, the circulating dye-bound albumin exits the capillaries and
concentrates in the surrounding tissue.
6. The dissection of the midgestation placenta requires practice. In order to main-
tain the appropriate orientation of maternal, extraembryonic, and embryonic tis-
sues, it is best not to disrupt the amniotic contents. It is also essential to identify
mesometrial vs antimesometrial poles. The mesometrial decidua is thicker than
the antimesometrial decidua. The shape of the mid-gestation conceptus can be
compared to an ice-cream cone (more evident on days 10–11 of gestation). Using
such an analogy, the mesometrial pole would be the location of the ice cream. We
13. In our hands, in situ hybridization is a reliable method for identifying cell types
contributing to the expression of a specific gene within the uteroplacental com-
partment. An appreciation of the dynamic changes in uteroplacental morphology
is essential to maximize the benefit of the approach.
14. Antibodies are effective tools for identifying and localizing proteins. Each antibody–
antigen interaction needs to be optimized for the specific technique employed.
Acknowledgments
We would like to thank past and current members of our laboratory for their
efforts in developing and characterizing the methods described in this chapter.
This work was supported by grants from the National Institutes of Health (NIH)
(HD20676, HD39878, HD48861) and the Hall Family Foundation.
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22
Analysis of the Structure of the Ruminant Placenta
Methods of Fixation, Embedding, and Antibody Localization
at Light and Electron Microscope Levels
F. B. P. Wooding
1. Introduction
The basic structure of all ruminant placentas described so far is cotyledon-
ary (or placentomal), with local proliferations of apposed fetomaternal mem-
branes forming placentomes that are linked by flat interplacentomal areas. In
vivo, the placenta consists of an intimate apposition of trophoblast to uterine
epithelium or derivative, with interdigitation of microvilli on both sides. Any
separation of these two surfaces is an artifact of preparation except in hemopha-
gous zones, where maternal blood is released between the surfaces and phago-
cytosed by the trophoblast.
Placentomes develop from a flat membrane apposition at implantation (20 d
post coitum [dpc] in cow and 16 dpc in sheep) by a mutual growth of
fetomaternal surfaces to form villi, remodeling the endometrium rather than
invading it. Claims of maternal crypts forming into which the trophoblast villi
grow are based on poor fixation and processing, the two surfaces separate very
quickly after death.
The trophoblast epithelium uninucleate cell (UNC) gives rise to binucleate
cells (BNC) with characteristic cytoplasmic granules at the earliest stage of
implantation. The BNC develop in the trophoblast epithelium out of contact
with the basement membrane or the tight junction. When mature (fully granu-
lated), they migrate up to and through the tight junction while maintaining its
barrier function. The BNC apex now fuses with the uterine cell or derivative to
which it is apposed and its cytoplasmic contents are injected into the uterine
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
315
cell (1). The BNC plasma membrane on the fetal side of the tight junction is
resorbed by the trophoblast UNC. The microvillar junction (MVJ) reforms and
in the cow/deer placental types, the trinucleate cell formed by the injection,
releases the BNC granules to the maternal side by exocytosis, then dies and is
resorbed by the trophoblast. In the sheep/goat placental types, the continuous
injection of BNC content forms a persistent fetomaternal syncytium (“uterine
derivative”) replacing the original uterine epithelial cells (2).
Throughout pregnancy in all ruminants, approx 20% of the trophoblast are
BNC, and approx 20% of these BNC are in the process of migration and injec-
tion. The number of BNC falls in the days before parturition, and this fall can
be induced prematurely by injection of cortisol into the fetal circulation in
vivo (3).
The BNC migration and injection process allows delivery throughout preg-
nancy of the fetal molecules in the granules (which contain placental lactogen
hormone and many different pregnancy-associated glycoproteins [PAGs]) to
the maternal circulation while maintaining the placental barrier between the
two circulations. The width of this barrier is reduced progressively by indenta-
tion of maternal and fetal capillaries into the respective trophoblast and uterine
epithelia, down to between 1 and 2 µm in many places, but the number of
membrane layers in the barrier stays the same throughout pregnancy.
It should be emphasized that the above analysis is the view of the author,
based on a relatively small sample of the enormous variety of ruminant spe-
cies. The author looks forward to more studies with a greater variety of modern
techniques to fully test the interpretation presented previously.
The two main problems to be addressed in investigating ruminant placental
structure and function are the preservation of the fetomaternal interface as it is
in vivo, and the maintenance of the antigenicity of the material during fixation
and embedding. The best (and probably the only) method of achieving the first
objective is to doubly perfuse the placenta via both fetal and maternal circula-
tions as quickly as possible after death of the animal. The uterus should be
removed and perfused via the uterine arteries, first tying or clamping off part
or the whole of one uterine horn if frozen and/or unfixed samples are required.
After 5 min or so of perfusion the uterus is opened and the placenta is perfused
via the umbilical arteries. Perfusion of different food colorings finally allows
identification of the best-fixed areas when the placenta is cut into smaller
samples for processing. Successful perfusion produces a tissue that is firm
enough to maintain its structure when cut into pieces small enough to process
for light microscopy (LM) and electron microscopy (EM).
One can start immersion fixation more rapidly, but cannot avoid crush-
ing and separating the initially soft tissues, and penetration of the fixative
is unavoidably slower and much more uneven. The process can produce useful
results but the drawbacks of the range of fixation quality and the lack of pres-
ervation of the volume relationship between blood and tissue must always be
borne in mind when assessing the results.
For optimal ultrastructure and membrane preservation, a fixative with more
than 2% glutaraldehyde is advisable, whereas for immunocytochemistry
(IMCYT), 4% formaldehyde is optimal. Because the cost of animals is high,
perfusing with 4% formaldehyde and, subsequently, cutting the placental
sample up into several different fixatives can help maximize limited animal
numbers. Fortunately, with most ruminants, the amount of tissue available from
a single placenta is considerable. The use of several types of embedding media
is also recommended: wax, acrylic, and epoxy nonosmicated blocks form a
useful basic resource for subsequent work.
To simplify the transition from LM to EM, use a pieces-of-coverslip (2 to
6 mm2) carrier system for LM IMCYT. It reduces antibody-volume require-
ments and storage problems. Then, one can apply in Petri dishes the same pro-
cedures as those used for EM IMCYT with grids. However, this is the
preference of the author, and produces no better results than does conventional
slide processing. To further simplify the number of procedures, the author uses
the immunogold–silver enhancement system for all IMCYT, but this is not
necessarily any more sensitive than more conventional ABC or peroxidase LM
IMCYT methods.
2. Materials
2.1. Fixatives
1. 4% Paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.2. To
depolymerize the paraformaldehyde:
a. Add 2 mL NaOH to 50 mL water in a 250-mL conical flask.
b. Add magnetic stirrer bar and stir on magnetic stirrer hotplate.
c. Add 20 g of solid paraformaldehyde (Agar Scientific, Stansted, UK).
d. Heat water to 60°C until solution is clear.
e. Cool and make up to 500 mL with PBS.
2. 1% Glutaraldehyde–4% paraformaldehyde in PBS (in step 1 above, with 21 mL
of 25% aqueous glutaraldehyde [Agar Scientific] added to 479 mL formaldehyde
solution).
3. 4% glutaraldehyde in PBS plus 5% sucrose.
3. Methods
3.1. Perfusion Fixation
1. Kill animal by shooting (e.g., at slaughterhouse) or barbiturate overdose via
injection.
2. Remove uterine tract as quickly as possible, leaving uterine arteries as long as
possible—trace them as far back to the dorsal aorta as you can before cutting.
3. Wear disposable plastic gloves for all manipulations with fixative. Tie a blunted
18-gauge (or as large as practicable) disposable needle into each uterine artery.
4. Inject fixative at 18 to 20°C through the needles with a 50-mL disposable
syringe. Do not flush the vasculature first with buffer or saline. Manually
apply as much pressure as is necessary to force blood out of the uterine vein until
it runs clear; do not worry about total pressure exerted. With sheep, this will take
approx 150 to 200 mL fixative for each side.
5. Clamp uterine vein outflow and inject a further 25 mL of fixative. This ensures
that as many arteries and veins are perfused as possible.
6. Cut uterus open just above cervix, avoiding obvious blood vessels, and remove
the fetus. Cannulate both umbilical arteries in the placental direction with blunted
needles. The arteries are thicker and more solid than the veins. Inject 100–200 mL
fixative down each, cutting the umbilical veins to allow outflow if necessary. Con-
tinue to trickle fixative through all cannulae for 10 min.
7. Inject 2 mL of aqueous 1% malachite green or green food dye down each uterine
artery and 2 mL of a different color (e.g., 0.1% nigrosin or blue food dye) down
the umbilical arteries. Push the dyes through with 25 mL of fixative (see Note 1).
8. Open uterus out flat, slice across the placentomes centrally to find the best double-
colored ones, and take a 2- to 3-mm thick whole slice across the center of the
placentome. Cut into 2- to 3-mm cross section “matchsticks” running the full
depth of the placentome. This should be accomplished in a pool of fixative on a
wax or Sylgard elastomer resilient layer in a Petri dish. The samples are suitable
in size for resin processing; wax processing can accommodate much larger pieces.
For interplacentomal samples, cut 1- × 1-cm squares through the full depth of the
uterine wall, but always cut from the fetal side, again selecting the best-dyed areas.
9. Divide the samples into a chosen range of fixatives and fix for a further 2 h or up
to overnight if it is more convenient.
10. Rinse all samples in buffer (PBS) and store at 4°C until processed. The samples
can be stored for several years at this stage, as long as a bacteriostatic/antifungal
(e.g. 0.05% Thimerosal) is incorporated.
3.3. Embedding
1. 2-mm cubes are best, but up to 4-mm cubes will embed satisfactorily. If you use
larger areas, keep one dimension down to 2 mm to allow sufficient liquid resin
permeation prior to curing.
2. For conventional ultrastructural studies only, with no IMCYT, use the glutaral-
dehyde-fixed material and, in a fume cupboard, transfer to freshly made 1% osmic
acid plus 1.5% potassium ferrocyanide in PBS for 1 h, rinse with distilled water,
and then incubate in 2% uranyl acetate in distilled water for 15 min, finally rins-
ing with distilled water.
3. Dehydrate in 50% alcohol, 75% alcohol, and 2X 100% alcohol for 15 min each.
The author uses disposable 14- × 50-mm glass tubes with plastic stoppers for all
embedding procedures.
4. For epoxy (Araldite): 100% Alco to propylene oxide (15 min); overnight in 66%
araldite/33% propylene oxide; 4 h in 100% araldite. Place in plastic (Beem) cap-
sules or trays (Agar Scientific) in fresh araldite, orienting as required. Cure at
60°C for 8 h or until the surface, when cold, is not sticky to touch.
5. For acrylic (K4M): 100% Alco to 33% K4M/66% Alco (40 min); 66% K4M/
33% Alco (40 min); 100% K4M overnight. Place in fresh K4M in plastic (Beem)
capsules with minimum air space, close tops, and cure at a distance of 30 cm
from an ultraviolet (UV) light (wavelength 375 nm) for 2 to 3 h at room tempera-
ture, or as long as it takes—check periodically with a mounted needle (see Note 2).
6. For wax: 100% Alco to 100% Xylene or equivalent (overnight), 100% wax at 55
–60°C (3X 60 min). Place into moulds at 60°C and, finally, cool rapidly to pro-
duce best cutting texture.
7. All of the above processing is performed at room temperature except where noted.
The K4M can also be processed at –20°C to preserve antigenicity (use a –20°C
freezer and dry ice in a polystyrene box for solution changes). The curing takes
much longer at –20°C.
3.4. Sectioning
1. Fill a rack with 22- × 22-mm coverslips or slides and put into 2% 3-Aminopropyl
trimethoxy silane (APTES; Sigma) in 100% alcohol for 10 min, rinse in 100%
alcohol, leave in distilled water for 1 min, blow dry or blot edges thoroughly, and
dry in oven at 60°C for 60 min. APTES ensures that the sections stay firmly stuck
to the glass during processing. Place a coverslip on a hard, flat surface, and
score in two directions at right angles with a diamond pencil to produce pieces
2–9 mm x 2–9 mm (in various sizes to accommodate a variety of section sizes).
Break into the individually scored pieces using gentle pressure with the “wrong”
end of a pair of forceps on filter paper.
2. Cut 0.5- to 1.0-µm thick resin sections or 4- to 8-µm thick wax sections for LM,
or 80- to 110-nm thick sections for EM, onto a water surface using an ultramicro-
tome. For LM, pick up at least two sections per cover slip piece or slide-transfer
each of the sections with a metal loop to a drop of water on a slide. LM IMCYT
is inherently variable—do not be satisfied with what is localized on only one of a
pair of sections without checking further. For EM, pick up on 300 mesh naked
or formvar film-coated nickel (not copper) grids or slotted supports. Dry all at
60°C for 30 min before further processing.
3.5. Immunocytochemistry
1. Initially, removal of wax or araldite from the sections is necessary. K4M is used
without any pretreatment.
a. Dewaxing. Immerse coverslip pieces (use one eppendorf tube for each piece)
or slides in xylene or equivalent for 10 min, transfer to 100% alcohol for
10 min, wash in running tap water for 15 min. Dry around the sections on
slides and encircle them with a wax pencil or PAP pen to minimize the vol-
umes of reagents required subsequently to cover the sections.
b. Resin removal. Float coverslip pieces section side down on a drop of sodium
ethoxide (dissolve 15 g of NaOH pellets in 15 mL of 100% alcohol using a
magnetic stirrer bar for 4 h; keep in the dark). Handle this carefully; it is a
very corrosive solution. On slides, use a drop on top of the sections. All float-
ing on drops is performed on parafilm sheets secured in Petri dishes by press-
ing along the film edges with the “wrong” end of a pair of forceps. Treat with
sodium ethoxide for 15 min, jet-wash (from a washbottle with a narrow jet)
the coverslip pieces or slides with 100% alcohol, float on (or drop on) 50%
alcohol, leave for 5 min, then wash with water for 5 min.
2. K4M sections start here with the dewaxed or deresinated sections. Float sections
on coverslip piece or grid (or place a drop on a slide) for 10 min on a drop of PBS
containing 1% bovine serum albumin (Sigma) and 0.05% Thimerosal (Sigma).
This solution is used for all antibody dilutions.
3. Sections on grids and coverslip pieces (subsequently referred to as sections) are
touched at the side to filter paper to draw off excess fluid (do not wash), floated
on the primary antibody (e.g., rabbit or mouse) (see Note 3) overnight at 4°C, jet-
washed with PBS, and incubated for 40 min on the relevant secondary gold col-
loid-labeled antibody (e.g., goat anti-rabbit or goat anti-mouse) using 4-nm gold
for the LM and 4-, 10-, or 15-nm gold for EM.
4. After the immunoreaction, the sections are jet-washed with PBS and distilled
water and left in distilled water for 10 min. The 4 nm gold is now intensified by
floating the sections on the intensification reagent for 7 min for grids (EM) and
15–20 min for LM (slides or coverslip pieces). The level of LM label can be
monitored continuously by eye or on a microscope. The reaction is stopped by
jet-washing with distilled water and the LM sections dried. For counterstaining the
EM grids can be transferred successively to uranyl acetate (5% aqueous, 15 min),
jet-washed with distilled water, then with lead citrate (0.1% lead citrate in 0.05%
NaOH), jet-washed, and finally dried. Reduce the staining times if the electron
density obscures the label. LM counterstains can be applied once the
immunoreaction has been assessed. Try 1% toluidine blue in 1% sodium borate,
and reduce the stain level (differentiate) with acid alcohol (1 mL 1N HCl per 15
mL 100% alcohol). Alternatively, try 0.2% Fast green in 0.03 N HCl and reduce
with warm water.
5. Permanently mount the LM sections using Biomount (BB-International, Cardiff,
UK). In conventional mountants (e.g., Depex), the metallic black silver depos-
ited in the intensification reaction oxidizes quite rapidly to an invisible silver
carbonate.
4. Notes
1. Perfusion fixation is rarely 100%; the use of dyes is essential to monitor this and
select the best areas of fixation.
2. The time required for curing resins can be unpredictable. With K4M, always cure
initially from underneath the Beem capsules, targeting the end with the speci-
men. Suspend the capsules in a perforated sheet of clear plastic above the UV
light source. If the top half/portion is slow to cure (check with a mounted needle),
suspend the UV light above the capsules to finish. When using Araldite, cure at
60°C until it resists a mounted needle and is not sticky on the surface after cool-
ing to room temperature.
3. Immunocytochemistry: start with one-tenth and one-hundredth dilutions of any
new antibody, then dilute according to results. Never trust localizations seen on
only one of a pair of sections—IMCYT results are inherently variable. The au-
thor has not found antigen retrieval (microwave, pressure cooking, heat treat-
ment) prior to IMCYT of the sections useful, but many people do—consult
Chaiwun et al. (4) and Groos et al. (5) for details.
References
1. Wooding, F. B. P. and Flint, A. P. F. (1994) Placentation, in Marshall’s Physiol-
ogy of Reproduction, 4th edition, Vol. 3, Pregnancy and Lactation (Lamming, G.
E., ed.). Chapman and Hall, London: pp. 242–460.
2. Wooding, F .B .P., Morgan, G., Brandon, M. R., and Camous, S. (1994) Mem-
brane dynamics during migration of placental cells through trophectodermal tight
junctions in sheep and goats. Cell Tissue Res. 276, 387–397.
3. Ward, J. W., Wooding, F. B. P. and Fowden, A. L. (2002) Effects of cortisol on
the binucleate cell population in the ovine placenta during late gestation. Placenta
23, 451–458.
4. Chaiwun, B., Shi, S. R., Cote, R. J., and Taylor, C. R. (2002) Major factors influ-
encing the effectiveness of Antigen Retrieval Immunohistochemistry, in Antigen
Retrieval Techniques (Shi, S. R., Gu, J., and Taylor, C. R., eds.). Eaton Publish-
ing, Natick, MA: pp 41–53.
5. Groos, S., Reale, E., and Luciano, L. (2001) Reevaluation of epoxy resin sections
for LM and EM immunostaining. J. Histochem. Cytochem. 49, 397–406.
23
Characterization of the Bovine Placenta
by Cytoskeleton, Integrin Receptors, and Extracellular
Matrix
Christiane D. Pfarrer
Summary
The cytoskeleton together with integrin receptors and proteins of the extracellular matrix
provide sensitive indices of the development and organization of the bovine placenta. The bo-
vine placenta is classified as synepitheliochorial because migrating trophoblast giant cells fuse
with single uterine epithelial cells. This phenomenon may be interpreted as a restricted tropho-
blast invasion. Bovine placentomes from early placentation until term can be characterized by
indirect immunohistochemical methods. In order to do so, placental tissues are snap-frozen in
liquid nitrogen or perfusion-fixed in formalin and embedded in paraffin. Depending on the
antibodies used, the different cell types within the cow placenta are identified either on frozen
sections or on paraffin sections according to the expression of different cytoskeletal filaments
(α smooth muscle actin, different cytokeratins, desmin and vimentin). The specific expression
of integrin receptors (subunits α1, α2, α3, α4, α5, α6, αv, β1, β3, and α4) as well as proteins of the
extracellular matrix (collagens type I and IV, fibronectin, and laminin) in the different cell
populations is also examined.
Key Words: Placenta; bovine; trophoblast giant cell; migration; cytoskeleton; integrin;
extracellular matrix.
1. Introduction
The cow placenta is cotyledonary and synepitheliochorial. That means fetal
cotyledons interdigitate with maternal caruncles, thus forming so-called
placentomes, where the chorionic epithelium (or trophoblast) is in intimate
contact with the uterine or caruncular epithelium. The bovine chorionic epithe-
lium consists of “normal” polarized cytotrophoblast cells and a second popula-
tion of trophoblast cells, namely nonpolarized, mostly binucleated trophoblast
giant cells (TGCs) (1). These TGC are generated by unidentified stem cells
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
323
through acytokinetic mitosis (2), and have migratory properties. TGC migra-
tion is characterized by a sequence of events: first, TGCs “squeeze through”
chorionic tight junctions (3) and, in a next step, fuse with single uterine epithe-
lial cells via specifically formed pseudopodia (1). Finally, the resulting
trinucleated feto–maternal hybrid cells degenerate (3,4), maybe partly by
apoptosis and/or through exfoliation toward the feto-maternal contact inter-
face, where they may be phagocytosed by mononuclear trophoblast cells. Be-
cause this TGC migration does not continue beyond the maternal basement
membrane (BM), it is regarded as a restricted type of trophoblast invasion (5).
Highly invasive trophoblast during hemochorial placentation of humans and
rodents is characterized by specific binding patterns of integrin receptors to
proteins of the extracellular matrix (ECM) (6), and other invasive processes
like tumor growth are associated with similar/comparable expression patterns
(7). Integrins are heterodimeric transmembrane glycoproteins serving as adhe-
sion receptors at the cell surface (8). Twenty-four different heterodimers that
are formed by the dimerization of various α- and β-chains have been identified
to date (9). Integrins interact with a variety of ligands, including ECM glyco-
proteins and molecules on the surface of neighboring cells (10,11). The β sub-
unit is linked to components of the cytoskeleton, and besides promoting cellular
conformational changes, integrin activation may also induce phosphorylation
cascades of signalling proteins (12). This results in bidirectional signal trans-
duction (“outside-in” and “inside-out” signaling) (8).
Prior to implantation, the integrin β1 subunit occurs basolaterally on fetal
mononuclear trophoblast cells and trophoblast giant cells of day 24 post insemi-
nation, which implicates β1 integrin in the process of trophoblast migration and
cell development (13). As attachment proceeds, implantation-associated changes
in the expression patterns of integrin subunits α1, α3, and α6, as well as extra-
cellular matrix proteins collagen IV and laminin, have been observed in the
bovine endometrium and in isolated binucleate trophoblast cells (14). The alter-
ation of integrin and ECM expression patterns may be induced by the fusion of
TGC with uterine epithelial cells (14). Implantation in the closely related goat
differs, because collagen type IV decreases in maternal BM and is conse-
quently interpreted as a modification of the BM composition rather than its
destruction (15).
In the definitive bovine placenta, around day 80 until near term (around day
270), the expression of the respective integrin subunits suggests the presence
of collagen receptor α2β1 and laminin receptor α6β1, which may be responsible
for the adhesion of fetal and maternal epithelial cells to BM (5) (Fig 2).
Coexpression of laminin, α2, α6, and β1 in nonpolarized TGC supports the
concept that TGC migrate along their own laminin matrix utilizing integrin
α6β1, and maintain cell–cell contacts with neighboring cells via α2β1 integrin
(5). In maternal stroma and fetal mesenchyme, a pool of integrins is present
which may be involved in the regulation of proliferation and differentiation of
maternal septa and fetal villi (5) (Fig 3).
During the estrous cycle of cows, changes in the localization of collagens I,
III, IV, and VI in the uterine wall are also observed (16). In contrast, the por-
cine uterine epithelium, lacking TGC, shows no detectable structural changes
in the basement membranes during epitheliochorial implantation (17,18).
Cell migration is dependent on the mechanical properties of the actin cytosk-
eleton, and changes in cell shape, anchorage, and motility are associated with the
dynamic reorganization of the actin-cytoskeleton (19,20). Migratory TGC pos-
sess specifically arranged actin filaments (21).
Integrin binding leads to cytoskeletal responses including induction of focal
accumulations of a variety of cytoskeleton-associated molecules, vinculin,
talin, α-actinin, and F-actin, which may lead to conformational changes of the
actin cytoskeleton and the transduction of signals to the nucleus and altered
gene transcription (22). In vitro, beads coated with the ECM glycoprotein
osteopontin induced a transmembrane accumulation of focal adhesion com-
plexes (talin and α-actinin) at the apical surface of ovine luminal epithelial and
conceptus trophectoderm cells, revealing functional integrin activation and
cytoskeletal reorganization and potentially simulating events occurring during
embryo implantation in sheep (23).
Identification of the tissue components within the bovine placentome (fetal
cotyledon and maternal caruncle—both consist of connective tissue and epi-
thelium) may be facilitated by the analysis of the expression of different
cytoskeletal filaments (Fig. 1). The method is well established, and has been
used for the characterization of other placental types, such as the endothelio-
chorial mink and hemochorial guinea pig, macaque, and human (24–29). In the
bovine placenta, cytokeratin is used for the identification of fetal and/or mater-
nal epithelial cells, whereas tissue of mesenchymal origin can be detected by
vimentin (connective tissue, including endothelial cells), α-smooth muscle
actin and desmin (smooth muscle cells of blood vessel walls and pericytes).
2. Materials
2.1. Tissue Collection
1. Liquid nitrogen (N2).
2. Tissue-Tek® O.C.T.™ Compound (Sakura, Torrance, CA).
3. Fixative: 4 % buffered formaldehyde (pH 7.4).
4. Washing step: phosphate-buffered saline for immunohistochemistry (PBS-IHC)
(see Subheading 2.2., step 1).
2.2. Immunohistochemistry
1. PBS-IHC stock solution: 41 g NaCl, 11 g Na2HPO4 · 2H2O, 2.75 g KH2PO4. Add
up to 1000 mL with distilled water, adjust to pH 7.2. Working solution: one part
stock solution and four parts distilled water give 0.1 M PBS-IHC.
2. Primary antibodies and their sources are listed in Table 1.
3. Secondary antibodies generally detect the species the primary antibody is raised
in. In this case: biotinylated horse anti-mouse/anti-rabbit secondary antibody
(Vector Laboratories, Burlingame, CA, USA).
Table 1
Antibodies
Antibodies Clone Host Dilution Supplier (Cat. No.)
α-sm Actin 1A4 mouse 1:100 Dako (M0851)
Collagen IV CIV 22 mouse 1:100 Dako (M785)
Cytokeratin AE1, AE3, Ks13.1 mouse 1:20a Linaris (E020)
Desmin DE-R-11 mouse 1:50a Dako (M0724)
Fibronectin IST-3 mouse 1:250b Sigma (F0791)
Integrin α1 polyclonal rabbit 1:800 b Chemicon (AB1934)
Integrin α2 polyclonal rabbit 1:400b Chemicon (AB1936)
Integrin α3 polyclonal rabbit 1:75b Chemicon (AB1920)
Integrin α4 polyclonal rabbit 1:25b Chemicon (AB1924)
Integrin α5 polyclonal rabbit 1:400b Chemicon (AB1928)
Integrin α6 NKl-GoH3 rat 1:25b Chemicon (MAB1378)
Integrin αv polyclonal rabbit 1:100b Chemicon (AB1930)
Integrin β1 polyclonal rabbit 1:100b Chemicon (AB1952)
Integrin β3 PM 6/13 mouse 1:80b Chemicon (MAB1381)
Integrin β4 polyclonal rabbit 1:1600b Chemicon (AB1922)
Laminin polyclonal rabbit 1:20b BioGenex (AR078)
Vimentin Vim 3B4 mouse 1:100 Dako (M7020)
Secondary Antibodies:
Anti mouse/ IgG (H+L) horse 10–20 µL/mL Vector (BA-1400)
anti rabbit
(biotinylated)
Anti-mouse FITC IgG donkey 1:200 Chemicon (AP192F)
Anti-rabbit FITC IgG donkey 1:100 Chemicon (AP182F)
Anti-rat CY3 IgG donkey 1:100 Chemicon (AP189C)
Anti-rabbit CY3 IgG donkey 1:300 Chemicon (AP182C)
2.3. Immunofluorescence
1. PBS for immunofluorescence (PBS-IF) stock solution: 16 g NaCl, 0.4 g KH2PO4,
2.28 g Na2HPO4. Add up to 1000 mL with distilled water, adjust pH to 7.3. Work-
ing solution: one part stock solution and one part distilled water give 0.02 M
PBS-IF.
2. PBS-IF/Tween: 0.3 % Tween (Polyoxyethylene-Sorbitan Monolaurate) in PBS.
3. Antibody dilution buffer (100 mL): 0.3 % Tween in PBS-IF, 1.0 g bovine serum
albumin (BSA), 45 mL glycerol (pH 8.0). Economize production of antibody
dilution buffer by dividing into aliqots: produce aliquots with 0.5 M Na2CO3 (pH
9.5) and freeze in portions of 5 mL.
4. Mounting media: e.g., Vectashield (H-1200, Vector Laboratories) or ProLong®
Antifade Kit (Molecular Probes Inc., Eugene, OR, USA) or Mowiol (Sigma)-
propyl gallate (Sigma) mounting medium.
3. Methods
3.1. Tissue Collection
1. After removal from the cow (see Note 1), the uterus is opened along the large
curvature with a pair of scissors. The fetus is removed and crown–rump length is
recorded (see Note 2).
2. Snap-freezing: select placentomes, gently excise whole placentomes (see Notes
3 and 4), cut into smaller pieces (around 1 cm3), wrap in aluminum foil or use
Tissue-Tek for embedding, snap-freeze in liquid nitrogen, and store at –80°C
until use.
3. Perfusion fixation: select placentomes (see Note 5), separate the artery, make a
small incision into wall of artery, enter a blunt cannula (see Note 6), secure with
clamp, and inject approx 80 mL of fixative with gentle and steady manual pres-
sure (see Note 7).
4. Excise placentomes, cut into slices of approx 0.5 cm, and postfix in the same fixa-
tive as mentioned above for 24 h and wash in PBS-IHC (three times for 10 min).
5. Cut into smaller pieces of around 1 cm2. Thickness of slices remains 0.5 cm.
Make sure that one tissue block includes the total height of the placentome from
allantochorion to caruncular stalk.
6. Paraffin-embed the tissue.
7. Depending on the properties of the antibodies used, either frozen or paraffin sec-
tions are used for the immunohistochemical detection of selected antigens.
8. Frozen sections: mount tissue blocks of approx 1 cm3 on specimen holders with
Tissue-Tek, produce cryostat sections of 10–12 µm at approx –22°C, and mount
sections on Superfrost Plus glass slides.
9. Paraffin sections: prepare sections of approx 3 µm, mount these on Superfrost
Plus glass slides, deparaffinize in xylene (once for 5 min at 60°C, twice for 5 min
at room temperature), and rehydrate in a series of graded alcohol (5 min in each
of 100 %, 100 %, 96 %, and 70 %).
6. Dilute primary antibodies with antibody dilution buffer (e.g., 1:200) and add to
sections and incubate at 4°C in a humidified chamber overnight.
7. Rinse in PBS-IF/Tween, three times for 10 min.
8. Dilute secondary antibodies (fluorescein isothiocyanate [FITC]- or CY3-conju-
gated) 1:200 in antibody dilution buffer and apply to sections (from now on, all
steps must be conducted in darkness) and incubate at room temperature in a humidi-
fied chamber for 1 h.
9. Rinse in PBS-IF/Tween, three times for 10 min.
10. Wash in distilled water (short).
11. Apply mounting medium (see Note 16) and seal with nail polish.
12. Examine within a week with fluorescence microscope (store sections at 4°C).
4. Notes
1. If the material is taken during routine slaughtering, it is very important to remove
the uterus from the cow as fast as possible to avoid separation of fetal and mater-
nal tissues. Excision and/or fixation should be done immediately.
2. If cows of defined gestational ages are not available, the measurement of the fetal
crown-rump length can be done. This will provide an approximation of the stage
of gestation.
3. Gentle manipulation of placentomes is essential to avoid separation of fetal and
maternal components of the placentome. Generally, it is very hard to prevent this
separation, especially when cutting the placentomes into smaller pieces. It can be
helpful to freeze complete placentomes and to split them with a knife and a ham-
mer when frozen.
4. Select and excise material for snap-freezing first to avoid contamination with
fixative.
5. If you wish to take material for examination with molecular biological methods
also, use gloves during the preparation and handling of the equipment and tissue
to decrease RNA degradation.
6. Try to select placentomes with only one supplying allantochorionic artery and
vein to avoid incomplete fixation and stay away from areas where placentomes
were already excised, because fixative will leak from cut blood vessels.
7. The use of butterfly cannulae is most convenient, because manipulation can be
done from a distance.
8. Successful fixation will be obvious, because placentomes will turn pale and hard,
and venous efflux will consist of pure fixative in the end. If this is not the case,
check for serial connection to other placentomes and clamp these. Then repeat
fixation.
9. Incubation with primary antibodies may be done either at room temperature (or
37°C) for 1 h or at 4°C overnight. Please note that the specificity of antibody
binding may be decreased at room temperature and thus can be associated with
Acknowledgments
The author dedicates this book chapter to her scientific mentor Professor Dr.
Rudolf Leiser, and gratefully acknowledges the generous donation of fluores-
cence pictures by Martina Zeiler (both Justus-Liebig-University Giessen, Ger-
many). The author further acknowledges the fruitful collaboration with Drs.
M. Guillomot (INRA, Jouy-en-Josas, France), G. Johnson (Texas A&M Uni-
versity, College Station, TX, USA), P. Hirsch, and C. Y. Lang (both Justus-
Liebig-University Giessen, Germany).
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24
Molecular Markers for Human Placental Investigation
Berthold Huppertz
Summary
The human placenta is a source for a variety of growth factors, hormones, and other pro-
teins. The cellular source of the proteins can be best determined by immunohistochemistry.
Furthermore, immunohistochemistry can also be used to identify a specific cell type and to
differentiate it from other types of cells. Thus, there is the need for specific markers of those
cell types that are present in the placenta. In this chapter, the basic protocols for the identifica-
tion of proteins in a tissue section are described. This chapter focuses on methods that are
available in the majority of laboratories, and therefore concentrates on methods that are used
together with light microscopy.
Key Words: Immunohistochemistry; morphology; antibody; M30; TUNEL; ssDNA;
marker; syncytiotrophoblast; cytotrophoblast; Hofbauer cell; macrophage; fibroblast;
myofibroblast; endothelial cell.
1. Introduction
With its specific location between mother and fetus, the placenta comes in
direct contact with maternal blood and endometrial tissues. Although defined
as an allograft that should be recognized as nonself by the mother, the placenta
is normally not rejected but remains within the uterus for 40 wk. The placenta
is composed of different tissues, comprising (1) the villous trophoblast as the
epithelial cover of the villous tree, (2) the villous stroma with mesenchymal
cells, fetal vessels, and free connective tissue cells such as macrophages
(Hofbauer cells), mast cells and plasma cells, (3) fetal blood that enters the
placenta via the two umbilical arteries and leaves the placenta via the umbilical
vein. Another tissue derived from trophoblast is the extravillous trophoblast,
which invades maternal tissues, finally reaching the walls of spiral arteries as
deep as the inner third of the myometrium.
Both trophoblast populations, villous and extravillous trophoblast, are de-
rived from the trophoblast layer of the blastocyst and maintain all characteris-
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
337
tics of epithelial cells. The villous stroma develops from the extraembryonic
mesenchyme, which is derived from the two-layered embryonic disk and fur-
ther develops a mesodermal phenotype. The first free connective cells, the
Hofbauer cells, directly derive from the placental mesenchymal cells within the
villous stroma, which is also true for the endothelial and first blood cells (1).
Basic protocols for the identification of proteins in a tissue section are
described in this chapter. In order to focus on methods that are available in
the majority of laboratories, this chapter will concentrate on methods that are
used together with light microscopy. In some cases, the use of electron micros-
copy is the better choice, e.g., if the localization of a membrane protein in the
plasma membranes of two adjacent cells must be defined.
Immunohistochemistry of placental tissues is often used to differentiate dif-
ferent cell types from each other. Thus there is a need for specific markers of
the cell types present in the placenta. In Table 1, a list of potential markers for
various cell types within the human placenta can be found. Protocols frequently
used to identify apoptotic cells within the human placenta are also provided
(2,3).
2. Materials
2.1. General Histology
2.1.1. Tissue Preparation
1. Transfer container for the transport of the placenta (isolated to keep the placenta
cool).
2. Plastic bags to keep a term placenta during transport.
3. Small plastic vial to keep a first-trimester placenta during transport.
4. Sterile scalpels, scissors and foreceps.
5. 250-mL bottles.
2.1.2. Fixation
1. 4% neutrally-buffered formalin solution. For 1 L: solve 4 g NaH2PO4 and 6.5 g
Na2HPO4 in 900 mL double distilled water, add 100 mL 37% formaldehyde solu-
tion (Merck, Darmstadt Germany; for analysis, stabilized with about 10% metha-
nol) and adjust pH to 7.0 with HCl or NaOH. Filter fixative and store at 4°C.
2. 250-mL bottles.
3. Embedding cassettes.
Table 1
Molecular Markers for Human Placental Investigation
Tissue/cell type Marker Reference
1. villous trophoblast cytokeratin 4 (review)
cytokeratin 18 neoepitope (M30) 2
syncytin 5
CD10 6
AFP (first trimester) 7
GB25 8
PLAP (third trimester) 9
CD133 10
A. syncytiotrophoblast hCG 11
cadherin-11 12
endoglin/CD105 13,14
β-microglobulin, HFE (third trimester) 15
Thomsen-Friedenreich antigen 16
mucin 1 16
PPAR-γ 17
hPL 4 (review)
B. cytotrophoblast HAI-1 14,18
E-cadherin 12
2. villous stromal cells CD9, CD45, vimentin 4 (review)
CTLA-4 19
A. mesenchymal cells Vimentin 20,21
B. fibroblasts vimentin, desmin, α-sm-actin 20,22
cytokeratin 8/18 (few cells) 23
C. myofibroblasts/ vimentin, desmin, α-sm-actin, α-sm-actin 20–22
smooth muscle cells sm-myosin 24
caveolin-1, -2 25
D. endothelial cells CD34 26,27
caveolin-1, -2 25
von Willebrand factor, Ulex europaeus lectin 28
1F10, PAL-E 29,30,31
E. macrophages CD68 32
CD14 (anti-leu-M3) 33,34
Occurrence of markers may be of importance for those who want to isolate and culture the respective cells or
simply need a marker to identify cells. Abbreviations and CD numbers: 1F10, monoclonal antibody recognizing an
unknown endothelial protein; AFP, alpha-fetoprotein; CD9, p24/motility-related protein-1 (MRP-1), DRAP-27, type
III membrane protein, 228 amino acids; CD10, common acute lymphoblastic leukemia antigen (CALLA), EC
3.4.24.11, neprilysin, enkephalinase, gp100, neutral endopeptidase metalloendopeptidase (NEP); CD14, lipopolysac-
charide receptor (LPS-R), anchored by glycosylphosphatidylinositol (GPI), 356 amino acids; CD34, gp105-120,
heavily glycosylated type I transmembrane protein, 385 amino acids; CD45, leukocyte common antigen (LCA),
tyrosine phosphatase (EC 3.1.34), long single chain type I transmembrane protein, 1120-1281 amino acids; CD68,
macrosialin, gp110, type I transmembrane glycoprotein, 354 amino acids; CD105, endoglin, type I integral mem-
brane protein, 633 amino acids; CD133, AC133, PROML1, hematopoietic stem cell antigen, pentaspan transmem-
brane glycoprotein, 865 amino acids; CTLA-4, cytotoxic T lymphocyte-associated protein-4; GB25, monoclonal
antibody recognizing an unknown trophoblast protein; HAI-1, hepatocyte growth factor activator inhibitor type 1;
hCG, human choriogonadotropin; HFE, hemochromatosis protein; hPL, human placental lactogen; PAL-E, mono-
clonal antibody recognizing an unknown endothelial protein; PLAP, placental alkaline phosphatase; PPAR-γ, per-
oxisome proliferator-activated receptor γ.
3. 250-mL bottles.
4. Incubator adjusted to 54°C.
5. Heating plate adjusted to 54°C.
6. Embedding molds.
7. Forceps.
8. Microtome.
9. Glass slides.
3. Humidified chamber.
4. 3% H2O2 (see Section 2.2.8.).
5. Acetic acid buffer. For 500 mL: solve 1 g acetic acid in 400 mL distilled water,
adjust pH to 6.0 with NaOH and add distilled water up to 500 mL.
6. Blocking solution: phosphate buffered saline (PBS) with 1% BSA and 0.1%
Tween 20.
7. PBS with 0.1% Tween 20.
8. Starting with the secondary antibody solution the materials are identical with the
materials for the standard immunohistochemistry (see Subheadings 2.2.11.–
2.2.15.).
3. Methods
3.1. General Methods
3.1.1. Tissue Preparation
1. Directly after delivery or termination of pregnancy, take the placental material
and put it into a plastic bag (third-trimester material) or into a small plastic vial
(first-trimester material). Place the bag/vial on ice in an isolated container and
transfer the placenta to the laboratory (see Note 1).
2. Place the placenta on a tray and cut out the tissues used for your experiments (see
Note 2). Take a scalpel or scissors and cut pieces with a maximal width of 5 mm
from the placenta. The other dimensions should be in the range of 1–3 cm (see
Note 3).
3.5. Specific Protocol for the TUNEL Test (see Note 16)
The use of a commercial kit with some minor adaptations is described (see
Note 17).
1. Rehydrate and deparaffinize the sections (on glass slides) in an alcohol series
with two times xylene (5 min each), two times 100% ethanol (5 min each), one
time 90% ethanol (3 min), one time 80% ethanol (3 min), one time 70% ethanol
(3 min), and a short washing step in Tris buffer.
For all of the following steps of the protocol, it is important not to over
incubate the specimens, otherwise false positives cannot be excluded (see Note
18). It is also very important not to let the specimens dry out during or between
any steps.
2. Permeabilize the specimens with proteinase K solution for 20 min at room tem-
perature and rinse specimens in TBS (see Note 18).
3. Inactivate endogenous peroxidases by incubating the specimens in 3% H2O2 for
5 min at room temperature and rinse them with TBS (see Note 19).
4. Cover the specimens with equilibration buffer for 20 min.
5. During this time prepare the TdT reaction mix on ice. Mix 57 µL TdT labeling
reaction mix with 3 µL TdT enzyme for each specimen and apply the solution
onto the specimens (see Note 20). The specimens are incubated in a humidified
chamber for 90 min at 37°C.
6. Rinse the specimens with TBS buffer and apply the stop solution for 5 min at
room temperature.
7. Rinse the specimens with TBS buffer again and cover them with blocking buffer
for 10 min at room temperature.
8. Apply the peroxidase-streptavidin conjugate onto the specimens for 30 min at
room temperature in a humidified chamber.
9. Wash the specimens in TBS buffer and apply 100 µL DAB solution onto the
specimens. A brown precipitate in apoptotic nuclei will appear in 10–15 min (see
Notes 21 and 22).
10. Rinse slides with distilled water and cover the specimens with 100 µL methyl
green for 3 min to counter stain the nuclei.
11. Wash the specimens with distilled water and cover the section with glycerin gela-
tin using a coverslip.
4. Notes
1. Normally, there is a delay between delivery of the placenta and availability and
usage of this material in the laboratory. There are a few scientists who are able to
obtain this material fresh from delivery and fix or freeze it within a few minutes.
But mostly, there is at least a 10- to 30-min gap between delivery room and labo-
ratory as a result of the distance between both rooms. A term placenta can be kept
on ice (without direct contact) for this time without additional solutions. A first-
trimester placenta is kept in a small vial and normally also does not need addi-
tional solutions for this duration of storage.
2. When cutting pieces of the placenta, take care not to destroy the fragile villi
inside the placenta. If you use foreceps to hold the tissue during cutting, take the
side that will not be used for fixation. When taking the piece of tissue of interest,
hold it at the edge without compressing the villous tissue.
3. Fixation in formalin solutions requires a minimal diffusion distance of the fixa-
tive. The same is valid if you freeze the samples in liquid nitrogen; the thicker the
sample, the longer it takes to freeze it; thus, with thicker samples, generation of
ice crystals will destroy the tissue. Therefore, samples obtained from the pla-
centa should have a maximal width of 5 mm. Other dimensions may be chosen
“without limitations,” e.g., if samples from a term placenta are required, then a
sample covering the whole thickness of the placenta can be obtained, but one
must keep in mind that the width of one side is restricted to 5 mm. One must keep
in mind that fixation of the samples is performed with embedding cassettes. This
will restrict the size of the samples to about 3 × 1–2 × 0.5 mm.
4. Tissues can be shock-frozen in liquid nitrogen or fixed in formalin for paraffin
embedding. Because most of the sections that are available in pathology depart-
ments and other archives are paraffin sections, this chapter concentrates on the
fixation and handling of those materials.
5. Fixation in formalin should not exceed 24 h. Longer fixation times lead to loss of
immunoreactivity; also, other fixatives may reduce the binding capacity of anti-
bodies used in immunohistochemistry. Smaller samples from a first-trimester
placenta or from villous explant cultures need much shorter fixation times. Very
small samples (up to 3 mm in diameter) only require 1 to 2 h of fixation.
6. After fixation, the samples must be dehydrated before embedding into paraffin.
The times for the alcohol series have to be adapted depending on the volume of
the samples. For small sample sizes, the following times are sufficient: 45 min in
70% ethanol (or storage for longer times), 45 min in 95% ethanol, three times
45 min in 100% ethanol, three times 45 min in acetone, 60 min in paraffin (or
overnight).
7. The higher the temperature during embedding into paraffin, the worse is the anti-
genic stability. Thus, antigenicity decreases, and some antibodies may not be
able to bind to the altered antigens. Therefore, the use of low-melting paraffin is
recommended. Paraffin with melting temperatures between 52°C and 54°C (e.g.,
Paraplast x-tra; Fluka, 76259) are recommended; however, paraffin with melting
temperatures up to 58°C may be used.
8. Any detection protocol or kit may be used here. A variety of protocols are used
for immunohistochemistry and it is always necessary to adapt a protocol to the
needs of the respective laboratory. A representative protocol used in our labora-
tory is presented.
9. Incubation times of the H2O2 solution to block endogenous peroxidase activity
have to be adapted according to the concentration of H2O2. Incubation with a 3%
H2O2 solution requires only 5–10 min incubation whereas a 0.3% H2O2 solution
requires an incubation time of 30 min. The H2O2 solution should always be pre-
pared and used fresh.
10. Normally, Tris buffer is used in all steps, but other buffers such as PBS may be
used. For some specific antibodies and staining procedures, alternative protocols
have to be used (see Subheadings 3.3. and 3.4.).
11. For most primary antibodies an incubation time of 60 min at room temperature is
sufficient to result in a clear staining with low background. But depending on the
antibody, changes of the times and temperature may be necessary. Another
often-used protocol combines incubation overnight at a temperature of 4°C.
Please note that conditions need to be optimized for every single antibody.
12. Incubation times for AEC may vary depending on the antibodies used. To stan-
dardize the protocol, a fixed incubation time (e.g., 10 min) should be used. But
sometimes it may be necessary to wait longer (up to 30 min) or to stop the incu-
bation already after a few minutes, depending on when the color development is
complete.
13. Two chromogens are classically used in immunohistochemistry (AEC and DAB)
although other substrates or fluorochromes can be used. In paraffin sections, espe-
cially from archives, the use of the classical chromogens is superior to the use of
other substrates. Both AEC and DAB are chromogens used for staining peroxi-
dase labeled compounds in immunohistochemistry. AEC produces an insoluble
end product that is red in color while DAB produces a brown water insoluble end
product.
14. The monoclonal antibody with the clone number M30 specifically recognizes an
epitope of the cytokeratin 18 protein generated during cleavage by caspases.
Thus, this antibody should recognize only cytokeratin 18 cleavage products.
However, under denaturing conditions (Western blots), it is apparent the M30
antibody also recognizes intact cytokeratin 18. Denaturation of native cytokeratin
18 protein may occur during from the increased temperatures used during embed-
ding into paraffin. Therefore, low temperatures are essential when using the M30
antibody. It must be kept in mind that cytokeratin 18 is only found in some epi-
thelia. Thus, this antibody cannot be used to detect apoptosis in cells of mesoder-
mal origin such as villous stromal cells.
15. Similarly to the M30 antibody that recognizes a caspase-generated cleavage prod-
uct of cytokeratin 18, the F7-26 antibody (Alexis Corporation, Lausen, Switzer-
land) specifically binds to apoptotic single-stranded DNA (ssDNA). The stability
of apoptotic DNA is decreased during thermal denaturation due to proteolysis of
DNA-bound proteins by effector caspases. The antibody is specific for ssDNA
and does not bind to double-stranded DNA. The advantage of this antibody over
the widely used TUNEL test (discussed later) is its high sensitivity to apoptosis
with nearly no staining of necrotic nuclei.
16. Similar to the antibodies M30 and F7-26, the TUNEL test is used to identify late
apoptotic cells. The TUNEL test is not based on an antibody recognizing its spe-
cific antigen. This time, an enzyme (TdT) recognizes nicks inside the DNA
strands and links nucleotides to the ends. Using an excess of labeled nucleotides,
the nicks can be visualized.
17. A representative protocol is provided that generally gives reproducible and con-
vincing results. The protocol uses the TdT-FragEl kit from Calbiochem (cat. no.
QIA33) with some slight adaptations. We have tested a variety of different kits
and have tried to create our own protocol. We have found that the protocol pro-
vided with the above mentioned kit is satisfactory, with some minor modifica-
tions.
18. Care should be taken using this assay since it is very easy to produce false posi-
tive results. Not only does the TUNEL test also stain necrotic DNA, but, depend-
ing on the protocol used, even mitotic cells or cells in interphase may be labeled.
Thus, close inspection of the morphology of the positive nuclei is always recom-
mended. A TUNEL positive nucleus should always display a morphology that is
condensed (higher density of chromatin), irregular in shape and smaller in size.
Large ovoid nuclei that show little densely packed chromatin but that show
TUNEL positivity are most likely a false positive. And even within one section
and one incubation, there may be areas where the staining is reliable, whereas the
area next to it shows 100% positive nuclei (which is clearly false positive).
19. The digestion with proteinase K is one of the crucial steps of this protocol.
Overincubation for only a few min results in a dramatic increase in false-posi-
tive nuclei. Thus, one must check first whether the time given in this protocol is
Acknowledgments
The author thanks Dr. Maria Kokozidou for critically reading the manu-
script and Uta Zahn for help with the exact reading of the protocols.
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25
Correlative Microscopy of Ultrathin Cryosections
in Placental Research
Summary
In this chapter, we describe procedures for correlative microscopy in immunocytochemical
studies on the human placenta. We have adapted ultrathin cryosections for use in high-resolu-
tion immunofluorescence microscopy (IFM) and for correlative immunocytochemical local-
ization using fluorescence and electron microscopy. High-resolution IFM of ultrathin
cryosections (50–100 nm in thickness) can be important because these physical sections mini-
mize the potential for false co-localization in the z-dimension. In addition, IFM of these sec-
tions affords greater sampling efficiency than does immunoelectron microscopy (IEM). These
ultrathin cryosections are compatible with conventional electron microscopy because a rela-
tively low-voltage electron beam can penetrate them. Thus, the same ultrathin cryosections of
placenta can be viewed in both fluorescence and electron microscopes. This latter point can be
of importance because it may be necessary to know the true size and shape of objects observed
by IFM; this can be determined best by IEM. Additionally, IEM can provide the “reference
space” lacking in IFM. The use of ultrathin cryosections is a powerful approach for placental
research, especially for the investigation of the in situ localization of antigens in the complex
structure of the human placenta.
Key Words: Placenta; villi; immunocytochemistry; correlative microscopy; immunofluo-
rescence; immunoelectron microscopy; ultrathin cryosections; Alexa; FluoroNanogold; silver
enhancement; caveolin-1α; early endosome antigen 1 (EEA1).
1. Introduction
Immunocytochemistry is a powerful and diverse set of methods directed
toward obtaining spatial and temporal information concerning the expression
and distribution of antigens in situ. Immunocytochemistry can provide unique
information that cannot be gained readily with biochemical, immunochemical,
or morphological methods alone. Ideally, the localization of antigens is highly
specific, with minimal background signal; this relies upon the inherent speci-
ficity of the antigen–antibody reaction. However, specificity can vary among
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
351
antibodies, ranging from those with exquisite specificity to those that cross-
react with molecules other than the one to which the antibody was generated. It
is therefore prudent to test the specificity of antibodies in as many ways as
possible to determine if a particular antibody will be useful for immunocy-
tochemistry. Another important consideration in all immunocytochemical
experiments is the use of proper control reactions. This is crucial in order
to prevent spurious immunolocalization so that the genuine antigen distribu-
tion is ascertained. A delineation of the range of controls for immunocy-
tochemical preparations will not be given due to space limitations. The reader
is referred to the work of other authors who have dealt with this issue directly
(1,2). In addition to antibody specificity and the use of proper controls, there
are conditions that should be met before completely successful immunocy-
tochemistry can be achieved. These include: (a) preservation of immunoreac-
tivity; (b) retention of antigens in the proper location; (c) retention of morphological
detail (this is especially the case for electron microscopy); (d) maintenance of
equal accessibility of antibodies to antigen molecules at different locations
within the specimen; and (e) ability to label multiple antigens in the same
sample (3).
The nature of the biological sample to be examined by immunocytochemis-
try can set constraints on the methods that must be employed. Single cells,
such as those grown in tissue culture, require minimal sample preparation for
light microscope level immunocytochemistry since they are relatively thin and
are directly amenable to certain kinds of optical microscopy. However, these
same cells require special preparative methods for immunoelectron micros-
copy (IEM). Typically, single cells must be embedded and subsequently cut
into extremely thin sections (50–100 nm in thickness) before examination in
an electron microscope. Various plastic resins have been used to embed cells
for the preparation of conventional thin sections. Alternatively, these cells can
be “embedded” in concentrated sucrose solutions and subsequently frozen for
the preparation of ultrathin cryosections (see Subheading 3.1.) (4).
Intact tissue such as human placenta, on the other hand, presents additional
problems for immunocytochemical analysis. Diffusion of antibodies into intact
tissue as well as subsequent visualization is problematic. These difficulties can
be overcome by sectioning the tissue into thin slices. Useful sections range
from 5–20 µm in thickness for light microscopy to 50–100 nm thickness for
electron microscopy (EM). In order to cut usable sections, the tissue is gener-
ally embedded in a matrix of some sort prior to sectioning; paraffin or plastic
resins are commonly used to embed tissues. For retention of antigenicity within
tissues, preparation of frozen sections is generally less intrusive than other
preparative methods. Tissue to be frozen is routinely fixed and subsequently
infused with sucrose solutions prior to freezing. The use of sucrose has two pur-
Ultrathin cryosections of cells and tissues have been used for IEM using
colloidal gold particles as the detection system for more than 20 yr. This approach,
sometimes called the Tokuyasu method, has been extremely valuable for detect-
ing the distribution of a number of antigens at the ultrastructural level (4).
Indeed, our understanding of some fundamental aspects of cell biology is
largely dependent on this methodology. However, this technique, like all tech-
niques, is subject to limitations. The exclusive use of ultrathin cryosections for
EM limits their utility as a result of the sampling limitations imposed by the
small amount of material that can be examined in the electron microscope at
any one time. The use of colloidal gold as the reporter system also imposes
limitations. Colloidal gold particles are discrete structures that can be counted
for quantitative analysis. However, various investigators have addressed the
question of whether the density of colloidal gold immunolabeling correlates
with antigen concentration. In such studies, the determination was that a one-
to-one relationship between colloidal gold particles and antigens did not occur
(5,6). It has been proposed that the greatest labeling efficiency achieved with
colloidal gold probes is 20% or less (7). The major reasons proposed for the
relative inefficiency of labeling with colloidal gold immunoprobes are: (a) poor
penetration of the colloidal gold into the section thus only the most superficial
antigens are detected and (b) steric hindrance effects (8). Another important
consideration in using colloidal gold is the recognition that labeling efficiency
varies with the size of the gold particles. Smaller gold particles lead to greater
labeling efficiency than do larger ones (i.e., 5-nm gold particles label more
efficiently than do 10-nm particles, and so forth) (9–11).
These limitations aside, ultrathin cryosections offer an excellent substrate
for immunocytochemical localization of antigens. One important consideration
is that they are thin enough for antibodies to readily penetrate throughout their
volume (in the absence of colloidal gold particles). We have used ultrathin
cryosections for high-resolution immunofluorescence microscopy (IFM) (Fig. 1
and Subheading 3.2.). These sections have a real advantage in eliminating out-
of-focus fluorescence since all of the fluorescence must come from within the
section. This is in contrast to using conventional sections of 5 µm in thickness
and then imaging them with a confocal microscope. In the confocal micro-
scope, the out-of-focus fluorescence signal is minimized by optical sectioning.
The resolution in the z-dimension usually achieved with confocal microscopy
of biological samples is about 500 nm (12). The ultrathin cryosections we
employ are about 100 nm in thickness (Figs. 2,3). Thus, the use of ultrathin
cryosections can minimize the possibility of false co-localization in two-color
IFM (Fig. 4) (13). In addition, in IFM the sampling efficiency is increased
when compared with IEM. These are real advantages that we have utilized in
our study of the distribution of a number of antigens in the human placenta.
Fig. 2 (see companion CD for color version). Diagram summarizing a model illus-
trating the advantages of using ultrathin cryosections for high-resolution immunofluo-
rescence microscopy (IFM). The left side shows a diagram of a terminal villus cut in
cross section. Three cell types are shown: endothelium (#1), pericyte (#2), and syncy-
tiotrophoblast (STB) (#3). The black and gray dots indicate two different structures
labeled with two different fluorochromes in IFM. The gray bar indicates the volume of
an ultrathin cryosection (about 100 nm). The right side shows the fluorescence signals
in the z-dimension (side view) and the x- and y-dimensions (top view). In 5 µm sec-
tions, the fluorescence signal from the individual structure may be stacked in the vol-
ume of the section; this may lead to false co-localization as indicated by the white
color. In confocal optical sections (approx 500 nm), the potential for false co-localiza-
tion is reduced when compared to thicker conventional cryostat sections. However,
separate small organelles (50–200 nm size range) that are labeled with two different
fluorochromes and that are stacked within the section may appear to be co-localized in
the same structure as indicated by the white color (arrowhead). In ultrathin
cryosections (100 nm or less in thickness), the possibility for false co-localization is
minimized further since small structures (50–200 nm in size) could occupy the entire
volume of the section as indicated by the gray color (arrowhead).
Fig. 4 (see companion CD for color version). Double-label immunofluorescence microscopy (IFM) detection
of caveolin (CAV)-1α and early endosome antigen (EEA)1 on an ultrathin cryosection. (A) In a terminal villus
of the placenta, CAV-1α is localized to capillary endothelium (a star indicates the capillary lumen) and adjacent
pericytes (P) and is seen as small punctate structures (13,16,21). In the syncytiotrophoblast (STB) (arrowheads),
CAV-1α is not detected. (B) EEA1 is primarily in the apical portion of the STB in vesicle-like structures (arrow-
heads) (13). EEA1-positive structures are also present in the endothelium and pericytes (arrows) but are less
8/29/05, 11:23 AM
abundant than in the STB (arrowheads). (C) The differential interference contrast (DIC) image of the same
section shown in A and B illustrates the morphology of the section. The fluorescence image of the 4',6-diamidino-
2-phenylindole (DAPI)-labeled nuclei has been merged with the DIC image to facilitate orientation. The lumen
of the capillary (star) and the STB (arrowheads) are indicated. (D) The merged image shows the distribution of
357
CAV-1α and EEA1 and illustrates the relationship between the two IFM signals. The same labels (arrowheads,
arrows, P, and star) used in each panel are presented to provide reference points. Bar = 10 µm.
358 Takizawa and Robinson
Ultrathin cryosections are also very useful for correlative fluorescence and
electron microscopy (Fig. 1 and Subheading 3.3.); that is imaging the same
exact structures (in the same ultrathin cryosection) by both fluorescence and
electron microscopy. This methodology is important becauses it can bridge the
resolution gap between fluorescence and electron microscopy. In our studies,
we have used a single reporter system that contains both a fluorochrome and a
gold-cluster compound. This probe is known as FluoroNanogold. In addition
to containing a fluorochrome and a gold probe in the same reagent, it has the
further advantage of being extremely small (approx 1.4 nm for the gold probe).
This probe appears to behave more like a fluorochrome-conjugated secondary
antibody than a colloidal gold-coupled immunoprobe. It appears to penetrate
fully into ultrathin cryosections (11). We have used this reagent in correlative
microscopy in which we first collect a fluorescence image and then following
a silver-enhancement reaction to enlarge the gold cluster compound, image the
same structures in an electron microscope (Fig. 5). This approach is valuable
when it is important to know the true size and shape of a structure and when it
is important to see the “reference space.” In IFM, the only structures evident
are those tagged with the fluorochrome; all other parts of the cell or tissue are
invisible under these conditions. In EM, on the other hand, all structures are seen
not just those positively labeled. Imaging this reference space may be vital for
understanding the positive immunolabeling pattern (13). The use of ultrathin
cryosections is essential in these types of experiments.
2. Materials
1. Carbon steel blades (Feather Safety Razor, Osaka, Japan).
2. Pink base plate wax (Coltene/Whaledent Inc., Cuyahoga Falls, OH).
3. 60 × 15 mm and 35 × 10 mm cell culture dishs (BD Falcon, Franklin Lakes, NJ).
4. Hardened filter paper (Grade 50, Whatman, Clifton, NJ).
5. SS-style tweezers (Ted Pella, Redding, CA).
6. Groove type specimen carrier pins (Mager Scientific, Dexter, MI).
7. Cryo P diamond knife (Diatome-US, Fort Washington, PA).
8. Reichert Ultracut E equipped with an FC 4D cryounit (Leica, Vienna, Austria).
9. Mouse anti-early endosome antigen (EEA)1 monoclonal antibody is available
from BD Transduction Laboratories (San Diego, CA). EEA1 is a 180-kDa coiled-
coil dimer that is crucial for endosome fusion (14).
10. Anti-peptide antibody specific for caveolin (CAV)-1α was generated in chickens.
Immunocytochemical characterization of this antibody has been reported (15).
11. Alexa Fluor 488 and 594 goat anti-chicken and goat anti-mouse immunoglobulin
(Ig)G as well as the ProLong antifade kit can be obtained from Molecular Probes
(Eugene, OR).
12. Biotin-labeled goat anti-mouse F(ab)' 2 antibody is available from Jackson
ImmunoResearch (West Grove, PA).
wrapped in aluminum foil and then stored in light tight box. When the silver
enhancement is ready, 1.5 mL of distilled water is added to a stock tube and then
mixed well in a darkroom with a sodium vapor safelight. This is freshly prepared
on the day of use.
28. NPG silver enhancement stock solution: 5 mL of Gum arabic stock, 2 mL of 0.5 M
MES stock, and 1.5 mL of NPG stock are combined and mixed well for 5 min.
29. Working NPG silver enhancement solution: 8.5 mL of NPG silver enhancement
stock solution is well mixed for 5 min under a room light and then 1.5 mL of the
silver lactate stock solution is mixed with the NPG silver enhancement stock
solution in a darkroom with a sodium vapor safelight. Immediately after mixing
for 1 min, the working NPG solution is applied to sections labeled with FNG.
30. Neutral fixer solution: 250 mM sodium thiosulfate (Sigma-Aldrich) and 20 mM
HEPES (Sigma-Aldrich), pH 7.4. The pH is adjusted with 1 N NaOH; the solu-
tion is stored at 4°C.
31. Reduced osmium fixative: 2% osmium tetroxide–1.6% potassium ferrocyanide
in 0.1 M cacodylate buffer, pH 7.4. This is freshly prepared on the day of use and
handled in a chemical fume hood.
32. Uranyl acetate (UA) solution: 4% uranyl acetate (Mallinckrodt, Paris, KY) in
distilled water. UA is wrapped in aluminum foil and then stored at 4°C. UA is
passed through a syringe filter with 0.2-µm pore size (Acrodisc, Pall Corp., Ann
Arbor, MI) before use.
33. 2% polyvinyl alcohol (PVA), and 0.0015% lead citrate–2% PVA (LC-PVA) so-
lutions: 120 mL of distilled water is boiled in a beaker and cooled at 4°C. 2.4 g of
PVA (MW 30-70k, Sigma-Aldrich, product no. P8136) is added to the beaker
and mixed well at 4°C. 20 mL of 2% PVA is taken to a 60 mL disposable syringe
and can stored at 22°C for at least 2 wk. 1.5 mg of lead citrate (trihydrate, carbon-
ate-free, Polysciences) is added to the remaining PVA, ultra-sonicated for 15
min, mixed with stirring for 5 min and then left unstirred for 15 min at 22°C.
Small amount of precipitates may be present in the bottom of the beaker. Super-
natant of the LC-PVA is taken into a 60-mL disposable syringe and can be stored
at 22°C for at least 2 wk. PVA and LC-PVA stock solutions as well as UA are
filtered before use.
34. 0.8% uranyl acetate-1.6% polyvinyl alcohol (UA-PVA) solution: 200 µL of UA
and 800 µL of PVA are mixed in a 1.5-mL microcentrifuge tube with a vortex.
35. Nikon Optiphot microscope equipped with a Photometrics Cool Snap fx charge-
coupled device (CCD) camera (Roper Scientific, Tucson, AZ).
36. MetaMorph image analysis software system (Universal Imaging Corp.,
Downingtown, PA).
37. Photoshop software (Adobe, Mountain View, CA).
3. Methods
The methods described below outline (1) the preparation of ultrathin
cryosections of human placenta, (2) the technique of high-resolution immuno-
fluorescence microscopy using ultrathin cryosections, and (3) the procedure of
correlative microscopy using FNG.
6. Silver enhancement. 1.4-nm FNGs bound to the cryosections are then subjected
to a silver enhancement procedure in order to render them visible in the sections
under an electron microscope (20). The grids are then floated on three droplets of
50 mM MES buffer for 1 min in each droplet on Parafilm. Immediately after
making working NPG silver enhancement solution, the grids are washed on a
droplet of the working NPG solution within a few seconds to minimize the dilu-
tion of the silver enhancement solution with MES buffer and then floated on
another droplet of the working NPG solution for 3–3.5 min at 22°C under a sodium
vapor safelight (see Note 12). The grids are immediately washed by floating them
successively on droplets of neutral fixer solution for 5 min at 22°C under a
sodium vapor safelight and three droplets of PBS for 6 min.
7. Positive contrast enhancement and PVA embedding. Visualization and preserva-
tion of ultrastructure of cryosections is achieved by the positive contrast enhance-
ment method (16). After silver enhancement, the ultrathin cryosections on grids
are postfixed on droplets of reduced osmium fixative on Parafilm for 15 min at
22°C in a chemical fume hood. The grids are washed on three droplets of distilled
water for 1 min each droplet and then floated on droplets of UA-PVA for 15 min
at 22°C. The grids are then washed on droplets of PVA for 10 s and droplets of
LC-PVA for 10 s and then floated on droplets of LC-PVA for 15 min at 22°C.
The EM grids on droplets of LC-PVA are collected with a wire loop (3–3.5 mm
in inner diameter) and the excess LC-PVA fluid is removed with a small piece of
hardened filter paper (see Note 13). The grids are then dried in air.
8. Electron Microscopic Observation. Grids are examined with a Philips CM-12
transmission electron microscope operated at 60 kV. The same regions examined
by fluorescence microscopy are relocated and then electron micrographs are col-
lected (Fig. 5).
4. Notes
1. We routinely cut a tissue block from tissue in the half or one third depths from
the maternal surface of the central region of placenta because it is rich in terminal
villi. Initial fixation during mincing on dental wax, as well as additional 2 h fixa-
tion, is important to meet the conditions for achievement of successful immunocy-
tochemistry as described in the Introduction. Fixative is freshly prepared on the
day of use. It should be noted that 2 h fixation in 4% paraformaldehyde at room
temperature is minimally essential for preservation of placenta ultrastructure for
immunoelectron microscopy; in other words, this fixation is needed for correla-
tive microscopy (16,21).
2. Before adding the 20% gelatin solution, a pellet of villi is resuspended with the
cacodylate buffer since the gelatin solution is viscous. In addition, for pipeting
of the 20% gelatin solution, 3–4 mm is cut from a 1000 µL tip to permit easy
mixing.
3. It is better to finish the necessary trimming of samples prior to sucrose infiltra-
tion since sucrose-infiltrated samples are very sticky. Furthermore, postfreezing
trimming may crack the frozen samples. For the prevention of sample dissocia-
tion from the pins during storage in liquid nitrogen, the mounting surface of the
pins is well scratched with a single edge blade and then ultra-sonicated in acetone
in a beaker for 15 min prior to use.
4. The cutting of ultrathin cryosections and transfer from a knife to either coverslips
or EM-grids is one of the most crucial steps in this technique. It is key to make a
flat (i.e., uncompressed and unwrinkled) thin section with a diamond knife and
then collect it on a droplet of the gelatin–sucrose pick-up solution. Many investi-
gators think it necessary to make sections stretch using the surface tension of a
2.3 M sucrose pick-up solution developed by Tokuyasu (22), because our samples
are fixed only with 4% paraformaldehyde, the higher surface tension of the 2.3 M
sucrose pick-up solution results in alterations in the ultrastructural integrity of
ultrathin cryosections (4,22,23). This leads to the failure of immunocytochemis-
try (i.e., poor labeling, increase of background, dislocation of antigen sites) as
well as poor visualization of cell ultrastructure (17). Another alternative pick-up
solution is 1% methylcellulose–1.15 M sucrose in order to reduce the surface
tension (24). For correlative microscopy, ultrathin cryosections are collected on
Maxtaform “finder” grids (200 mesh, nickel; Graticules, Tonbridge, Kent, UK)
in order to facilitate location of specific structures when going from the optical to
the electron microscope. Nickel grids are better than copper ones since nickel
grids are stronger.
5. The ability to store cryosections in pick-up solutions contain 0.05% sodium azide
at 4ºC greatly increases efficiency. When we examined the in situ distribution of
CAV-1α in human placenta, there was no significant difference in its immunore-
activity and the ultrastructure of placenta between immediately processed sec-
tions and ones stored for 1 yr (unpublished data).
6. This step is designed to gently dissolve the gelatin–sucrose solutions that cov-
ered sections on coverslips. It is convenient to use a 24-well plate for the treat-
ment (i.e., washing and blocking) of coverslips during immunocytochemistry.
You can discard used PBS from a 24-well cell culture plate using a 200-µL tip
attached to a suction tube and add new PBS with a disposal transfer pipet.
7. Each coverslip is carefully picked up from a well with a pair of No. 5 tweezers,
excess MFBS-PBS on the side of the coverslip opposite the section is wiped with
a piece of Kimwipes XL Delicate Task Wiper (Kimberly-Clark, Neenah, WI),
and then the coverslip is floated on a droplet of primarily antibody (approx 25–
50 µL/coverslip) on Parafilm in the cell culture dish. Beginners may need to
practice handling coverslips with tweezers. A piece of wet filter paper is attached
on the inside surface of the lid of the dish in order to prevent evaporation of
primary antibody solution. The use of these small coverslips for immunocy-
tochemistry minimizes the amount of primary antibody solution required.
8. A research-grade fluorescence microscope equipped with the proper fluorescence
filter sets, high magnification objective lens, and differential interference con-
trast optics is required for the imaging shown in this chapter. In addition, the
microscope should have a high quality electronic camera and an image analysis
system. It should take CCD camera features into consideration, including: quan-
tum efficiency, scan mode (bit and MHz), noise, sensitivity, resolution, and price.
9. For washing and blocking of the grids, the use of a 0.5-mL microcentrifuge tube
rack (50-place capacity; Ieda Trading Corp., Tokyo, Japan; cat. no. 9901) would
be helpful. The rack is covered with a Parafilm sheet and then dents are made on
the Parafilm at the openings with the bulb of a disposal transfer pipet.
10. Three microliters of the antifading medium is applied to the center of both a
coverslip and a microscope slide. The grid is carefully placed face-up on the
droplet of the antifading medium on the microscope slide not to leave air bubbles
in the medium. The coverslip then overlies the grid to keep the grid in the center
of the coverslip. The use of an 18-mm coverslip is large enough to minimize
contamination with immersion oil under optical microscopic observation using a
×100 objective lens. The choice of antifade reagents is important for correlative
microscopy with FNG. When 1,4diazabicyclo[2.2.2]octane (DABCO) is employed
instead of NPG, the gold signal from FNG is dramatically diminished but the
fluorescence signal is unaffected (19). The gold signal of DABCO-treated
samples decreases to approx 30% of that of the sample that is treated with NPG
(19). We recommend that NPG be used and that DABCO be avoided as an anti-
photobleaching reagent for this technique.
11. When the coverslip is removed with a pair of SS-style tweezers, a few droplets of
PBS are carefully added to the temporary slide to float the coverslip. It is neces-
sary to keep damage of ultrathin cryosections at a minimum.
12. Each batch of gum arabic stock solution, NPG, and silver lactate should be pre-
liminarily tested to determine the best enhancement incubation time. FNG are
adhered to 0.25% poly-L-lysine (Sigma-Aldrich)-coated formvar grids. These
grids are exposed to the silver enhancement procedure for various time (e.g.,
1–5 min) and then examined by electron microscopy (11).
13. Removal of excess of LC-PVA is important to attain adequate morphological
detail in ultrathin cryosections. As Tokuyasu reported (25), the removal of the
excess is terminated immediately after the remaining amount of LC-PVA forms a
concave meniscus with its center barely touching the center of the grids. The
transfer loop is then left in air. The embedding medium begins to dry from the
center, becomes a thin film in the loop and then shows a gold-blue color in
reflected light. The dried film in the loop is carefully cut along the rim of the
grids with a pair of SS style tweezers prior to picking up the grids.
Acknowledgments
This work was supported in part by grants from the National Institutes of
Health (HD38764 [JMR]). We are indebted to Ms. Heather Richard and the
Campus Microscopy and Imaging Facility at Ohio State University for assis-
tance. We thank Drs. Shigeki Matsubara and Takeshi Takayama of Jichi Medi-
cal School for their technical support. We also thank Dr. Fumimaro Takaku,
President of Jichi Medical School for his encouragement.
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26
Vascular Corrosion Casting of the Uteroplacental
and Fetoplacental Vasculature in Mice
Summary
This chapter describes methods for making vascular corrosion casts of the uteroplacental
and fetoplacental vasculature of the mouse placenta. A catheter placed in the ascending tho-
racic aorta of a pregnant mouse permits the introduction of a methyl methacrylate casting com-
pound into the lower body vasculature, including the uterus and placenta. A fine-tipped glass
cannula attached to a double-lumen catheter is used to instill the same casting compound in the
fetoplacental vessels of mouse placentas. Following polymerization of the casting compound,
tissue is digested off of the placental casts using 20% KOH. The washed and dried casts are
then available for light or scanning electron microscopy. The methods described have been
used to cast the mouse uteroplacental vasculature from 5.5–18.5 d gestation and the
fetoplacental vasculature from 12.5 d gestation to term.
Key Words: Mouse; placenta; umbilical vessels; uterus; pregnancy; embryo; decidua; cir-
culation; microvasculature.
1. Introduction
The availability of mutant strains of mice with placental insufficiencies has
emphasized the need for methods to investigate the normal and abnormal vascu-
lar anatomy of the mouse placental circulation. One method we found invaluable
was vascular corrosion casting of the uteroplacental and fetoplacental vasculature.
We used this method to help establish the normal maternal and fetal circula-
tions in the mouse placenta in the last half of pregnancy (1). Vascular casts
permitted three-dimensional visualization of the structure of the blood spaces
in the placenta, and by partially filling the circulations from the arterial or
venous sides, we were able to conclusively identify and separately describe the
arterial and venous supply. We found that blood spaces so obvious in vascular
casts often collapsed during tissue dissection and processing for histology and
were barely detectable on histological sections. Qualitative and quantitative
information on vessel number and diameter can also be obtained from the casts.
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
371
372
2. Materials
2.1. Vascular Corrosion Casting of the Uteroplacental Vasculature
in Mice
1. Pregnant mouse.
2. Warming mattress and/or warming light (VWR International, Inc., West
Chester, PA).
34. 20% KOH (20% KOH is corrosive. Wear protective gloves and eyewear and
avoid contact with skin and clothing).
35. Distilled water.
36. Pasteur pipets with bulb.
35. Intracardiac heparin stock solution (100 IU/mL): 0.1 mL of 10,000 IU/mL hep-
arin and 10 mL of 0.9% NaCl. If prepared and stored using sterile techniques,
this solution can be stored at 4°C for up to 1 mo.
36. Perfusate: 10 mL of 2% xylocaine (20 mg/mL), 10 mL of 0.9% NaCl, and 0.2 mL
of intracardiac heparin (100 IU/mL). Mix in a small beaker. Draw up 10 mL
through a 0.2-µm syringe filter into each of two 10-cc syringes. Cap the syringes
and heat to 40–45°C.
37. Warm PBS: 0.14 M NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4,
pH 7.2, at 40–45°C.
38. 10-cc syringes (eight syringes are required).
39. Warm 3% paraformaldehyde in a labeled 3-cc syringe with a 23-gage needle.
40. 20% KOH (20% KOH is corrosive. Wear protective gloves and eyewear and
avoid contact with skin and clothing).
41. Distilled water.
42. Pasteur pipets with bulb.
3. Methods
3.1. Vascular Corrosion Casting of the Uteroplacental Vasculature
in Mice
The methods described below outline (1) the surgical procedure, (2) the per-
fusion and casting of the lower body vasculature (including the uterus and pla-
centa), and (3) the processing of the vascular casts. The casting methods
detailed here are used to cast the entire uteroplacental vasculature (arteries,
microcirculation, and veins). However, we have used modifications of these
methods to cast the veins in blue and arteries in red or to prepare partial casts of
the venous or arterial circulations by infusing via the vena cava or aorta, respec-
tively (see Note 1).
3.1.1. Surgical Procedure
1. Anesthetize the mouse (see Note 2).
2. Lay the mouse on its back on a warmed tray to maintain body heat during the
surgery (see Note 3). Tape the mouse’s front paws to the tray to secure the animal.
3. Inject 0.05 mL of intracardiac heparin (see Note 4) combined with 0.1 mL of
2% xylocaine (see Note 5) into the mouse’s heart using a 1-cc syringe and a
27-gauge needle (see Note 6).
4. Open the mouse’s chest using Toughcut Metzenbaum scissors, taking care to leave
the diaphragm intact (i.e., do not enter the abdominal cavity) (see Note 7). Cut
through the sternum near the fifth rib. Cut along the rib towards the back of the
mouse on each side. Cut across the ribs as far dorsally as possible in a rostral direc-
tion. Once cut, the rib cage can be deflected rostrally to expose the heart and lungs
(Fig. 2). Using gauze and cotton swabs, clear any blood from the chest cavity.
5. The heart should still be beating (allowing the heparin to circulate). Xylocaine
will stop the breathing so the mouse will die quickly. When the heart stops, the
anesthesia is discontinued.
Fig. 2. Placement of the catheter in the aorta of the pregnant mouse which allows
perfusion and casting of the lower body vasculature (including the uterus and pla-
centa). (A) Retracted heart. (B) Lung. (C) Inferior vena cava with loose tie. (D) Intact
diaphragm. (E) Esophagus. (F) Ribs. (G) Azygos vein. (H) Aorta. (I) Catheter tied in
place in the aorta.
8. Attach a 10-cc syringe filled with warmed (45°C), filtered perfusate to the cath-
eter (see Note 8). Fill the catheter with perfusate and expel a small volume of the
perfusate into the chest cavity. Lay the catheter alongside the aorta with the tip of
the catheter in the expelled perfusate to prevent air from travelling up the catheter.
9. Using the two ties around the aorta, extend the aorta using a small amount of
tension. Cut a small hole in the aorta, between the two ties, using extra fine or
delicate scissors. Take care not to cut through the vessel.
10. Using forceps, insert the catheter in an anterograde direction through the hole
into the aorta. Advance the catheter until its tip is lying just inside the abdominal
cavity. Secure the catheter in place with the downstream tie.
11. Ensure that the catheter is in the aorta and has not perforated the vessel by gently
pulling back on the plunger of the 10-cc syringe (see Note 9). Some blood should
come back in the catheter. Flush this blood back into the mouse.
12. When satisfied that the catheter is positioned correctly, secure it in place using
the upstream tie. Wrap the tie around the catheter and tighten it without occlud-
ing the catheter (Fig. 2).
13. Tether the catheter to the ribcage using 6'0' suture with a three-eighths-inch
tapered, curved needle.
14. Trim the aortic catheter ties.
15. Locate the right atrium and, using iris scissors, make a cut through the wall of the
chamber to act as a vent.
16. Gently infuse approx 1 mL of warmed perfusate through the catheter and ensure
that the blood being pushed by this influx of perfusate is exiting through the vent
in the right atrium. Ensure that the tie around the inferior vena cava is still loose
and not occluding the vessel.
17. Transfer the mouse preparation on its tray to a fumehoood where an infusion
pump, the casting compound supplies, and a pressure-head system (for maintain-
ing a constant 20 mmHg pressure during curing of the casting compound) have
been set up.
3.1.2. Perfusion and Casting of the Lower-Body Vasculature, Including
the Uterus and Placenta
The following steps are carried out in a fume hood.
It is critical that air is not allowed to enter the vascular system during perfu-
sion and casting. Air bubbles will decrease the quality of the vascular casts.
Preliminary experiments are important to measure femoral arterial blood pres-
sure during infusion of the perfusate and casting compound. Ensure that pres-
sures do not exceed normal systolic arterial blood pressure. Rates and times
given below are suitable for our pregnant ICR (CD-1) mice (1). It may be nec-
essary to adjust infusion times and/or rates depending on the size and/or strain
of mouse.
1. Infuse approx 9–10 mL of warmed (45°C), filtered perfusate using an infusion
pump (see Note 8). Begin at a rate of 0.5 mL/min increasing gradually to a final
rate of 4 mL/min. It should take approx 2–2.5 min to infuse 9–10 mL of perfusate.
within 24–48 h depending on the size of the samples. Digestion can be aided by
removing the old KOH (with a Pasteur pipet) and adding fresh KOH if digestion
is not completed in the first 24 h.
2. Following complete tissue digestion, wash the cast with distilled water. It is
imperative that this washing is done very carefully to avoid damaging the deli-
cate vascular cast. With a Pasteur pipet, add and remove at least three cycles of
distilled water from the vial containing the cast. Ensure that all tissue is washed
off of the cast.
3. The cleaned cast can be stored in distilled water (see Note 11). For viewing with
light or scanning electron microscopy (SEM) (see Note 12), casts must be air-
dried. It may be necessary to dissect away portions of the cast to expose the
vessels of interest (Fig. 1A). Dissection can be done under a light microscope
using Iris scissors. Dried casts should be stored covered to protect them from
damage and dust.
fresh for each placenta being cast. The casting compound is prepared once an
embryonic heartbeat has been observed and while the umbilical vessels are
being prepared for infusion. The surgeon prepares the sample for casting while
the assistant prepares the casting compound. The surgeon inserts the cannula
into the umbilical vessel and holds it in place while the assistant acts as an
infusion pump and pushes the casting compound into the vessel. With the
exception of the removal of the pregnant uterus and the preparation of the
casting compound, all work is carried out using a surgical microscope with an
observer port allowing both the surgeon and the assistant to view the sample.
The methods described as follows outline the surgical procedure, perfusion
and casting of the placenta, and processing of the casts.
7. Using fine forceps and iris scissors, cut through the yolk sac and amniotic mem-
branes along the placental margin. Cut one-third to one-half of the way around
the placenta. Choose the side with the least yolk sac vasculature for the incision.
8. Put down the sharp instruments to avoid damaging the embryo and placenta. We
use a blunt Hayman-style microspatula to gently manipulate the embryo and pla-
centa. Gently lift the embryo out from within the membranes. Position the embryo
so it lies next to but faces away from the placenta (Fig. 3). Use the body of the
embryo to support the umbilical vessels at one end, and the position of the pla-
centa to put tension on the umbilical vessels. The vessels should be extended but
not stretched.
9. Revive the selected embryo by gradually warming it. Position the stream of warm,
humidified air over the embryo. It is important to keep the preparation warm
from this point on. Prior to being warmed, the umbilical vessels will be empty of
blood. As the embryo is warmed, its heart will begin to beat and the umbilical
vessels will fill with blood. When blood first appears in the vessels, the umbilical
vein will be a bright red color. Very quickly, the color of the blood in both ves-
sels will appear identical. A pulse will be visible in the umbilical artery. The
branches arising from the umbilical vein usually overlay those from the umbili-
cal artery on the placental surface (Fig. 4A) (see Note 14).
10. As soon as there is a visible heartbeat, drop 1–2 drops of warm 3% paraformalde-
hyde onto the umbilical vessels (using a 3-cc syringe and 23-gauge needle). The
paraformaldehyde helps to prevent vasospasm of the umbilical vessels during
handling and helps maintain the hole that will be cut in the vessel for cannula
insertion.
11. As soon as a heartbeat is detected, the assistant should begin mixing the casting
compound. The casting compound is a mixture of Batson’s No. 17 monomer
base solution (0.5 mL at 4°C), catalyst (0.15 mL at 20°C), and promoter (10 µL at
4°C) with the addition of jet liquid (0.24 mL at 4°C). All of the components of
the casting compound (except the catalyst), the syringes to measure the compo-
nents and the test tubes used to mix the casting compound in are kept at 4°C on
crushed ice. The first two components can be premeasured and stored in their
respective syringes. The promoter is measured as required. The jet liquid must be
measured immediately prior to use. If left in a plastic syringe, the jet liquid will
digest the syringe. If desired, pigment can be added to the casting compound (see
Note 15). The amount of pigment added to the casting compound depends on the
desired darknesss of the color in the cast. Start with the least amount of pigment
paste possible and adjust accordingly (see Note 16). Mix the components on ice
in a 12-mm × 75-mm glass test tube in the order described previously. Mix the
components thoroughly but gently with a wooden applicator stick. Avoid intro-
ducing air bubbles or ice chips into the mixture, because they will decrease the
quality of the casts. Let the casting compound stand on ice for 30 s once it is
completely mixed. Draw up the casting compound into a chilled 3-cc syringe.
Expel any air and attach the syringe to the catheter (see Note 17). Return to the
mouse preparation.
Fig. 3. Cannulation of the umbilical vessel for perfusion and casting of the
fetoplacental vasculature. The body of the embryo supports the umbilical vessels while
the position of the placenta places tension on the vessels. Perfusate and casting com-
pound are infused into one umbilical vessel with the second umbilical vessel acting as
a vent.
12. In order to slow down the setting of the casting compound, rest the catheter sys-
tem on an ice pack or a bag of crushed ice. Keep the catheter system as cool as
possible during infusion of the casting compound.
13. While the assistant is preparing the casting compound, the surgeon must prepare
the sample for infusion of the casting compound. Ensure that the umbilical ves-
sels are stable and under slight tension. Position the sample by rotating the Petri
dish so that the work can be done comfortably. The casting compound will be
infused into the placenta through a hole cut in one of the umbilical vessels. A
hole cut in the other umbilical vessel will act as a vent. Cut holes in both the
umbilical artery and the umbilical vein approximately halfway between the em-
bryo and the placenta but a distance apart so that leakage from one hole does not
obscure the other hole (see Note 18). Do not sever either vessel completely. Blood
will flow out of the vessels as holes are cut. It is crucial that the surgeon keeps
track of the location of the holes in the vessels. It is often difficult to see the holes
once the blood has drained out of the vessels. After cutting both holes, drop 1–2
drops of warm 3% paraformaldehyde onto the umbilical vessels. This helps to fix
the holes in an open position.
Fig. 4. Vascular corrosion casts of the fetoplacental vasculature in mice. The cast of
the embryonic circulation of the placenta shown in A and B was prepared by infusing
plastic into one umbilical vessel until it drained out the other. The casts in C and D
were prepared by partially filling the circulation via the umbilical artery (C) or umbili-
cal vein (D). (A) A scanning electron microscopy (EM) photograph of a complete,
undissected cast. The cast shows a central circular region and a peripheral labyrinthine
ring when viewed from the embryonic side. The branches arising from the umbilical
vein (v) usually overlay those from the umbilical artery (a) on the placental surface.
(B) The “tuft-like” capillaries of the labyrinth drain into veins at the edge of the cen-
tral circular region. (C) The umbilical artery branches into many widely distributed
arteries that branch a few times while traversing the labyrinth. At the mesometrial side
of the placenta, these arteries branch abruptly to form the capillaries of the labyrinth.
(D) The capillaries drain into evenly distributed, superficial veins located in the cen-
tral circular region.
casting compound into the preparation through the cannula via an attached
double lumen catheter (Fig. 5). The perfusate will flush the blood out of the
placental vessels prior to casting.
1. The glass cannula has a tapered tip. The surgeon should hold the cannula with its
tip next to the umbilical vessel that the cannula will be inserted into in order to
compare their diameters. The glass tip of the cannula should then be broken at the
appropriate spot along its length leaving a tip with a diameter that can be inserted
into the hole made in the umbilical vessel. Ideally, the cannula is easily inserted
into the vessel and the taper along the length of the cannula will seal the cannula
within the vessel. To break the tip of the cannula, hold the glass tip in the end of
a pair of Dumont forceps and close down around the tip. This will break off the
tip where it is held. It may be necessary to tap the tip of the cannula against the
edge of the forceps to remove jagged edges in the glass.
2. The assistant should then attempt to flush first perfusate and then casting com-
pound through the cannula to clear all air and to ensure that the tip of the cannula
is large enough to allow their passage. The casting compound is the more viscous
of the two solutions and will ultimately determine how large the tip of the can-
nula must be. The tip must be large enough to allow passage of the casting com-
pound but small enough that it can still be inserted into the hole in the umbilical
vessel. If the tip is not large enough to allow passage of the solutions, repeat
Step 1. Avoid contaminating the sample with casting compound during this
process.
3. After the cannula tip has been broken to an appropriate diameter, flush the cast-
ing compound out of the cannula using the perfusate.
4. The surgeon inserts the tapered tip of the cannula into the hole cut in one of the
umbilical vessels and advances the cannula in the direction of the placenta until
the vessel fits snugly around the cannula (Fig. 3) (see Note 19). The assistant
may exert a very slight pressure on the perfusate syringe to expel a minimal
amount of perfusate from the tip of the cannula during its insertion. This will help
to open up the hole in the vessel and aid in the insertion of the cannula. Often
there is already pressure built up within the catheter system from the flushing
procedure and it is unnecessary for the assistant to exert any additional pressure
during cannula insertion. It is critical that all pressures exerted with this system
are very slight to prevent damage to the delicate placental vessels. Pressure can
be added as required but it is impossible to reverse damage done by initially
using too much pressure.
5. When the cannula is in place, the assistant very gently pushes perfusate through
the catheter. The perfusate is expelled through the tip of the cannula and into the
umbilical vessel and the placenta. The perfusate will flush the blood out of the
placental vasculature via the vent in the other umbilical vessel. Carefully monitor
the size of the placenta during the infusion of the perfusate. If the placenta
appears to swell, immediately reduce the infusion pressure. When the fluid
flowing out of the vent is colorless, one can assume that the blood has been
26_Whiteley_371_392_F
386
Fig. 5. The double lumen catheter system used for casting the fetoplacental vasculature. (A) The glass cannula and the outer
tubing through which the casting compound passes. Note the tapered tip on the glass cannula and the distance between the end of
the inner tubing and the tip of the cannula in an assembled catheter, as shown in the detail. (B) The inner tubing through which the
perfusate passes. (C) The assembled catheter system.
8/29/05, 11:23 AM
Whiteley, Pfarrer, and Adamson
Vascular Corrosion Casting in Mice 387
flushed out of the placenta. The vessels on the surface of the placenta should now
be cleared of blood.
6. Remove the syringe containing the perfusate from the catheter system. This pro-
vides a pressure vent within the system during infusion of the casting compound.
7. Start infusing the casting compound very slowly. A very slight pressure is all that
is required to begin moving the casting compound through the catheter. It should
take 10–20 s for the casting compound to begin to enter the placenta. Do not
become impatient waiting for the casting compound to begin moving down
through the cannula. When the casting compound begins to enter the umbilical
vessel, the assistant should stop pushing on the casting compound syringe momen-
tarily to ensure that excessive pressure is not being exerted on the placental vascu-
lature. Gradually begin to increase the infusion pressure to control the rate of
entry of the casting compound. Specks of color in the casting compound are moni-
tored to ensure the infusion pressure is high enough to sustain flow into the pla-
centa. Carefully monitor the size of the placenta during infusion of the casting
compound. If the placenta appears to swell, immediately reduce the infusion pres-
sure. The appearance of leaked plastic under the surface of the placenta is indica-
tive of a vessel rupture caused by the use of excessive pressure.
8. Continue infusing the casting compound until either the casting compound runs
out, the casting compound becomes too viscous to infuse, casting compound is
seen exiting through the vent, or until a rupture is observed. Ideally, casting com-
pound should be seen exiting through the vent before one is finished infusing the
casting compound and before it becomes too viscous to infuse. If a rupture is
observed, stop infusing the casting compound to avoid excess artefact caused by
plastic curing outside of the placental vessels. To obtain casts of the venous or
arterial sides of the circulation, infuse via the umbilical vein or artery respec-
tively and stop infusing the casting compound shortly after it enters the placental
microcirculation (Fig. 4C,D).
9. After infusing the casting compound, carefully remove the cannula from the um-
bilical vessel. Tighten a 7'0' silk tie around the umbilical vessels if there is con-
cern that the casting compound is still fluid enough to flow out of the placenta. In
order to reduce the amount of tissue to be digested, the embryo may be removed
at this time. Transfer the placenta to an appropriately sized vial or test tube and
allow a minimum curing time of 2 h.
damaging it. With a Pasteur pipet, add and remove at least three cycles of dis-
tilled water from the vial containing the cast. Ensure that all tissue is washed off
of the cast.
3. The cleaned cast can be stored in distilled water (see Note 20). For viewing with
light or SEM (see Note 21), casts must be air-dried. Dried casts should be stored
covered to protect them from damage and dust.
4. Notes
1. The casting methods detailed here are used to cast the entire uteroplacental vas-
culature (arteries, microcirculation, and veins). However, we have used modifi-
cations of these methods to cast the veins in blue and arteries in red. We
simultaneously infused red casting compound into the maternal aorta and blue
casting compound retrogradely into the maternal intra-abdominal inferior vena
cava. The uterus was exposed in a bath of warm PBS so that the clearing and
filling of the uterine vasculature could be visually monitored. A dual infusion
pump was used to simultaneously infuse the two colors of casting compound at
the same rate. The infusion was stopped once both colors were observed entering
the intrauterine vasculature. We also prepared partial casts of the venous or arte-
rial circulations by infusing via the vena cava or aorta respectively and stopping
before the casting compound exited from the microcirculation.
2. We use isoflurane inhalation anesthetic (5% for induction and 1.5% for mainte-
nance), but injectable anesthetics could be used. We use a Vaporstick Small Ani-
mal Anesthesia Machine with a Flush Valve and a Modified Jackson Rees
breathing circuit (Anesco, Georgetown, KY). Excess anesthetic is scavenged.
We have made a mouse facemask using the end connector from an endotracheal
tube. The mouse’s face fits snugly inside the larger end of the connector.
3. We lay our mouse on a small metal tray resting on a warming mattress. Our work
surface is heated from above with a warming light. The intent is to maintain the
mouse’s body temperature at 37–38°C and to maintain a normal blood pressure
and heart rate. This will help to ensure that the intracardiac heparin is circulated
throughout the mouse’s body before death.
4. Heparin is injected into the mouse to prevent clotting of the blood. It is critical
for the production of good quality casts to flush the blood from the vessels to be
cast. Injecting the heparin directly into the heart allows for quick circulation of
the heparin throughout the mouse’s body prior to death.
5. Xylocaine is also injected into the heart for immediate circulation. We have found
that xylocaine causes the cessation of respiration but the heart continues to beat,
circulating the heparin and xylocaine. The inclusion of xylocaine in the perfusate
enhanced the clearance of blood from the uterine vessels and improved vascular
filling possibly by “anesthetizing” the vascular musculature. Nevertheless, incom-
plete vascular filling of the uteroplacental circulation occurred in at least some
placentas of each mouse. Incomplete filling may be due to constriction of the
sphincter-like structures visible around the extrauterine arteries; they are some-
times partially constricted even in well filled casts (Fig. 1G).
6. Intracardiac injection can be performed using two methods. Both are performed
with the mouse lying on its back with its front paws secured to the metal tray.
Both methods are done before the chest cavity is opened. The intracardiac hep-
arin and xylocaine are combined and injected using a 1-cc plastic syringe and a
three-quarter-inch, 27-gauge needle. The needle is inserted through the mouse’s
skin either through the diaphragm lateral to the xiphoid cartilage and directed
forward and medially toward the heart or between the fifth and sixth ribs on the
left side and directed forward toward the heart. When the needle has been inserted,
aspirate a small volume of blood from the heart to ensure that the needle is in the
heart before injecting the heparin and xylocaine. Breathing will stop almost
immediately if this has been successful.
7. By not entering the abdominal cavity, one can check for punctures in the aorta
because the appearance of casting compound in the abdominal cavity would then
be the result of a leak through a vessel puncture or tear.
8. The perfusate is filtered through a 0.2-µm syringe filter to remove any particulate
matter that may be in the perfusate. Particles introduced into the blood vessels
during perfusion may interfere with the passage of the casting compound through
the catheter. Particles can also interfere with the polymerization of the casting
compound and can cause weak points or breakages in the casts.
9. The catheter tip must be cut blunt. If the tip of the catheter has sharp edges or if it
is cut to a point, it is likely to perforate the aorta as it is passes through the vessel.
10. The Jet Acrylic Liquid was added to the casting compound to decrease the overall
viscosity of the casting compound. Decreasing the viscosity improved the flow
of the casting compound and the filling of the placental microvasculature.
11. If a dried cast floats when returned to distilled water for storage, apply one to two
drops of 70% ethanol to the cast to reduce surface tension. We use a plastic
syringe and a 23-gauge needle to drop the alcohol onto the cast. The cast will
then sink in water. Use a minimal amount of alcohol to avoid having the alcohol
soften the cast.
12. To view the casts using SEM, we mount the casts on SEM stubs using 5-min
epoxy glue. The glue must dry overnight before the casts are sputtercoated with
gold. We have found that the casts require extensive coating with gold to elimi-
nate problems of charging when viewing the casts with the scanning electron
microscope. In order to adequately coat the casts with gold, we initially
sputtercoat the casts in an upright position. We then tip the stubs on their sides
and rotate the stubs in one-third turns through three cycles of sputtercoating.
13. We have also used a continuous drip of warm PBS to warm the embryo and
restore a heartbeat. We prefer the humidified warm air, because it does not cause
a build-up of liquid at the surgical site. Also, the force of the drops of PBS falling
onto the embryo can damage younger embryos. We do keep a supply of warm
PBS at the work site and have the option of using it along with the humidified
warm air to revive the embryo.
14. It is helpful to have the assistant record the orientation of the umbilical vessels
(e.g., which vessel is rostral) because it can be difficult to distinguish them once
the vessels are cut and the blood drains out.
15. We have found it useful to add a very slight hint of pigment (red) to our casting
compound. The color aids in the visualization of the casting compound as it passes
through the cannula and the placenta. The amount of pressure used to infuse the
casting compound is critical. Infusion pressures that are too high can rupture the
delicate vessels within the placenta, causing a leakage of the casting compound
throughout. With pressure that is too low, the casting compound sets before the
vasculature has filled. Being able to track the casting compound as it moves
through the cannula and the placenta allows for greater control over the rate and
pressure at which the casting compound is infused. Pigment is available in red,
white, yellow, blue, and green (Polysciences, Inc., Warrington, PA).
16. An excessive amount of pigment in the casting compound can interfere with the
curing of the casts. Add the least amount of pigment possible to achieve the desired
effect. Preliminary trials mixing small amounts of the casting compound with
varying amounts of pigment are useful to determine the amount of pigment
needed and the effect of varying amounts of pigment on the curing of the casting
compound.
17. Much of the refinement of this technique involved perfecting the double-lumen
catheter system used to infuse the perfusate and casting compound in sequence
(Fig. 5). The double lumen eliminated the need to remove and reinsert cannulae;
we found this extremely difficult because of the viscous, sticky properties of the
casting compound. The system is comprised of a double-lumen catheter and a
glass cannula with a tapered-tip.
a. To create the cannula, a borosilicate glass capillary tube (0.75 mm inner
diameter [I.D.], 1.0 mm outer diameter [O.D.]; F.H.C., Inc., Bowdoinham,
ME) is drawn to a fine point with a pipet puller (Model P-97 Brown-Flaming
Micropipette Puller, Sutter Instrument Company, Novato, CA). The large end
of the glass cannula is attached to 28 cm of Tygon microbore tubing (0.04 in.
I.D., 0.07 in. O.D., Cole Parmer Instrument Company, Vernon Hills, IL), and
glued in place with 5-min epoxy glue. This tubing is referred to as the outer
tubing and is the tubing through which the casting compound flows. An 18-gauge
needle is inserted into the other end of the outer tubing and is glued in place with
5-min epoxy glue. A 2-cm length of Silastic medical grade tubing (0.062 in.
I.D., 0.125 in. O.D., Fisher Scientific Ltd., Ottawa, Ont.) covers the junction
between the glass cannula and the outer tubing and is glued in place with 5-min
epoxy glue. The Silastic tubing helps to strengthen the junction between the
glass cannula and the microbore tubing.
b. The inner tubing of the catheter system is comprised of 40.5 cm of PE 10
tubing (Intramedic®, Becton Dickinson and Company, Parsippany, NJ). This
is the tubing through which the perfusate flows. The inner tubing is threaded
through a double male leur lock adapter (Cobe Canada, Ltd., Scarborough,
ON, Canada) with approximately 3 mm of the PE tubing left sticking out one
end of the adapter. The tubing is glued in place by filling the lumen of the
adapter with Silastic Medical Adhesive-Silicone Type A (Dow Corning Cor-
poration, Midland, MI). The Silastic adhesive must cure overnight. The PE 10
tubing is then threaded through a clear, four-way stopcock with male leur lock
(Cobe Canada, Ltd., Scarborough, ON, Canada) with ports that are all open. The
double male leur lock adapter is attached to the female port on the stopcock.
c. To assemble the catheter, the long end of the PE 10 tubing is threaded through
the 18-gauge needle on the outer tubing and passed down through the lumen
of the outer tubing. It may be necessary to trim the length of the PE 10 tubing
so that the tip of the PE 10 tubing sits approx 1–1.5 cm from the tip of the
glass cannula. The hub of the 18-gauge needle is then attached to the male
port on the stopcock. The 10-cc perfusate syringe is attached to the double
male leur lock adapter using a female-to-female leur adapter (Cole Parmer
Instrument Company, Vernon Hills, IL). The 3-cc casting compound syringe
is attached to the free port on the single stopcock.
18. We have used two methods to cut the holes in the umbilical vessels. The first is to
tear holes in the vessels using a pair of No. 55 Dumont forceps. Alternatively,
holes can be cut in the vessels using a pair of very delicate Vannas or spring
scissors.
19. We have produced excellent placental casts by infusing the casting compound
either into the umbilical vein or the artery. However, the umbilical vein seems to
provide more reliable filling.
20. If a dried cast floats when returned to distilled water for storage, apply one to two
drops of 70% ethanol to the cast to reduce surface tension. We use a plastic sy-
ringe and a 23-gauge needle to drop the alcohol onto the cast. The cast will then
sink in water. Use a minimal amount of alcohol to avoid having the alcohol soften
the cast.
21. To view the casts using SEM, we mount the casts on SEM stubs using 5-min
epoxy glue. The glue must dry overnight before the casts are sputtercoated with
gold. We have found that the casts require extensive coating with gold to elimi-
nate problems of charging when viewing the casts with the scanning electron
microscope. In order to adequately coat the casts with gold, we initially
sputtercoat the casts in an upright position. We then tip the stubs on their sides
and rotate the stubs in one-third turns through three cycles of sputtercoating.
Acknowledgments
We thank Dr. Bradley Smith for his valuable advice on fetal perfusion tech-
niques, Dora Chan for assisting in the establishment of embryonic casting
methods in our laboratory, Yong Lu for his work dissecting casts, and Doug
Holmyard for his SEM expertise. This work was supported by a grant from the
Canadian Institutes of Health Research.
References
1. Adamson, S. L., Lu, Y., Whiteley, K. J., et al. (2002) Interactions between tropho-
blast cells and the maternal and fetal circulation in the mouse placenta. Dev. Biol.
250, 358–373.
2. Smith, B. R. (2000) Magnetic resonance imaging analysis of embryos, in Devel-
opmental Biology Protocols, Volume 1 (Tuan, R. S. and Lo, C. W., eds.). Humana,
Totowa, NJ: pp. 211–216.
3. Pfarrer, C., Winther, H., Leiser, R., and Dantzer, V. (1999) The development of
the endotheliochorial mink placenta: light microscopy and scanning electron mi-
croscopical morphometry of maternal vascular casts. Anat. Embryol. (Berl.) 199,
63–74.
4. Gannon, B. J. (1978) Vascular casting, in Principles and Techniques of Scanning
Electron Microscopy, Volume 6 (Hayat, M. A., ed.). Van Nostrand Reinold, New
York, NY: pp. 170–193.
27
Analysis of Fetal and Maternal Microvasculature
in Ruminant Placentomes by Corrosion Casting
Summary
Vascular corrosion casting is a useful tool for studying the vascular architecture of complex
organs. The synepitheliochorial placenta of ruminants is composed of two closed blood cir-
cuits, a fetal and a maternal one. The microvasculature of each circuit has the shape of the
corresponding cotyledon (villous trees) and caruncle (crypts). These two compartments inter-
digitate with each other in a complementary fashion. Understanding three-dimensional vascu-
lar arrangements is facilitated by scanning electron microscopy of vascular corrosion casts.
Methods to be used in the generation of vascular casts from fetal and maternal placentomal
blood vessels are described, with special emphasis on casting resins and corrosion using potas-
sium hydroxide. The procedure of splitting larger casts following gelatin embedding and freez-
ing is also presented.
Key Words: Placenta; ruminants; corrosion cast; microvasculature.
1. Introduction
The application of scanning electron microscopic analysis to microvascular
corrosion casting (1) has enormously widened the scope of morphological
blood vessel research. Novell and coworkers first applied this technique to the
avian lung (2). In the placenta, this method was introduced by MacDonald in
the epitheliochorial pig 1976 (3), then, it was modified by Leiser and cowork-
ers for many other species with different placental types in comparative pla-
cental research (4–6).
The placenta is a unique organ with maternal and fetal blood vessels devel-
oping in close proximity. The coordinated establishment of these vascular sys-
tems is essential for materno–fetal exchange (7). Three-dimensional
materno–fetal capillary interrelationships vary among species (4,6). Vascular
organization at the materno–fetal interface influences diffusion efficiency and
thus nutrient delivery to the developing fetus (8–10).
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
393
2. Materials
2.1. Anesthetics for Small Ruminants—Goat and Sheep
1. During the initial phases of the experimentation the animals are administered a
tranquilizer, Rompun® (2 mL, intramuscularly, Bayer, Leverkusen, Germany)
and a barbiturate (Narcoren®, Merial, Hallbergmoos, Germany, intravenously).
2. Barbiturates are used for prolonged anesthesia (20–30 mg/kg body mass) and
eventual euthanasia (50–60 mg/kg body mass) during perfusion of the placenta
(intravenously).
3. Methods
3.1. Preparation of the Animals
1. For each pregnant animal it is essential to have an accurate history, including
age, breed, feeding records, health status, as well as the exact gestational age,
which should be recorded as day and h of insemination and/or, later in the study,
by measuring the crown–rump length (CRL) of the fetus (13).
2. The pre-experimental treatment of animals is different for cows vs goats or sheep.
In cows, which are generally slaughtered for commercial purposes, the pregnant
uterus must be excised as soon as possible after killing of the animal. The uterus
is opened and depending on the stage of gestation the fetus may be delivered
alive or is euthanized. Goats and sheep are usually sacrificed exclusively for
experimental purposes. Initially, the animal is tranquilized and anesthetized
(see Note 2) and the uterus is left inside the body of the animal at the beginning
of the experiment. As buffer perfusion (discussed later) is initiated, the anesthe-
sia is increased, and finally the animal is euthanized by an intravenous overdose
with the same anesthetic, and the uterus (including the placenta) excised (dis-
cussed later).
area should not be too large, because injected fluids (buffers and liquid plastic)
may be lost in too big a volume of blood vessels. On the other hand, too small
of an area may favor extravasation of these solutions, caused by rupture of
vessel walls, due to high and/or uncontrolled pressure (see below) in the selected
area. The vascular casting of ruminant placentomes may be applied to maternal
blood vessels, fetal vasculature, or both maternal and fetal systems.
1. The method is initiated by perfusion with phosphate buffer. To do so, well visible
arteries (which are preferred to veins) with a caliber not less than 0.3 mm are
cannulated. The maternal arteries at the external side of uterus must be carefully
prepared.
2. The fetal vasculature is casted, after opening the uterus, through the umbilical
arteries or their distinct branches.
3. For combined materno–fetal casts overlapping areas must be perfused in parallel.
4. The vessels should be cannulated as close as possible to the selected area. The
selected areas are perfused with phosphate buffer (38°C) by syringe (20 mL) and
cannula using manual pressure (about 10 mL/min) for a short time (about 1 min
until “blanching”; discussed later), then, the buffer is changed to ice-cold (4°C)
phosphate buffer to retard postmortem degenerative changes of the vessel wall
and too slow polymerization of the instilled liquid plastic resin. Putting the whole
uterus on ice or into an ice-cold water bath can also enhance the cooling effect.
5. Successful perfusion is visible as “blanching” of the placentome, because the
buffer replaces the blood in placental areas, which are tributaries of the cannu-
lated vessel(s). Buffer perfusion of the maternal system may not be visible,
because of the highly anastomosed vasculature; therefore the selected area must
be delimited by clamps.
3.3. Instillation of Liquid Plastic
The instillation of liquid plastic into the buffer-perfused vascular areas of
ruminant placentomes may be conducted with two different resins, either
Batson/Sevriton (5) or Mercox. Both resins work well in vascular casting ex-
periments but require different protocols (14,15).
3.3.1. Batson/Sevriton Protocol
1. Batson No. 17 compound/Sevriton has a hydrophilic quality resulting in very
detailed casts, mirroring exactly the luminal side of the vessel wall by scanning
electron microscopy (including endothelial cell impressions, discussed later). Its
viscosity, however, is relatively high; therefore, it cannot be used with cannulas
below 0.7 mm diameter and an instillation rate of more than 5 mL/min by manual
pressure (see Note 5).
2. The different components of the resin are mixed immediately before use (see
Section 2).
3. The instillation of the liquid plastic takes up to several min for filling the buffer-
perfused placental area through the same arteries (veins) as used for the buffer
perfusion until the venous outflow consists of pure plastic. Instillation of this
plastic resin is limited to 3 to 5 min before polymerization of the plastic starts.
4. Because this period of time may be too short for a complete filling of the per-
fused vascular system, the plastic and all other materials (buffer), tools (syringes,
cannulas, scissors, clamps, knives) and the tissue itself (discussed earlier) must
be cooled down (4°C) before use. This procedure extends the time for instillation
up to approx 8 min.
state, all the little plastic branches are fixed and are then cut with a knife.
3. To achieve the smoothest fracture lines, the gelatin-embedded casts can be fur-
ther hardened in liquid nitrogen (N2) and cracked with hammer and knife. How-
ever, the disadvantage of this method is that it is not as easy to control (see Note 6).
4. After thawing, the gelatin is removed by a second corrosion procedure, which
follows the same regime like the first (see Subeading 3.4.).
5. Although the gelatin treatment facilitates cleaning, the casts, again, need a very
thorough and repeated washing in distilled water at room temperature, followed
by washes in 5% Extran solution, and distilled water again in order to have all
remnants of tissue removed from very dense vascular casts.
6. The cut or cracked casts are air-dried and suitable specimens are selected by
means of stereomicroscopy and mounted onto stubs using fast-drying conductive
adhesive carbon cement.
7. The specimens are dried again and stored in a dust-free dessicator until used.
8. Finally, the casts are sputter-coated with gold or platinum (30 nm) and examined
with a scanning electron microscope (see Note 7).
4. Notes
1. Air bubbles must be avoided. Air bubbles can enter the vascular system during
the buffer and resin perfusion, especially when cannulas are incorrectly inserted
into the vessels or when the resin is mixed or stirred too vigorously. Once air is
trapped inside the vascular system there is no way to remove the air. Air bubbles
cause incomplete formation of vascular casts. Vascular casts with air bubbles do
not represent a replica of the vessel and may be more susceptible to fracture or
damage. Air bubbles tend to be a greater problem when using Mercox.
2. The slaughter process and anesthesia require careful attention. Anesthesia during
in situ perfusion of the placenta in goats and sheep should be maintained as long
as possible, since the solutions are driven through the body with the help of the
heart. Animals should be euthanized at the time of infusion of the toxic com-
pounds (liquid plastic resin and fixatives; see Note 3).
Fig. 4. Appearance of casts with (A) and without (B) extravasations. (A) Corrosion
cast of bovine fetal cotyledonary villous tree (day 220 of pregnancy) where only the
villous tip shows the vasculature (lower left). All other parts have a sculpture-like
appearance resulting from extravasation of Batson resin. The complex ramification of
intermediate villi into terminal villi is clearly visible (arrows). (B) Ovine fetal cast
with Mercox-filling; day 140 (near term) of pregnancy. The stem vessels, fetal stem
artery (FSA), and vein (FSV), are usually found within the center of a vascular tree.
Few arterioles branch off the stem arteries (arrows) to enter the capillary system, where
it is easy to follow single vessels until they enter numerous venules (arrowheads),
which run towards the stem of the vascular tree in a parallel manner. The images in A
are from ref. 16, with permission; the images in B are from ref. 18, with permission.
3. Perfusion-fixation with formalin (2%) or with other fixatives may minimize post-
mortem degeneration of placental vessel walls and enhance their rigidity, which
allows casting of early fetal placental stages. Extravasation during casting with
Mercox may be decreased following fixation. Fixation prior to resin instillation
has its limiations. It takes time and increases handling stress of the delicate tis-
sue. Consequently, the most successful approach is often a short buffer perfu-
sion, quickly followed by the instillation of plastic.
4. In order to achieve good casting results, the size of the selected area should be
restricted to less than 10 × 10 cm (Figs. 4 and 5). The vasculature at the periphery
of the selected area must be adequately clamped. It is important that care is taken
Fig. 5. Bovine fetal BatsonR cast showing serially linked capillary loops of termi-
nal villi day 270 (near term) of pregnancy. Highly anastomosing capillaries in the
neck region (top area) of three terminal villi are serially bridged (dashed lines). A
fourth terminal villus is obviously still growing (arrow). Note the increase of the cap-
illary diameter towards the terminal loops, which form sinusoidal dilations (stars) at
the tips of the loops. The image is from ref. 12, with permission.
403
405
Superficial
Network/coiling Size/form capillary Materno–fetal
Classification of capillary of capillary structure interhemal
of placental type Architecture (vascular) system cross-section (roughness) distance
macroscopic microscopic
Fetal and Maternal Microvasculature
Multiple placentomes Maternal Caruncles Branched crypts Irregular-coarse- Irregular Smooth Highly variable
(Fig. 1) (Figs. 3A, 6A) (Fig. 3A) network (Fig. 2B,C) (Figs. 2B, 5) (Fig. 2C)
(Fig. 2B,C)
Epitheliochorial Fetal Cotyledons Branched Terminal Roundish Rough
(10,16) (Fig. 6B) Villi (Fig. 4) coilings approx 8 µm (Fig. 2B,C)
(Figs. 2B, (Figs. 2B
4B, 5) 4B, 5)
8/29/05, 11:24 AM
405
406 Leiser and Pfarrer
References
1. Aharinejad, S. H. and Lametschwandtner, A. (eds.) (1992) Microvascular Corro-
sion Casting in Scanning Electron Microscopy. Techniques and Applications.
Springer-Verlag, Wien.
2. Nowell, J., Pangborn, J., and Tyler, W. S. (1970) Scanning electron microscopy
of the avian lung. Scan. Electron Microsc. 1, 249–256.
3. MacDonald, A. A. (1976) Uterine vasculature of the pregnant pig: a scanning
electron microscope study. Anat. Rec. 184, 689–697.
4. Leiser, R. and Koob, B. (1992) Structural and functional aspects of placenta mi-
crovasculature studied from corrosion casts, in Scanning Electron Microscopy of
Vascular Casts: Methods and Applications (Motta, P. M., Murakami, T., and
Fujita, H., eds.). Kluwer, Boston: 261–277.
5. Leiser, R., Dantzer, V., and Kaufmann, P. (1989) Combined microcorrosion casts
of maternal and fetal vasculature. A new method of characterizing different pla-
cental types, in Developments in Ultrastructure of Reproduction (Motta, P. M.,
ed.). Alan R. Liss, New York: 421–433.
6. Leiser R., Pfarrer C., Abd-Elnaeim M., and Dantzer V. (1998) Feto-maternal an-
chorage in epitheliochorial and endotheliochorial placental types studied by his-
tology and microvascular corrosion casts. Troph. Res. 12, 21–39.
7. Moll, W. (1981) Theorie des plazentaren Transfers durch Diffusion, in Die
Plazenta des Menschen (Becker, V., Schiebler, T. H., Kubli, F., eds.). Thieme,
New York: 140–152.
8. Carter, A. M. (1975) Placental circulation, in Comparative Placentation (Steven,
D. H., ed.). Academic, New York: 108–160.
9. Faber, J. and Thornburg, K. (1983) Placental Physiology. Structure and Function
of Feto-Maternal Exchange. Raven, New York.
10. Leiser, R. and Kaufmann, P. (1994) Placental structure: in a comparative aspect.
Exp. Clin. Endocrinol. 102, 122–134.
11. Mossmann, H. W. (1987) Vertebrate Fetal Membranes. Macmillan, Basingstoke, UK.
12. Leiser, R., Krebs, C., Ebert, B., and Dantzer, V. (1997) Placental vascular corro-
sion cast studies: a comparison between ruminants and humans. Microsc. Res.
Tech. 38, 76–87.
13. Richter, J. and Goetze, R. (eds.) (1978) Tiergeburtshilfe. Parey, Berlin.
14. Abd-Elnaeim, M. M., Miglino, M. A., Pfarrer, C., and Leiser, R. (2003) Microvas-
cular architecture of the fetal cotyledons in water buffaloes (Bubalus bubalis) dur-
ing different stages of pregnancy. Ann. Anat. 185, 325–334.
15. Pfarrer, C., Ebert, B., Miglino, M. A., Klisch, K., and Leiser, R. (2001) The three-
dimensional feto-maternal vascular interrelationship during early bovine placen-
tal development: a scanning electron microscopical study. J. Anat. 198, 591–602.
16. Leiser, R., Krebs, C., Klisch, K., et al. (1997) Fetal villosity and microvasculature
of the bovine placentome in the second half of gestation. J. Anat. 191, 517–527.
17. Grosser, O. (1927) Frühentwicklung, Eihautbildung und Placentation des
Menschen und der Säugetiere, in Deutsche Frauenheilkunde, Geburtshilfe,
Gynäkologie und Nachbargebiete in Einzeldarstellungen (Jaschke, R. T., ed.).
Bergmann, Munich.
18. Krebs, C., Longo, L. D., and Leiser, R. (1997) Term ovine placental vasculature:
comparison of sea level and high altitude conditions by corrosion cast and
histomorphometry. Placenta 18, 43–51.
V
MOLECULAR ANALYSIS AND GENE TRANSFER
TECHNIQUES
28
Microarray Analysis of Trophoblast Cells
Summary
A complex repertoire of trophoblast gene products governs the multifaceted functions per-
formed by the placenta during the relatively short period of pregnancy. Cloning and sequencing
the human as well as other mammalian genomes allow investigators to gain better insight into
the function of trophoblast genes. Our ability to identify transcripts by their nucleotide sequences
and determine their expression patterns enables us to glean information on gene function.
Although the molecular principles underlying microarray are not new to biology, the high
throughput, low reaction volumes, fluorescent labeling, accurate detection, and robust analysis
software makes this approach most appealing to today’s researchers, when compared with stan-
dard filter blotting techniques. This chapter focuses on DNA microarray of the human placental
transcriptome as a means to identify alterations in gene expression in different physiological or
pathological conditions.
Key Words: Placenta; trophoblasts; oligonucleotide microarray; cDNA microarray;
transcriptome; RNA; variability; precision; normalization; replicates.
1. Introduction
Several techniques have been available for analysis of differential gene
expression. These include Northern analysis, S1 nuclease protection, differ-
ential display, subtraction hybridization, representational difference analysis
(RDA), library screens, and serial analysis of gene expression (SAGE). Over
the recent years, additional technologies, including microarray, have been
added to the arsenal of the investigator, allowing research into the presence of
a large number of transcripts in a tissue or a dynamic probe into quantitative
changes in expression between different tissues, normal or diseased tissue, or
among cells exposed to single or several conditions (1). Array technology has
recently improved to enable a greater number of elements per chip surface
area. Currently, the entire human transcriptome is represented on the surface
area of modern chips (2–6 cm2). Arrays are available for more than 100 organ-
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
411
isms, and arrays from one organism are currently used to examine expression
in phylogenetically related species.
DNA microarray is based on the fact that matrix-immobilized DNA sequences
bind their complementary transcripts in a highly sensitive and specific manner
(recently reviewed in refs. 2–5). Manufacturing approaches to DNA arrays
include either “on slide synthesis,” in which DNA sequences are assembled
directly on the array substrate, or delivery approach, in which pre-assembled
oligonucleotides or cDNAs are attached to the microarray. Most noncommer-
cial array producers utilize the second approach, as long as a contaminant-free
chamber with regulated temperature and humidity levels can be assured. Hybrid-
ization takes place between the glass-bound arrayed polynucleotide sequences
and fluorescently labeled cellular mRNA, or after RNA conversion into cDNA
or cRNA using in vitro transcription (IVT). After extensive washings, the rela-
tive abundance of every transcript from each of the two populations is imaged
with a digitally operated confocal-scanning microscope, and analyzed using
image analysis algorithms.
Expression profiling is based on one-dye or two-dye approaches (Fig. 1). In
the one-dye approach, each RNA sample is labeled with a single label (e.g.,
phycoerythrin), and signals from the two experiments are compared for deter-
mination of gene activation or repression. In the two-dye approach, each of the
two RNA populations is labeled with a different fluorescent dye, such as cya-
nine-3 and cyanine-5 (Cy3 and Cy5), applied to a single chip. Cy3 and Cy5
dyes are stable and easy to incorporate into the probe during reverse transcrip-
tion. In addition, these dyes emit easily separable spectra. A fluorescent two-
channel dye detector, coupled to photomultiplier tubes (PMT) or a charged-coupled
device (CCD), scans each array for emitted signals. The expression level of a
gene is determined by the ratio of Cy3/Cy5 signals. Detector elements are
designed to yield a clear signal from robotic imaging of the arrayed probes.
Sampling of signal-to-noise ratio defines the threshold above which a signal is
determined positive. All experiments are typically pseudo first-order reactions,
in which there is a large excess of the immobilized probe relative to the labeled
target, and thereby eliminating the problem of probe competition. Overall, dif-
ferences in signal intensity represent the amount of applied target mRNA, tar-
get labeling, hybridization efficiency, efficiency of fluorescent excitation and
emission, and detection efficiency. Signals are converted to an expression level,
which is saved in a tab-delimited format and can be imported into spreadsheets
for further analysis. Results are reported as relative changes in a set of ex-
pressed genes, defining one transcriptome as control.
Commonly used microarray technologies include oligonucleotide arrays and
cDNA arrays (1,6). In oligonucleotide array technology, short (15–25) or long
(50–120) nucleotides are attached to the glass matrix. Sequences can be designed
Fig. 1 (see companion CD for color version). Differential gene expression can be
determined using a one-dye or two-dye approach. Computerized analysis of signal
intensity between two arrays (one-dye) or of combined signals in one array (two-dye)
indicates differences in gene expression between two experimental paradigms.
digms. The validity of this assumption, however, has been questioned (11). An
alternative approach is adjustment to total mRNA expression, under the assump-
tion that the total mRNA in the cell is constant. However, divergent expression
levels among transcripts may increase the chance that global normalization
will result in false estimates.
Although the procedure described in this chapter is based on a one-dye sys-
tem, the use of a two-dye system introduces an additional source of bias. It has
been observed that different dyes can display differential efficiencies in label-
ing and detection, and are influenced by signal intensity (12–14). Such differ-
ences can be normalized based on the assumption that overall gene expression
is the same between experimental and control samples. A preferred method of
normalization utilizes locally weighted regression (LOESS), where local esti-
mates of mean ratios are found over small intervals of total signal intensity.
This process is repeated along a sliding scale of intensities (15,16).
Researchers seek to answer diverse questions with microarray experiments.
These questions commonly center on finding genes whose expression changes
from control to experimental paradigms, or exhibit an expression profile that
fits a certain pattern (e.g., time course or disease progression). A common
approach to identification of expression changes between two experiments
depends on an arbitrary fold change as a threshold for expression difference.
Although simple, this approach has no associated level of statistical confidence,
and depends on the assumption of a constant coefficient of variance (17). When
this assumption is violated (as typically happens in microarray data), the fre-
quency of false positive results increases dramatically. This is particularly true
for transcripts that exhibit low expression intensities, where fold differences
can be amplified by a denominator’s low expression intensity. Moreover, these
signals tend to be near noise level, which may mask expression differences.
Newer approaches to decrease such bias adjust fold-change threshold to signal
intensity (17–21).
The use of replicate paradigms allows investigators to estimate experimen-
tal variance (22). Methods that are based on a t-test or analysis of variance
(ANOVA) can assign a confidence level to each change in gene expression
(23,24). These effective approaches have been further developed to adjust for
intensity-specific variance (17–21), and even for variance that is specific for
each probe-set (21). With the use of a large set of replicates, one can estimate
the expected variances from these factors, and use them as reference values
(“correction factors”) in future experiments, when samples are compared with-
out replicates (21).
Identification of expression profile patterns can be achieved using cluster-
ing programs, which are based on the premise that similar expression changes
may imply similar functions. For example, when compared with all other genes
in the experiment, all genes that exhibit particular expression values are clus-
tered. Algorithms such as k-means and self-organizing maps cluster transcripts
by expression values based on a pre-defined number of expression patterns.
Hierarchical clustering defines “gene-tree” based on relative expression values
such that genes with the most similar expression values are clustered further
down on the tree (16,25). Techniques are available with which to utilize infor-
mation from array analysis in characterization of a new sample or paradigm.
Diverse methods can define a set of predictor genes, selected based on their
defined and discriminatory behavior in a known type of sample. This set of
predictor (signature) genes can be used to define an unknown sample or bio-
logical response.
Procedures presented in this chapter are based on prefabricated high-density
oligonucleotide arrays (Affymetrix) using a single dye (info can be found at
www.affymetrix.com). We therefore focus on sample preparation for array
hybridization and provide tips for data analysis. The experimental principles
provided here are applicable to other array platforms. It should be noted that
rapid progress in microarray technology is likely to impact many of the proce-
dural and technical notes presented in this chapter. The reader is therefore
advised to obtain platform-specific updates prior to performance of the experi-
ments. Additional research based on placental microarray has been published
(17,21,26,27).
2. Materials
1. Cell culture. Standard growth medium: Earl’s Medium 199 (M199), Ham’s/
Waymouth (H/W; composed of equal volumes of Ham’s F12 medium and
Waymouth medium), or Dulbecco’s modified Eagle’s medium containing 10%
fetal bovine serum (Hyclone, Logan, UT), 20 mM HEPES (Sigma, St. Louis,
MO), pH 7.4, 0.5 mM L-glutamine, penicillin (10 U/mL), streptomycin (10 µg/
mL), and Fungizone® (0.25 µg/mL).
2. RNA preparation and cleaning. Tri Reagent® (Molecular Research Center, Inc.,
Cincinnati, OH), chloroform (without isoamyl-alcohol or other additives), iso-
propanol, diethylpyrocarbonate (DEPC)-treated dH2O. RNA is cleaned using
QIAgen’s RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). It is noted that
β-mercaptoethanol must be added to Buffer RLT before use. β-Mercaptoethanol
is toxic, and should be dispensed in a fume hood with protective clothing. In
addition, Buffer RPE should be diluted in 4 vol of ethanol (96–100%).
3. cDNA synthesis and cleaning. SuperScript™ Choice System for cDNA Synthe-
sis from Invitrogen Life Technologies (Carlsbad, CA) and a T7T21 oligonucle-
otide primer (GenSet, La Jolla, CA). For clean up: Phase Lock Gel® Light
(1.5 mL Eppendorf/Brinkmann, Westbury, NY), Phenol:chloroform:isoamyl
alcohol (25:24:1) saturated with 10 mM Tris-HCL pH 8.0/1 mM ethylenediamine
tetraacetic acid (EDTA), NH4Ac, absolute ethanol and 80% ethanol.
3. Methods
3.1. Cell Culture or Tissues
Microarray analysis can be performed on transcriptomes derived from any
tissue or cell type. Analysis of placental tissues can be typically performed
using whole placental tissue samples (biopsies), placental explants, plated pri-
mary placental trophoblasts or trophoblast cell lines (see Note 2).
1. To prepare primary human cytotrophoblasts for our experiments, we typically
digest normal term human placentas using the trypsin-DNAse-Dispase/Percoll
method as described by Kliman (28), with modifications (29,30).
2. Cultures are routinely plated at a density of 350,000 cells/cm2 and maintained in
Earl’s M199 with additives as noted under Subheading 2.
3. To modulate trophoblast differentiation, we also culture cells in H/W, supple-
mented as described previously for M199 (31,32).
4. All cultures are maintained at 37°C, in a 5% CO2 atmosphere. Medium is changed
every 24 h.
10. The ethanol is removed, the RNA pellet air-dried, and then dissolved in 50–100 µL
of RNase-free (DEPC-treated) dH2O.
11. Finally, the RNA yield should be quantified. RNA yield and purity is determined
by spectrophotometry at 260 and 280 nm. A260/A280 ratios between 1.9 and 2.2
are acceptable. If lower, repeat cleanup may be needed (see Note 5). We also
recommend checking RNA quality by running 2 µL of each sample on 1% agar-
ose gel. There should be two distinct bands (28S and 18S), without smearing (see
Note 4).
12. It is essential to clean the RNA samples before cDNA synthesis (see Note 5). For
RNA cleanup, we use QIAgen’s RNeasy Mini Kit, following the manufacturer’s
protocol. Because the binding capacity of each RNeasy mini column is 100 µg of
RNA, we usually use 50–100 µg of RNA per column. At the end of the proce-
dure, we elute RNA with 30 µg RNase-free dH2O (see Note 6).
Fig. 3. An agarose gel analysis of purified in vitro transcription (IVT) product and
fragmented product. Fragment size (kb) is indicated in the margin.
before the wash and elution steps, and wait 5 min after adding water to the col-
umn for RNA elution, prior to centrifugation.
4. Note that the protocols from Affymetrix provide a formula for calculating the
relative amount of labeled cRNA yield, needed prior to array hybridization. The
difference between unpurified and purified RNA can be assessed by electrophore-
sis using a 1% agarose gel.
4. Notes
1. A GeneChip Sample Cleanup Module is available from Affymetrix, and may be
suitable for sample cleaning as an alternative to the described procedure.
2. Because biological variability is directly related to sample homogeneity, results
from homogenous samples exhibit lower variability, whereas results from tissue
biopsies (e.g., whole placental samples) exhibit greater variability, and therefore
require more replicates.
3. Although mRNA can be extracted and used for subsequent processing and analy-
sis, this is not necessary. Furthermore, it is more difficult to isolate mRNA, as all
precipitates are practically invisible. If needed, mRNA can be isolated using kits
such as Qiagen’s Oligotex mRNA Midi Kit (Cat. No. 70042). If mRNA is used,
amounts should be adjusted according to protocols from Affymetrix or other chip
manufacturers.
4. The placenta is a rich source of RNAse, necessitating careful isolation and puri-
fication procedures in order to avoid RNA degradation. RNA degradation can be
determined using 28S/18S rRNA signal ratio. If needed, capillary electrophore-
sis allows quantification of additional degradation species of RNA (33). Mini-
mizing differences in RNA degradation among the samples is essential for
comparison of transcript expression. This issue is particularly relevant for stud-
ies of apoptosis, where 28S rRNA is cleaved more rapidly than 18S (34). Note
that alternative RNA preparation protocols can also be used for extraction of
placental RNA.
5. The amount of RNA is critical for adequate microarray experiments. Low
amounts of RNA my result in signals at the lower limit of fluorescence detection,
with a low signal to noise ratio. At least 10 µg of RNA are needed for fluorescent
signal detection without amplification. Typically, the initial amount of RNA
needed for an experiment is 50 µg of total RNA or 2 µg of poly(A) mRNA.
Therefore, in studies using the human placenta, this is unlikely to be a limiting
factor. We usually extract 30–50 µg of RNA from 20 × 106 human term tropho-
blast cells or from 50 mg human placental tissue. When the amount of RNA is
insufficient, amplification using T7 polymerase, which exhibits linear amplifica-
tion (thereby sustaining relative RNA amounts), can be used. RNA purity is also
critical for signal reproducibility. Contaminants such as lipids, proteins, or sug-
ars can affect target hybridization to the slide surface. Microarray experiments
are based on the assumption that the level of RNA in the sample closely repre-
sents its relative amount in the tissue or cells.
6. To obtain a higher RNA concentration, a second elution step using the first eluate
can be performed. Although only a small amount of RNA will be used, it is diffi-
cult to obtain a high enough concentration of RNA if initial RNA quantity is less
then 50 µg. If the final concentration of RNA is too low, RNA can be precipitated
and resuspended in a smaller volume, but up to 50% of the product may be lost
during the process. The remaining RNA can be stored and used in other essays.
Acknowledgments
This research was supported by National Institutes of Health (NIH) R01
ES11597-01 and the Siteman Cancer Center GeneChip Core Facility, Wash-
ington University School of Medicine, St. Louis, MO, USA. We thank Elena
Sadovsky and Lori Rideout for technical assistance.
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29
Gene Expression Microarray Data Analysis of Decidual
and Placental Cell Differentiation
Summary
Gene expression analysis using DNA microarray approaches have provided new insights
into the physiology and pathophysiology of many biological processes. These include identifi-
cation of genetic programs and pathways that underlie cell and tissue differentiation and gene
expression programs responsive to genetic perturbations, drugs, toxins, and infectious agents.
In this chapter, we present methods for the analysis of microarray data using earlier investiga-
tions from our laboratory as examples of how gene expression patterns for cellular differentia-
tion may be detected and analyzed for biological significance and how regulated genes may be
classified into functional categories and pathways.
Key Words: Gene expression; clustering; microarray; decidualization; trophoblast; pla-
centa; differentiation.
1. Introduction
DNA microarray technology permits qualitative analysis of mRNA expres-
sion of multiple genes in a single specimen. Because large numbers of genes
can be assessed, microarray studies have provided considerable insights into
physiological processes such as cell proliferation, differentiation, apoptosis,
and malignant transformation. In addition, microarrays have provided insight
into the cellular responses to drug treatment, environmental toxins, and infec-
tious agents.
There are two main types of microarray technologies: the single-channel
array and the two-channel array. Two-channel arrays employ a reference RNA
in which the relative signal of a given gene is detected as the ratio of the signal
for the reference vs that of the sample. In contrast, single-channel arrays mea-
sure relative intensities of each gene per array and per RNA sample. Thus,
single-channel arrays require that each RNA (e.g., experimental or control
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
425
2. Materials
1. Microarray Suite/GeneChip Operating System (GCOS) software (Affymetrix,
Inc., Santa Clara, CA).
2. RMAExpress (written by Ben Bolstad, University of California, Berkeley) http:/
/stat-www.berkeley.edu/~bolstad/RMAExpress/RMAExpress.html
3. GeneSpring software (Silicon Genetics, Redwood City, CA) (see Note 1).
3. Methods
The general scheme for DNA microarray analysis is illustrated in Fig. 1.
Each of the individual steps of the analysis is described in greater detail below.
sion is then determined from the Affymetrix .cel files by either the Affymetrix
algorithm contained in MAS5 (which, among other things, subtracts the mis-
match signals) or the Robust Multi-array Analysis protocol (7) using the
RMAExpress program with default settings. The RMA program uses the per-
fect match data and not the mismatch data, but provides background adjust-
ment, quantile normalization, and then a summarization of gene-specific
relative signal strength. The probe set signals are then saved into a text file that
can be imported into GeneSpring. Using a series of dilution and calibration
experiments (8), we earlier observed that RMA reduces spurious signals and
eliminates false positive estimations of induced and repressed genes as com-
pared to signal strength estimated through MAS5.0.
3.2. Loading Data into GeneSpring
1. Importing data. GeneSpring recognizes the format of data files obtained from
most expression analysis programs. Although RMAExpress summary data is not
directly recognized by GeneSpring, a column editor permits a custom format to
specify gene identifier and signal columns in a tab-delimited text file.
2. Create experiment. Upon importing the data files, a new data set for each
microarray (referred to as new sample in GeneSpring) is created and saved. If
necessary, the new data set can be easily combined with another newly created
data set or an older data set using the GeneSpring sample manager that is part of
the software package.
into GeneSpring, RMA signal strength measures are first transformed from
log base 2 to linear values and then normalized to the median or mean of all
measurements for each gene across all samples or sometimes just to the con-
trol samples as a function of the experiment design.
b. Two-color experiment: In experiments that use two-channel microarray data,
a control is performed to account for dye-based gene labeling differences.
This is done by reversing the signal channel and control channel measure-
ments for selected samples and calculating a correction factor that shifts the
relative gene expression ratio as a function of which dye was used for which
sample. Then a combined per-Spot and per-Chip normalization is performed
with intensity-dependent (locally weighted regression [LOESS]) normaliza-
tion. To counter intensity-dependent labeling and hybridization differences, a
LOESS fit at each point is calculated and a LOESS curve is fit to the log-
intensity vs log-ratio plot to adjust the control value for each measurement.
3. Set up an experimental interpretation: Further data analysis is dependent on the
specific questions being addressed. For example, the numerical display mode
permits data within an experiment to be represented as ratio, log of ratio, or fold-
change. In log of ratio mode, normalized intensity values are plotted against a log
scale so that underexpressed and overexpressed genes are considered equally sig-
nificant. The display setting may also be customized by defining a parameter to
be continuous, noncontinuous, or not displayed. When a parameter is not dis-
played, samples with the same parameter values will be grouped together, which
can be important in the following statistical analysis techniques. Another option
in the interpretation setup is whether to turn on the cross-gene error model (see
Note 3).
the RMA algorithm compresses the expression range, a fold factor of 1.2, in our
experience, can indicate a significant change in expression level.
2. Statistical analysis. Statistical analysis (analysis of variation [ANOVA]) is a fil-
tering tool that can be applied to a gene list obtained from other filters such as
fold change. This filter compares mean expression levels between two or more
groups of samples (conditions) to detect subsets of genes that show statistically
significant differences in mean normalized expression levels. The interpretation
is important as it defines the data mode and the grouping of the samples. Both
one-way ANOVA and two-way ANOVA are permitted in GeneSpring. Compari-
sons can be performed with parametric or non-parametric methods at a specified
p-value cutoff, with or without multiple testing corrections.
Student’s t-test/ANOVA assumes variances to be equal while Welch t-test/
ANOVA assumes variances not equal across groups. The specific test to choose
depends on the variance across the data and practically the number of replicates
in the experiment. In our experience, Student’s t-test gives better results when
there are only a few replicates with the multiple testing correction option to con-
trol the false positive rate.
When testing the statistical significance of group comparisons for many genes,
a certain number of genes will pass the filter by chance alone and be considered
statistically significant. Multiple testing corrections can adjust the individual
p-value to account for this effect. The Benjamini and Hochberg test controls
the false discovery rate, defined as the proportion of genes expected to be identi-
fied by chance relative to the total number of genes called significant. However,
with too few replicates, the test itself may not have enough power to differentiate
false-positives or -negatives. That is, by applying multiple testing correction,
some potentially interesting genes could be incorrectly labeled false-positives
and removed because of a lack of statistical power. Figure 2A shows the differ-
ence in gene lists identified with different comparison options. Some clusters
identified statistically significant by Student’s t-test did not pass the Welch t-test,
although different expresison profiles are detected vissually. Figure 2B shows
the genes that passed the Student’s t-test but were not identified when using mul-
tiple testing correction. No obvious difference can be seen between the decidua
and trophoblast groups for these genes, many of which are false positives that
made the list by chance alone.
3. Combining filtering and statistical analysis. In earlier versions of GeneSpring, a
sequential maneuver was used to identify significance and magnitude of change
in expression of a set of genes between two conditions. With Bioscripts imple-
mented in the current release of GeneSpring V6.2, filtering and statistical analy-
sis can be combined and genes identified with one script by generating a volcano
plot (see Note 4). A volcano plot displays the negative log of p-values from a t-test
on one axis and the log2 of fold change between two conditions on the other axis
on the Scatter Plot view. Figure 3 shows a volcano plot that presents upregulated
(yellow) and downregulated (blue) genes that are also statistically significant in
one plot
Figure 2
Figure 3
Figure 4
4. Other analyses. GeneSpring is a rich source of analysis tools to assist in the iden-
tification of biologically meaningful expression data (see Note 5). One other op-
tion is to find genes with similar expression patterns to a selected gene generated
from the average of a group of genes. If a gene or group of genes is selected based
on expression level filtering and/or statistical tests, other genes whose expression
profiles are similar but did not pass the filters may be identified. Figure 4 shows
an example of genes selected because of similarities in expression pattern to a
specific gene.
3.5. Clustering
GeneSpring’s clustering algorithms are designed to form groups of genes or
conditions with similar expression patterns. GeneSpring supports a variety of
clustering methods—K-means, Gene Tree, Condition Tree, Self-Organizing
Map, and QT Clustering. Each method uses a series of different distance
metrics to define relative similarity, such as Pearson Correlation, Standard
Correlation, Distance, and others. These are useful tools to identify genes that
are potentially co-regulated as well as to reveal coordinated responses shared by
sets of samples to various experimental treatments. Figure 5A is an example of
Figure 5
gene tree clustering, where the closeness of the branches in the trees is a mea-
sure of the correlation of the genes’ expression. Clusters of genes (Fig. 5B–E)
that are co-regulated can be identified from the tree and further analyzed.
K-means clustering divides genes into groups with a high degree of similar-
ity based on their expression levels. In the time series experiment of
decidualization shown in Fig. 6, K-means clustering was used to identify 9
unique classes of genes that are upregulated or downregulated in a time-depen-
dent manner. Using a lower number of clusters resulted in groups with less
consistent patterns of expression, whereas using a higher number of clusters
resulted in groups that appeared to overlap with patterns of expression ob-
served in other groups.
3.6. Functional Classification
Genes can be categorized using shared attributes in the description of their
function or structure. This allows for genes to be grouped within common cat-
egories that can then be combined or contrasted to other categories. These cat-
egories include biological process, cellular component, and molecular function.
A useful approach to gene categorization is provided by the Simplified Gene
Ontology tool in GeneSpring using information stored in the annotations fields
of the genome features file. Combined with annotations retrieved by the
GeneSpider tool within GeneSpring, which updates gene annotations from
Unigene, LocusLink and Genbank based on Genbank accession numbers, we
Figure 6
have added annotations from Affymetrix annotation releases and Stanford pub-
lic database SOURCE (http://genome-www5.stanford.edu/) (see Note 6). The
Build Simplified Ontology tool groups genes hierarchically into biological cat-
egories (gene lists) based on the Gene Ontology Consortium Classifications
(http://geneontology.org/). GeneSpring’s ontology tool parses all of the anno-
tations in the genome and then assigns each gene to one or more ontology
groups. Additional gene categories can be constructed using selected annota-
tions in the program, such as chromosome location and pathways (see Sub-
heading 3.7.). The scripting environment of GeneSpring allows for automation
of the process of comparing a list of regulated genes in an experiment to each
of the gene categories. Many scripts are included in the BioScripts library
(BioScript Library 2.0\Biological Queries\Gene Ontology (GO) analysis)
released by SiliconGenetics.
3.7. Pathway Analysis
A pathway is a graphical representation of the interaction between gene
products in a biological system. User-drawn pathways as well as publicly avail-
able pathways such as Kyoto Encyclopedia of Genes and Genomes (KEGG,
http://www.genome.ad.jp/kegg/) can be imported to GeneSpring (see Note 7).
The expression change of the genes participating in a pathway can be viewed
on the graphical representation. This analysis can be very useful if you are
trying to identify a class of genes that are associated with a particular step or
regulatory element within a pathway. Androgen and estrogen metabolism path-
way, which is important in decidualization, is illustrated in Fig. 7.
3.8. Publication of Microarray Data-Based Experiments
A consistent procedure for the description and publication of microarray
data-based experiments is critical. It is important that a sufficiently detailed
description of the experiment and its analysis accompanies a microarray-based
publication so as to allow corroboration and re-analysis. It is also extremely
useful that there is the web-accessible release of primary microarray data. The
advantage of microarray data release is that it can permit others to corroborate
the authors’ interpretations as well as to permit additional questions to be posed
of the data set by different investigators. In order to accomplish this, the
Microarray Gene Expression Data Society (MGED) has produced a general
guideline document to aid authors in the presentation of relevant details that
can allow another investigator to understand the experiment and how it was set
up and analyzed using microarray technology. The guideline is called the Mini-
mal Information About Microarray Experiment (MIAME) checklist, and it is
currently available at the MGED website at http://www.mged.org/ as the
MIAME 2.0 document.
4. Notes
1. Other software such as Spotfire (Spotfire, Inc., Somerville, MA) and Genetraffic
(Iobion Informatics LLC, La Jolla, CA) are alternatives for analyzing microarray
data.
2. Quality control may be applied before analyzing the microarray data by checking
significant parameters in the report file generated from Affymetrix chip analysis.
Within GeneSpring software, the “All Samples” interpretation and clustering by
condition tree can also be used to check the quality of the data obtained from
microarray experiment.
Figure 7
436
3. The GeneSpring error model can be used to estimate either measurement varia-
tion or sample-to-sample variation. The estimates of these two components of
variation are used to estimate standard errors and compare mean expression lev-
els between experimental conditions. In case there are no replicates for a condi-
tion, statistical analysis can still be performed with the GeneSpring error model
turned on. However, using sufficient biological replicates is recommended in
microarray studies to obtain the most statistical power.
4. If multiple testing correction is not applied, the order of filtering and statistical
analysis is not important. However, using previously filtered gene lists in t-test/
ANOVA with multiple testing correction can result in a larger gene list due to
less false positives and smaller variance.
5. We only chose to cover the most basic and important tools that are frequently
used in our laboratory in this chapter. Many other tools implemented in
GeneSpring can be very useful in the process of identifying of significant genes.
6. The annotations for the ontology tool are regularly updated by SiliconGenetics.
Combining annotations from different public data sources provides the most com-
plete ontology analysis.
7. Stand-alone applications for pathway analysis with more functionality than
GeneSpring are available. We have used Ingenuity Pathways Analysis (Ingenu-
ity, Mountain View, CA).
Acknowledgments
We thank Cherie Kessler, Anoop Brar, and You-Hong Cheng for their con-
tributions to the DNA microarray studies cited in this chapter. Supported by
National Institutes of Health (NIH) grant HD-15201.
References
1. Yang, Y. H. and Speed, T. (2002) Design issues for cDNA microarray experi-
ments. Nat. Rev. Genet. 3, 579–588.
2. Brown, P. O. and Botstein, D. (1999) Exploring the new world of the genome
with DNA microarrays. Nat. Genet. 21, 33–37.
3. Cheng, Y. H., Aronow, B. J., Hossain, S., Trapnell, B., Kong, S., and
Handwerger, S. (2004) Critical role for transcription factor AP-2 in human tro-
phoblast differentiation. Physiol. Genomics 18, 99–107.
4. Brar, A. K., Handwerger, S., Kessler, C. A., and Aronow, B. J. (2001) Gene
induction and categorical reprogramming during in vitro human endometrial fi-
broblast decidualization. Physiol. Genomics 7, 135–148.
5. Aronow, B. J., Richardson, B. D., and Handwerger, S. (2001) Microarray analy-
sis of trophoblast differentiation: gene expression reprogramming in key gene
function categories. Physiol. Genomics 6, 105–116.
6. Handwerger, S. and Aronow, B. (2003) Dynamic changes in gene expression
during human trophoblast differentiation. Recent Prog. Horm. Res. 58, 263–281.
7. Irizarry, R. A., Bolstad, B. M., Collin, F., Cope, L. M., Hobbs, B., and Speed, T.
P. (2003) Summaries of Affymetrix GeneChip probe level data. Nucleic Acids
Res. 31, e15.
8. Freudenberg, J., Kong, S., Jegga, A., et al. Experimental design, data analysis,
and quality evaluation approaches to maximize cross-platform and cross-proto-
col inter-comparability of gene expression microarray data, Manuscript in
preparation.
9. Sartor, M. A., Medvedovic, M., and Aronow, B. J. (2003) in A Beginner’s Guide
to Microarrays (Blalock, E. M., ed), Technical Books, San Diego: pp. 151–178.
30
Assays to Determine Allelic Usage of Gene Expression
in the Placenta
Paul B. Vrana
Summary
Mammalian placentas express a large number of so-called imprinted genes. Imprinting refers
to mono-allelic or biased expression based on which parent contributed the allele. Many of these
imprinted loci encode factors involved in growth and cell-cycle regulation, as well as maternal
behavior. In general, paternally expressed genes tend to enhance growth, whereas maternally
expressed genes inhibit growth. Methods are described for developing assays to test the allelic
usage of a gene. The approaches described are best utilized within a system where multiple
strains are available, and it is possible to perform reciprocal crosses. Only polymerase chain
reaction-based methods are examined in any detail.
Key Words: Imprinting; mono-allelic gene expression; Peromyscus; placenta; polymerase
chain reaction.
1. Introduction
The last 20 yr have revealed that a number of autosomal mammalian genes
are primarily expressed from one allele. Typically, this mono-allelic expres-
sion is dependent on which parent contributed the allele. This phenomenon is
termed genomic imprinting (1). The mammalian placenta and extraembryonic
tissues are particularly rich in the expression of these imprinted genes, many of
which regulate growth (2,3). Also, much of the X chromosome is expressed
from the maternal allele exclusively in extraembryonic tissues in various mam-
malian species including rodents and cows. Whether or not this placental X
imprinting occurs in any human trophoblast cells is controversial, but it is clear
that the entire human placenta is not subject to this phenomenon (4). However,
skewing of X-inactivation, such that one allele is preferentially expressed occurs
regularly. Such skewing is often associated with spontaneous abortions (5).
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
439
2. Materials
1. Acrylamide and agarose electrophoresis apparatuses and reagents.
2. Dissection equipment and tissue homogenizer.
3. DNA sequencing reagents and apparatus or access to sequencing service.
4. Oligonucleotide primer synthesis and design programs.
5. Polymerase chain reaction (PCR) thermocycler.
6. Gel documentation system with quantification software and/or phosphorimager.
7. PCR reagents.
8. Reagents for RNA isolation, e.g., lithium chloride/urea or kit.
9. Reverse-transcription (RT) and PCR and associated reagents and/or kit.
10. Restriction enzymes and associated reagents.
11. Phosphorimager and/or gel documentation system with quantification software.
3. Methods
The methods described outline various strategies and considerations in devel-
oping allelic expression assays using placental tissues. Standard molecular biol-
ogy procedures, such as RNA purification and cDNA synthesis, are not covered
in any detail.
3.1. General Considerations
Because imprinting may be tissue-specific, if the gene effect is thought to be
associated with the placenta, one must examine placental RNA for allelic expres-
sion status (see Note 1). A parental polymorphism is generally necessary to
determine parent-of-origin allelic expression, although fluorescence in situ
hybridization (FISH) techniques may also be used to determine mono-alleic
from the two samples, using amounts that have yielded roughly equivalent PCR
products.
The assumption one makes when performing these allelic usage assays is
that the ratio of one allele to the other on the gel will reflect the actual usage on
the two parental chromosomes. Another danger is that as the PCR goes beyond
the linear amplification range, the lower expressed allele may “catch-up” with
the dominant allele. This again appears more likely with more rare transcripts.
First, the assay should be repeated several times, and preferably in both direc-
tions—that is, if one has alleles A and B, once in which A is the maternal
allele, and once in which B is the maternal allele. (This is a desirable experi-
ment regardless; there may be leakier imprinting in one direction.)
One way to prevent the “catching-up” phenomenon is to lower the number of
PCR cycles to approx 18–20, so that one is well within the linear range. To
visualize the PCR products after such low cycle numbers, one can add P32-
labeled CTP to the PCR cocktail. One can then use a phosphorimager to quan-
tify the ratio of one allele to the other. I should note here that one could use this
trick for quantification if a gel documentation system with software for ethidium
bromide-stained gels is not available. Often, imprinting will not be an all-or-
none situation, but rather a pronounced bias. It is advisable to quantify the ratio
between alleles on several different experiments to ascertain consistency.
bases from the polymorphism. This distance is to ensure that the polymorphic
base is read by the sequencing polymerase. Occasionally, PCR primers need to
be modified for sequencing purposes, particularly if one is not using a PCR-
based sequencing reaction. The mixing control in this case may be the genomic
DNA of a heterozygote, or an artificial mix. For certain model organisms such
as humans and mice, for which there are SNP databases, microarray based
allelic usage assays will likely emerge in the next decade.
Another method often used by human geneticists is the single-stranded con-
formation polymorphism (SSCP) assay. This method utilizes the fact that
single-stranded DNA of the same length, but different base composition, will
migrate differently in certain matrices. Even SNPs can result in mobility dif-
ferences between alleles. After the RT-PCR product is denatured, it is run on
long gels to allow ample room for separation. Typically these gels are either
acyrlamide or a matrix designed for this procedure termed mutation detection
enhancement gel solution. Protocols are readily available on the Internet (e.g.
http://www.cambrex.com/Content/Documents/Bioscience/MDE(18153).pdf).
The SSCP assay is often done “hot,” which adds the cost of the P32 nucleotide
and radioactive waste disposal.
Another option is the single nucleotide primer extension (SNuPE) assay
(20). In this method, an oligonucleotide primer is designed to bind immedi-
ately adjacent to a known SNP. This primer is then extended by a polymerase
in the presence of a complementary radio-labeled nucleotide to one of the two
bases present at the polymorphic site. The amount of each of the two DNTPs
incorporated from the extension of the RT-PCR of the message is then quanti-
fied after gel electrophoresis. Alternatively, fluorescently labeled DNTPs may
be combined with capillary electrophoresis.
The future of PCR-based allele-specific assays will likely see more allele-
specific real-time reverse-transcribed (or AS-RT2, as we refer to it) PCR. This
technique is a variation of the common “TaqMan” strategy. Normally, a probe
is hybridized to nascent RT-PCR products. This probe has both a reporter fluo-
rescent dye molecule and a quencher. The quencher acts only when it is in
close proximity to the dye. As the product is used as a template for the next
round of amplification, the quencher is detached, and the reporter fluoresces.
The amount of the fluorescence is then quantified. Applied Bio Systems (ABI
PRISM) machines (Foster City, CA) are among the most commonly used for
such detection. An excellent diagram of the process is viewable at http://
www.med.unc.edu/anclinic/Tm.htm.
In this allele-specific TaqMan reaction, oligonucletide probes (with differ-
ent fluorescent labels attached) must be designed so that they bind to the
amplicons in an allele specific fashion. AS- RT2 would seem optimal in that
the levels of message coming from each allele can be quantified. This tech-
nique has only recently been implemented (21), but its use will likely grow
rapidly.
3.6. Non-PCR-Based Methods of Detecting Differences in Allelic Usage
There are at least two other methods that may be used for allelic usage. The
first allele-specific FISH is certainly the most visually compelling method in
that one can see the expression of the actual chromosome. This technique
requires a polymorphism sufficient to prevent the probe (which, in the case
of FISH, tends to be large) from hybridizing to both alleles. Again, this is tech-
nically quite demanding.
Another method that can show allele usage and is quantitative is the RNase
protection assay (RPA). The RPA method uses a labeled probe, which binds to
a region of the message of interest, which then “protects” it from subsequently
added RNases. The mix is then run out on an acyrlamide gel, dried, and the
intensity of the band can be used to estimate the amount of message originally
present. The difficulty with this assay comes with developing two probes, each
of which are allele-specific, and this explains why, with certain exceptions
(e.g., there is an excellent H19 allele-specific assay in house mice [22]), it is
not used often in imprinting studies. General RPA protocols can be found on
the web (http://micro.nwfsc.noaa.gov/protocols/) and kits are available from
such companies as Ambion Inc. (Austin, TX).
3.7. Assessing Allelic Usage in Cases of Gene “Knockouts”
and Uncharacterized Genes
Two cases where typical allelic usage assays may not be possible are when
(1) the gene itself has been localized, but not identified, and (2) the gene in
question has been deleted, either by targeted mutation or via other means.
Imprinting of genes in these cases may be inferred depending on the inherit-
ance patterns. For example, the imprinting of Igf2 was discovered when it was
the subject of one of the first mouse targeted mutations. While the resulting
growth retardation phenotype was expected with reduction in Igf2 expression,
the genetics of the phenotype were not. When the mutation was passed mater-
nally, there was no effect on growth, while paternal inheritance gave a dra-
matic growth reduction (23). The homozygous null animals correspondingly
showed a phenotype equivalent to those offspring who had only received the
null mutation paternally.
Passing a mutant or novel allele both paternally and maternally is certainly
prudent in cases where the gene may not be directly assayed. The results of a
genetic test may not be as clear-cut as was the Igf2 situation. That is, although
one parental inheritance will likely yield more severe phenotypes than the
other, neither will result in wild-type offspring. Such a result is likely due to
the fact that many imprinted genes do not exhibit complete (all or none)
monoallelic expression. Such genetic tests are sometimes termed “functional
imprinting assays” in that they demonstrate the effects and/or consequences of
an imprinted gene. Assaying allelic usage of the endogenous gene, however, is
still necessary to confirm the effect.
3.8. Summary Flow Chart of Developing a PCR-Based Assay
to Determine Allelic Usage
Typically, this process starts with a notion that a gene might be imprinted
because of it is located near an imprinted domain, it is a candidate for involve-
ment in a parent-of-origin effect, or it is expressed in a tissue where imprinted
genes are known to be very abundant (e.g., the placenta).
1. Work out a protocol for amplifying the gene of interest.
2. Amplify as large a piece of the mRNA as possible from individuals likely to have
genetic differences (e.g., different strains). If a length difference is apparent, you
are set!
3. Digest these RT-PCR products with various restriction enzymes, particularly
those with nonspecific or four-base recognition sequences.
4. If no RFLP is apparent in several tries, ascertain an expressed polymorphism in
the gene of interest through (re-)sequencing multiple individuals.
5. Analyze the sequences for polymorphisms, and potential RFLPs.
6. If potential RFLPs are present, test them with the appropriate enzymes.
7. If there are no potential RFLPs, but multiple SNPs, one may try pairs of allele-
specific primers.
8. Alternatively, if there is only one SNP, or good primer designs are not possible,
sequence the RT-PCR products.
9. If there appears to be mono-allelic (or biased) expression, carefully test for inher-
ent amplification bias by performing a “mixing experiment.”
10. Quantify the ratio of one allele to the other.
4. Notes
1. One potential problem with these assays that is unique to the placenta is the poten-
tial contamination with maternal tissues. In the case of paternally expressed genes,
this is of course not an issue. One easy way to rule out maternal contamination is
to ascertain whether the gene of interest is expressed in (pregnant) uterine tissue.
If the gene is not expressed here, it makes a contamination artifact unlikely
(unless the gene is only expressed in uterine tissue in close contact with fetal
tissue, or is expressed in blood). Showing that the gene is imprinted in other
tissues (e.g., yolk sac or embryonic tissue) also strengthens a placental imprint-
ing argument. Finally, if the gene is only expressed/imprinted in the placenta,
one must be particularly careful about dissection. Use as late-stage a placenta as
possible, and utilize tissue farthest from the maternal surface.
2. Most of the potential pitfalls in the process have been previously described. How-
ever, perhaps the most common and (most difficult) problem in developing these
assays is finding a polymorphism. If the gene of interest has many small exons, it
is advisable to sequence across as many as possible by sequencing RT-PCR products
rather than utilizing genomic DNA. Again, focusing on the untranslated regions is
advisable. If no polymorphisms are present, one should examine alternative
strains or individuals. Indeed, if one has this luxury (i.e., multiple strains or very
polymorphic population), start the process by examining 3–4 strains/individuals.
3. Another problem that may occur is co-amplification of a closely related gene.
One must then redesign primers such that only the gene of interest is amplified,
or choose a time point when the contaminating family member is not expressed.
This situation can be vexing, as we realized while characterizing an imprinted
placental lactogen (PL), and then testing other PLs for imprinting (10).
References
1. Tilghman, S. (1999) The sins of the fathers and mothers: genomic imprinting in
mammalian development. Cell 96, 185–193.
2. Haig, D. (1996) Placental hormones, genomic imprinting and maternal-fetal
comunication. J. Evol. Biol. 9, 357–380.
3. Moore, T. and Reik, W. (1996) Genetic conflict in early development: parental
imprinting in normal and abnormal growth. Rev. Reprod. 1, 73–77.
4. Zeng, S. M. and Yankowitz, J. (2003) X-inactivation patterns in human embry-
onic and extra-embryonic tissues. Placenta 24, 270–275.
5. Sangha, K. K., Stephenson M. D., Brown, C. J., and Robinson, W. P. (1999) Ex-
tremely skewed X-chromosome inactivation is increased in women with recurrent
spontaneous abortion. Am. J. Human Genet. 65, 913–917.
6. Constancia, M., Hemberger, M., Hughes, J., et al. (2002) Placental-specific IGF-
II is a major modulator of placental and fetal growth. Nature 417, 945–948.
7. Wake, N., Takagi, N., and Sasaki, M. (1978) Androgenesis as a cause of hydatidi-
form mole. J. Natl. Cancer Inst. 60, 51–57.
8. Judson, H., Hayward, B. E., Sheridan, E., and Bonthron, D. T. (2002) A global
disorder of imprinting in the human female germ line. Nature 416, 539–542.
9. Rogers, J. F. and Dawson, W. D. (1970) Foetal and placental size in a Peromyscus
species cross. J. Reprod. Fertil. 21, 255–262.
10. Vrana, P. B., Matteson, P. G., Schmidt, J. V., et al. (2001) Genomic imprinting of
a placental lactogen gene in Peromyscus. Dev. Genes Evol. 211, 523–532.
11. Vrana, P., Guan, X.-J., Ingram, R. S., and Tilghman, S. M. (1998) Genomic imprint-
ing is disrupted in interspecific Peromyscus hybrids. Nature Genet. 20, 362–365.
12. Moglabey, Y. B., Kircheisen, R., Seoud, M., El Mogharbel, N., Van den Veyver,
I., and Slim, R. (1999) Genetic mapping of a maternal locus responsible for famil-
ial hydatidiform moles. Human Mol. Genet. 8, 667–671.
13. Vrana, P., Fossella, J. A., Matteson, P., del Rio, T., O’Neill, M. J., and Tilghman,
S. M. (2000) Genetic and epigenetic incompatibilities underlie hybrid dysgenesis
in Peromyscus. Nat. Genet. 25, 120–124.
31
Adenoviral-Mediated Gene Delivery to Trophoblast
Cells
Summary
This chapter focuses on technology for construction of recombinant adenoviruses contain-
ing reporter genes under the control of putative regulatory regions of the human (h)CYP19
(aromatase) gene, as well as expression vectors. These recombinant adenoviruses have been
used in primary cultures of human placental cells to characterize regulatory regions of the
hCYP19 gene and to analyze the function of transcription factors on hCYP19 expression and on
trophoblast differentiation.
Key Words: Trophoblast; recombinant adenoviruses; CYP19 gene; placenta; aromatase.
1. Introduction
Cytotrophoblast proliferation and differentiation to syncytiotrophoblast is
key to implantation and human placental development. As cytotrophoblasts
mature, they stop dividing and spontaneously fuse to form the terminally dif-
ferentiated syncytiotrophoblast layer that functions in gas and nutrient exchange
and in biosynthesis of steroid and polypeptide hormones (1). In previous studies
using trophoblast cells from human mid-gestation placenta in primary culture,
we observed that differentiation of cytotrophoblasts to syncytiotrophoblast was
associated with a marked induction of aromatase activity and of CYP19
(aromatase P450) gene expression (2). In humans, aromatase P450—the key
regulatory enzyme in estrogen biosynthesis—is expressed in a number of tis-
sues, including ovary and testis, brain, adipose stromal cells, and the syncy-
tiotrophoblast cells of the placenta (3). CYP19 gene expression in these tissues
is driven by tissue-specific promoters upstream of tissue-specific alternative
first exons, which encode the 5'-untranslated regions of CYP19 mRNA tran-
scripts. These alternative first exons, which are located from approx 110 to
From: Methods in Molecular Medicine, Vol. 121: Placenta and Trophoblast: Methods and Protocols, Vol. 1
Edited by: M. J. Soares and J. S. Hunt © Humana Press Inc., Totowa, NJ
451
approx 100,000 bp upstream of the CYP19 translation initiation site in exon II,
are alternatively spliced onto a common site just upstream of the translation
start site in exon II so that the protein encoded in each of these tissues is identi-
cal. In placenta, the majority of the CYP19 mRNA transcripts contain sequences
encoded by exon I.1, which lies approx 100,000 bp upstream of the start site of
translation in exon II (4). To analyze the genomic regions and response ele-
ments that mediate syncytiotrophoblast-specific hCYP19 gene expression, we
have transfected human trophoblast cells in primary culture with fusion genes
containing various amounts of DNA upstream of placenta-specific exon 1.1
(with or without mutations of putative response elements), fused to the human
growth hormone (hGH) structural gene, as a reporter. In some cases, expres-
sion vectors containing transcription factors that play a potential regulatory
role in hCYP19 gene expression and trophoblast differentiation are co-trans-
fected. Primary cultures of human trophoblast cells are highly resistant to stan-
dard gene transfection methods, such as DEAE-dextran, calcium phosphate,
lipofection, and electroporation. To circumvent this barrier, we have incorpo-
rated the fusion genes and expression vectors of interest into the genome of a
replication-defective human adenovirus and introduced these DNA constructs
into the human placental cells by infection (2,5). In our earlier studies, the
recombinant adenoviral particles were produced by in vivo recombination in
293 cells (2,5). In vivo recombination in mammalian cells is relatively ineffi-
cient and time-consuming. Therefore, more recently, we have utilized a highly
efficacious method developed by Vogelstein and colleagues (6) in which the in
vivo recombination step is carried out in bacteria rather than in mammalian
cells (7,8). The recombinant adenoviral plasmids are then transfected into 293
cells for production of recombinant adenoviral particles, which are titered to
ascertain the concentration of infectious viral particles (multiplicity of infec-
tion [MOI]) and used to transfer gene constructs of interest into the placental
cells in primary monolayer culture by infection. In experiments in which an
expression vector containing a transcription factor or other putative regulatory
factor is introduced into the placental cells, a control adenovirus containing the
gene for bacterial β-galactosidase under control of the human cytomegalovirus
(hCMV) promoter is used to infect a parallel set of dishes at the same MOI to
control for nonspecific effects of the adenoviral infection. In this chapter, we
describe our methods for isolation and primary culture of human trophoblast
cells, preparation of recombinant adenoviral particles, and their use for intro-
duction of gene constructs into the primary cultures of trophoblast cells by
infection.
2. Materials
2.1. Generation of Recombinant Adenoviruses
1. Shuttle vectors: pShuttle, pShuttle-CMV, pAdTrack, and pAdTrack-CMV
(kindly provided by Dr. Bert Vogelstein, Johns Hopkins Oncology Center, Balti-
more, MD; also available from American Type Culture Collection [ATCC,
Manassas, VA] and from Stratagene [La Jolla, CA]).
2. Adenoviral plasmids: pAdEasy-1 and pAdEasy-2 (kindly provided by Dr. Vogelstein;
also available from ATCC and from Stratagene).
3. Escherichia coli BJ5183 (kindly provided by Dr. Vogelstein), E. coli DH5α
(Invitrogen, Carlsbad, CA; also available from ATCC and from Stratagene).
4. Restriction enzymes Pme I and Pac I (New England Biolabs Inc., Beverly, MA).
5. Ampicillin and kanamycin (Sigma, St. Louis, MO).
6. Luria-Bertani (LB) medium (BD Biosciences, Franklin Lakes, NJ).
7. 10% glycerol (Fisher Scientific, Fair Lawn, NJ).
8. Bio-Rad Gene Pulser electroporator (Bio-Rad Laboratories, Hercules, CA).
9. LB/kanamycin plates.
10. Promega Miniprep kits (Promega Corporation, Madison, WI).
11. SuperFect transfection reagent (Qiagen Inc., Valencia, CA) or other transfection
reagent.
12. Phosphate-buffered saline (PBS).
13. Hank’s balanced salt solution (HBSS), pH 7.4 (GIBCO, Grand Island, NY).
14. 293 cells (ATCC, Manassas, VA) are cultured in Dulbecco’s modified Eagle’s
nedium (DMEM; Mediatech, Inc. , Herndon, VA).
15. Overlay Agarose (BioWhittaker Molecular Applications, Rockland, MD), a ster-
ile solution of melted 1% agarose in 25 mM HEPES, pH 7.4 (50°C).
16. 2X DMEM (GIBCO, Grand Island, NY) is used for titration of recombinant
adenoviruses.
17. Neutral red (Sigma, St. Louis, MO).
3. Methods
The methods described below outline: (1) modified adenoviral generation
method diagrammed schematically in Fig. 1; and (2) isolation and infection of
trophoblast cells.
3.1. Generation of Recombinant Adenoviruses
3.1.1. Preparation of Electrocompetent Bacterial Cells
1. Inoculate a fresh colony of BJ5183 into 10 mL LB medium. Shake the cells over-
night at 37°C.
2. Add 1 mL of cells into 1000 mL of LB medium in a 5-L flask. Grow for 4 to 5 h
at 37°C, until A550 is approx 0.8.
3. Collect cells in two 500-mL conical centrifuge bottles and incubate on ice for 10 min
to 1 h (the longer the cells are incubated the higher the competency).
4. Centrifuge at 2600g at 4°C for 10 min to pellet cells.
5. Resuspend the cell pellet in 1000 mL of sterilized, ice-cold 10% glycerol.
6. Centrifuge and pellet the cell suspension at 2500g for 30 min.
7. Repeat steps 5 and 6.
8. Pour off most of the supernatant; gently pipet off the remaining supernatant, leav-
ing about 20 mL. Resuspend the cells in the remaining supernatant and transfer
the cell suspension to a 50-mL tube. Spin at 2600g for 10 min, and pipet off all
but 5 mL of the supernatant.
9. Resuspend the cell pellet in the remaining supernatant. Aliquot in 50-µL aliquots
and store at –80°C.
Fig. 1. (opposite page) Schematic outline of the AdEasy system. The gene of inter-
est is first cloned into a shuttle vector, e.g., pAdTrack-CMV. The resultant recombi-
nant plasmid is linearized by digesting with restriction endonuclease Pme I, and
subsequently cotransformed into Escherichia Coli BJ5183 cells with an adenoviral
backbone plasmid, e.g., pAdEasy-1. Recombinants are selected for kanamycin resis-
tance, and recombination is confirmed by restriction endonuclease analyses. Finally,
the linearized recombinant plasmid is transfected into adenovirus packaging cell lines,
e.g., 911 or 293 cells. Recombinant adenoviruses typically are generated within 7–10 d.
The ‘’left arm’’ and ‘’right arm’’ represent the regions mediating homologous recombi-
nation between the shuttle vector and the adenoviral backbone vector. A,
polyadenylation site; Bm, BamHI; RI, EcoRI; LITR, left-hand ITR and packaging
signal; RITR, right-hand ITR; Sp, Spe I. (Reprinted from ref. 6, Copyright 1998
National Academy of Sciences, USA).
Fig. 2. Shuttle vectors and adenoviral plasmids. (Reprinted from ref. 6, Copyright
1998 National Academy of Sciences, USA).
2. The insert is sequenced using appropriate primers. Two microliters of the recom-
binant miniprep DNA is re-transformed into DH5α (or a comparable plasmid
propagation strain). Plasmids are then purified for adenoviral production in 293
cells.
(70%–5%).
4. The gradients are centrifuged at 1200g for 20 min at room temperature, and cells
in the middle layer (density 1.045–1.062) are collected, washed, and counted.
5. The cells are then resuspended in DMEM supplemented with 10% FBS and 1.2%
antibiotic/antimycotic solution and plated at a density of 2 × 106 cells per dish in
35-mm culture dishes or 15 × 106 cells per dish in 100-mm dishes.
4. Notes
1. In the generation of recombinant adenoviruses, Pme I and Pac I are used to lin-
earize the final constructs for transformation and transfection. Therefore, use of
inserts containing these sites will be problematical.
2. It is critical that recombinant adenoviral plasmids be generated in bacteria using
electroporation rather than other methods of transformation.
3. All the constructs (including recombinant adenoviral plasmids) are resistant to
kanamycin (NOT ampicilin) except pAdEasy-1 and pAdEasy-2.
4. It is essential that the cytotrophoblasts be exposed to recombinant adenovirus
within 1 h of plating, because the cells become resistant to adenoviral infection
upon differentiation to syncytiotrophoblast (10).
5. It is always important to infect parallel cultures with a “control” recombinant
Acknowledgments
We thank Vickey Chau and Jo Smith for isolation of cytotrophoblast cells.
Research using these techniques was supported by National Institutes of Health
(NIH) R01 DK-31206.
References
1. Ringler, G. E. and Strauss, J. F., III (1990) In vitro systems for the study of human
placental endocrine function. Endocr. Rev. 11, 105–123.
2. Kamat, A., Alcorn, J. L., Kunczt, C., and Mendelson, C. R. (1998) Characteriza-
tion of the regulatory regions of the human aromatase (P450arom) gene involved
in placenta-specific expression. Mol. Endocrinol. 12, 1764–1777.
3. Kamat, A., Hinshelwood, M. M., Murry, B. A., and Mendelson, C. R. (2002)
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Index
463
464 Index
fibronectin, 41, 42, 46-50, 53, 324, 327 derivation of trophoblast cell lines,
binding assay, 48, 53 220–226
FISH, fluorescence in situ hybridization, embryonic stem cells, 5, 189–201
447 endometrial cells, 79–83
fixation, tissue, 318, 319, 328, 338, 342 primary trophoblast cell culture,
Flk-1, 243 203–216
flow cytometry, 128, 141–143, 197–200 3β hydroxysteroid dehydrogenase
FluroNanogold, 353, 359–361, 363, 364 (3βHSD), 164, 302
17β hydroxysteroid dehydrogenase
G (17βHSD), 113, 114, 116–120
β-galactosidase staining, 231, 236–238,
252, 253, 268 I
gap junction, 149–152 Id-1, 164
GATA, 200 image analysis, 47–51, 53, 361, 363
GB25, 339 immortalization, cell, 79–83
Gcm1, 145, 172, 200, 286 immunocytochemistry, 5, 115–117,
gene delivery, 231, 233–236, 451–461 303, 307, 320, 321, 325–333,
GeneSpring, 428, 433 338–348
glycogen cells, 159, 160, 298 protocol
glycosylated cell adhesion molecule-1 bovine placentome, 325–333
(GlyCAM-1), 89 human
goat, vascular corrosion casting, embryonic stem cells, 197
393–406 placenta, 337–348
marmoset uterus, 115–117
H ruminant placenta, 320, 322
HAI-1 (hepatocyte growth factor immunoelectron microscopy,
activator inhibitor type 1), 351–367
206, 339 immunofluorescence, 328, 329, 363
HAND1, 164, 286 immunomagnetic cell isolation, 205,
hemochorial, placentation, 4 207–210, 213–215
heparan sulfate, 49 implantation, 4, 9–34, 35–53, 101–109,
heparin, 126–128, 153 315–322, 323–333
Hertz, Roy, 229 analysis, baboon, 101–109
HFE (hemochromatosis protein), 339 induction of delayed implantation,
histotroph, 86, 89 14, 15, 22–24
HLA, 206, 207 in vitro, 35–53
HLA-G, 200, 223, 224 procedures, mouse, 9–34
Hoechst 33342, 128, 184 morphological analysis
Hofbauer cell, 338 bovine, 323–333
human, 5, 79–83, 189–201, 203–216, ruminant, 315–322
220–226, 229–238, 451–461 imprinting, genomic, 6, 439–448
adenoviral-mediated gene delivery, Indian hedgehog, 145
451–461 infection, adenoviral gene delivery,
choriocarcinoma cell culture, 229–238 451–461
Index 467
Joan S. Hunt
University Distinguished Professor, Vice Chancellor for Research, Department of Anatomy and Cell Biology,
University of Kansas Medical Center, Kansas City, KS
Placenta research has progressed rapidly over the past several decades by taking advantage of
technical advances, such as microarray analysis, reverse transcriptase polymerase chain reaction,
protein analysis, and in situ hybridization. In Placenta and Trophoblast: Methods and Protocols,
Volumes 1 & 2, internationally recognized investigators describe cutting-edge laboratory techniques
for the study of the trophoblast and placental biology. The techniques presented range from
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differentiation, and function. Volume 1 provides readily reproducible protocols for studying embryo–
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Comprehensive and state-of-the-art, Placenta and Trophoblast: Methods and Protocols, Volumes
1 & 2 provide researchers a firm foundation for successful cellular and molecular analysis of the
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Features
• Readily reproducible methods for • New ides for investigating gene imprint-
studying the trophoblast and placental ing and gene transfer via viral vectors
biology • Step-by-step instructions to ensure
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• Strategies for the isolation and culture shooting and avoiding known pitfalls
of trophoblast cells
• Expert guidance on the molecular and
cellular analysis of the placental
phenotype