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Stem Cell Niche
Stem Cell Niche
Stem Cell
Niche
Methods and Protocols
METHODS IN M O L E C U L A R B I O LO G Y ™
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
Edited by
Kursad Turksen
Sprott Centre for Stem Cell Research, Regenerative Medicine Program,
Ottawa Hospital Research Institute, Toronto, ON, Canada
Editor
Kursad Turksen
Sprott Centre for Stem Cell Research
Regenerative Medicine Program
Ottawa Hospital Research Institute
Toronto, ON, Canada
The idea that there was a specialized microenvironment or “niche” regulating hemopoietic
stem cell function was first proposed by Ray Schofield over three decades ago. As interest
in stem cell biology has exploded over the last 10 years, so too has the interest in the stem
cell niche. This explosion of interest and, more recently, cellular-molecular-biochemical
characterization of not only the hemopoietic stem cell niche but the niches for other stem
cells was the driving force for putting together a volume of protocols for investigating stem
cell niches. As always, it is not possible to collect all the different protocols in one volume;
however, I have attempted to select a subset of representative protocols that hopefully will
provide both a flavor of the field and hopefully stimulate new approaches and methodolo-
gies by those interested in the stem cell niche.
The protocols gathered here are faithful to the mission statement of the Methods in
Molecular Biology series: They are well established and described in an easy-to-follow step-
by-step fashion so as to be valuable for not only experts but also novices in the stem cell
field. That goal is achieved because of the generosity of the contributors who have carefully
described their protocols in this volume, and I am grateful for their efforts.
My thanks as well go to Dr. John Walker, the Editor in Chief of the Methods in
Molecular Biology series, for giving me the opportunity to create this volume and for sup-
porting me along the way.
I am also grateful to Patrick Marton, the Editor of Methods in Molecular Biology and
Springer’s Protocol series, for his continuous support from idea to completion of this
volume.
Finally, I would like to thank Tamara Cabrero for her outstanding editorial work during
the production of this volume.
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
vii
viii Contents
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Contributors
ix
x Contributors
Abstract
Stem cells have the ability to switch between proliferative (self-renewal) and differentiation modes. The
Drosophila germarium is a well-established in vivo model for the study of communication between stem cells
and their niche. One commonly used technique for such study is immunostaining that allows examination of
protein localization at a fixed time point. This chapter provides a detailed protocol for immunofluorescence
staining of Drosophila ovaries. This protocol has been optimized to enable explicit visualization of the niche
structure, as well as to maximize the degree of multiplexing for protein labeling and detection.
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_1, © Springer Science+Business Media, LLC 2013
1
2 Lichao Luo et al.
2 Materials
2.2 PBS pH 7.4 Dissolve 7.6 g NaCl, 0.99 g Na2HPO4, and 0.41 g NaH2PO4·H2O
(1,000 ml) in 950 ml sterile water. Adjust the pH value to 7.4 using 10 M
NaOH (prepared in Subheading 2.1). Finally, bring the volume to
1,000 ml with sterile water.
2.5 1 M HEPES Weigh 238.30 g HEPES and dissolve it in 900 ml sterile water. Use
10 M NaOH (prepared in Subheading 2.1) to adjust the pH to
7.5. Finally, bring the volume to 1,000 ml with sterile water.
2.6 Fixative Buffer Mix 700 μl PBS, 200 μl 20 % PFA, and 100 μl 1 M HEPES (see
Subheading 2.5) (see Notes 2 and 3).
Fig. 1 The needle used for dissection. Black arrow shows the tip of wolframium
wire that has been sharpened by electrolysis
2.8 Needle Insert a wolframium wire through the hole of a needle attached to a
syringe and secure one end of the wire with the plunger. The length
of the wire extending out of the needle can be adjusted by moving
the plunger. Sharpen the tip of the wire by electrolysis (Fig. 1).
3 Methods
3.1 Fatten the Flies 1. Collect newly eclosed flies (within 24 h after eclosure) into
vials of fresh food with a dough of yeast paste and transfer
them daily into new vials with freshly prepared yeast paste (see
Note 4).
3.2 Dissecting All steps are performed at room temperature unless otherwise
and Staining stated. Steps 6–14 are carried out on a nutating mixer (see Note 5).
Take special care to ensure that the samples do not dry up all
the time.
1. Prepare a petri dish filled with PBS.
2. Anesthetize the flies on a carbon dioxide flowbed.
3. Use a forceps to hold the thorax of the anesthetized fly and
move it into the dish.
4. While grabbing the fly by one forceps, use another forceps to
tear the skin of the dorsal abdomen until the ovaries are visible
4 Lichao Luo et al.
under the microscope. Remove all other parts of the fly body
(see Note 6, Fig. 2).
5. Repeat steps 3 and 4 until enough ovaries are collected and
immediately transfer the ovaries into a microfuge tube contain-
ing fixative buffer (see Note 7).
6. Fix the ovaries for 20 min (see Note 8).
7. Remove all fixative, and wash the ovaries in PBT for 5 min.
Repeat this washing step for another three times (see Note 9).
8. Remove PBT and block the ovaries in 3 % BSA for 30 min.
9. Remove the blocking buffer and add the primary antibodies
diluted in 3 % BSA.
10. To label the niche and stem cells, incubate the sample for 2–4 h
with the following primary antibodies: (a) mouse monoclonal
anti-α-Spectrin [3A9, 1:100, Developmental Studies
Hybridoma Bank(DSHB)] and (b) anti-Lamin C (LC28.26,
1:40, DSHB) (see Notes 10–12).
11. Remove the primary antibodies, and wash the sample using
PBT for 5 min. Repeat this step for another three times.
12. Add secondary antibodies diluted in 3 % BSA and allow incu-
bation for 2–4 h in the dark (see Notes 13–15).
13. Remove the secondary antibodies, and wash the sample with
PBT three times. Each wash should last for at least 5 min.
14. Remove PBT and incubate the ovaries with DNA-staining dye,
such as TO-PRO-3 Iodide (1:5,000–1:10,000, Invitrogen) or
Hoechst (1:5,000–1:10,000, Invitrogen), dissolved in PBT,
for 20–30 min.
15. Remove the dye completely and add one drop of mounting
medium (Vector Laboratories, Inc.) into the tube such that the
ovaries are completely submerged. The fluorescence can be
stably maintained at −20 °C for 2 weeks.
3.3 Mounting and 16. Cut the end of a yellow pipette tip and use it to transfer the
Result Presentation stained ovaries onto a clean glass slide with minimal volume of
mounting medium (see Note 16).
Immunostaining of Germline Stem Cells and the Niche in Drosophila Ovaries 5
Fig. 3 The process of removing muscle sheath. (a) The anterior structure of the germarium (yellow arrow) is
hidden from view before removal of the muscle sheath (white arrow). (b) After removing the muscle sheath
(white arrow), the niche structure is revealed. Yellow arrow indicates the stack of TF cells
Fig. 4 Examples of fluorescence-labeled adult ovaries. The nuclei are stained by To-pro3 (Invitrogen). (a) Anti-
LamC (red, DSHB) strongly labels TF cells and CCs (white arrowheads). Anti-α-Spectrin (red, DSHB) marks
spectrosome in GSCs and cystblast (white arrows) and fusome in differentiated germ cells (yellow arrows).
Anti-Vasa (green) labels germline cells. ECs are pointed out by yellow arrowheads. (b) Anti-LamC (green) out-
lines TF cells and CCs (white arrowheads); anti-α-Spectrin (green) labels spectrosome in GSCs and cystblast
(white arrow) and fusome in differentiated germ cells (yellow arrows). Anti-pSMAD1-5 (red, Cell Signaling)
labels GSCs. Scale bar: 10 μm
17. Place a needle at the posterior part of the ovary to hold it. Use
another needle to dissociate it into individual ovariole.
18. Place one needle onto a late-stage egg chamber to hold the
ovariole stably while using another needle to peel off the mus-
cle sheath by sliding the needle along the slit between the mus-
cle sheath and the ovariole (Fig. 3) (see Note 17).
19. Place a cover glass directly over the sample. Now the sample is
ready to be examined under the fluorescence microscope
(Fig. 4).
4 Notes
Acknowledgments
References
1. Kirilly D, Xie T (2007) The Drosophila ovary: nizes encapsulation by somatic support cells.
an active stem cell community. Cell Res Development 129:4523–4534
17:15–25 9. Liu M, Lim TM, Cai Y (2010) The Drosophila
2. Lin H (2002) The stem-cell niche theory: les- female germline stem cell lineage acts to spa-
sons from flies. Nat Rev Genet 3:931–940 tially restrict DPP function within the niche.
3. Losick VP, Morris LX, Fox DT, Spradling A Sci Signal 3:ra57
(2011) Drosophila stem cell niches: a decade 10. Eliazer S, Shalaby NA, Buszczak M (2011)
of discovery suggests a unified view of stem Loss of lysine-specific demethylase 1 nonauto-
cell regulation. Dev Cell 21:159–171 nomously causes stem cell tumors in the
4. Xie T, Spradling AC (2000) A niche maintain- Drosophila ovary. Proc Natl Acad Sci USA
ing germ line stem cells in the Drosophila 108:7064–7069
ovary. Science 290:328–330 11. Streit P, Reubi JC (1977) A new and sensitive
5. Xie T, Spradling AC (1998) Decapentaplegic staining method for axonally transported
is essential for the maintenance and division of horseradish peroxidase (HRP) in the pigeon
germline stem cells in the Drosophila ovary. visual system. Brain Res 126:530–537
Cell 94:251–260 12. Goetsch JB, Reynolds PM, Bunting H
6. Song X, Wong MD, Kawase E, Xi R, Ding BC, (1952) Modification of Gomori method for
McCarthy JJ, Xie T (2004) Bmp signals from alkaline and acid phosphatase avoiding arti-
niche cells directly repress transcription of a fact staining of nucleus. Proc Soc Exp Biol
differentiation-promoting gene, bag of mar- Med 80:71–75
bles, in germline stem cells in the Drosophila 13. Watanabe S, Kimura Y, Honda M, Sasaki J
ovary. Development 131:1353–1364 (1994) Selective staining of the superficial cells
7. Song X, Xie T (2002) DE-cadherin-mediated of mouse urinary bladder epithelium by
cell adhesion is essential for maintaining horseradish peroxidase (HRP). J Electron
somatic stem cells in the Drosophila ovary. Microsc (Tokyo) 43:119–121
Proc Natl Acad Sci USA 99:14813–14818 14. de Cuevas M, Spradling AC (1998)
8. Schulz C, Wood CG, Jones DL, Tazuke SI, Morphogenesis of the Drosophila fusome and
Fuller MT (2002) Signaling from germ cells its implications for oocyte specification.
mediated by the rhomboid homolog stet orga- Development 125:2781–2789
Chapter 2
Abstract
Stem cells have an enormous capacity of self-renewal, as well as the ability to differentiate into specialized
cell types. Proper control of these two properties of stem cells is crucial for animal development, growth
control, and reproduction. Germline stem cells (GSCs) are a self-renewing population of germ cells, which
generate haploid gametes (sperms or oocyte) that transmit genetic information from generation to genera-
tion. In Drosophila testis and ovary, GSCs are anchored around the niche cells. The cap cells cluster in
females and hub cells in males act as a niche to control GSC behavior. With highly sophisticated genetic
techniques in Drosophila, tremendous progress has been made in understanding the interactions between
stem cells and niches at cellular and molecular levels. Here, we provide details of genetic, immunofluores-
cence labeling, and in situ hybridization techniques in identification and characterization of stem cells in
Drosophila male and female germline niches.
1 Introduction
In recent years, the stem cell field has opened a new venue in
regenerative and reproductive medicine. Stem cells have an enor-
mous ability to self-renew as well as produce diverse types of dif-
ferentiated cells [1–4]. Stem cells provide an opportunity to dissect
the cellular and molecular mechanisms controlling embryonic
development, cellular differentiation, and organ maintenance and
also have great potential in developing novel cell-based therapies.
In order for stem cells to function properly, a tight balance between
proliferation and differentiation should be maintained because
over-proliferation of stem cells results in tumor formation [5],
while under-proliferation results in loss of stem cell population,
which results in an inability to form the specific tissue or organ
[1, 6]. A large body of research suggests that stem cells are regu-
lated by specific microenvironments, known as niches, which is a
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_2, © Springer Science+Business Media, LLC 2013
9
10 Shree Ram Singh et al.
a Spermatogonia b c DAPI
Cyst cells
GSCs
CPCs
Hub
cells
VASA
Sox100B
Gonialblast
Stage 15
b-gal Arm g h i
1B1 Dapi *
Fig. 1 Immunostaining and in situ hybridization of Drosophila testes. (a) Schematic diagram highlights the tip
of the testes, which usually contain five to nine GSCs; only two GSCs are shown in this diagram, surrounded
by about twice as many cyst progenitor cells (CPCs). Both GSCs and CPCs anchor around the hub cells, which
act as niche cells. The testis proliferation center consists of hub, GSCs, CPCs, gonialblasts, and 2- to 16-cell
spermatogonia. (b) Wild-type stage 15 embryonic testes stained with anti-Vasa (red ) to mark the germline and
anti-Sox100B (green) representing the male- specific somatic gonadal precursor (msSGP) cells. (c) Wild-type
testes stained with DAPI. (d) Wild-type testis stained with anti-Arm (green) to mark the hub cells, 1B1 to mark
the spectrosomes and fusome (green), and anti-Vasa (red) marks all germ cells including GSCs. (e) Wild-type
testis stained with anti-BamC to mark the spematogonial cells (white color positive cells inside black dotted
lines) and anti-FasII to mark hub cells (black arrow ). (f) Wild-type testis stain with anti-Zfh-1 (red) to mark cyst
stem cells. (g) Wild type 6 days after heat shock GSC clones highlighted by dotted lines. Testis stained with
anti-β-galactosidase (red ), anti-Arm and anti1b1 (green). (h) Wild-type testis with Gef26 mRNA expression.
(j) Wild-type testis with upd mRNA expression (red ) using Fast red FISH. Dapi marks DNA (blue) in (d, f, g).
Scale bars: 20 µm (b); 10 µm (d-G, I)
12 Shree Ram Singh et al.
CC
TF
IGS CB
f g h
FSC
FC
GFP
DE-Cad DAPI
VASA DAPI
Fig. 2 Immunostaining and in situ hybridization of Drosophila ovary. (a) Wild-type ovary stained with DAPI.
(b) Schematic diagram highlights the tip of the ovary, which contains 2–3 GSCs and types of cells, depicted in
the figure. (c) Wild-type ovary stained with anti-Arm (green) to mark the cap cells, anti-1B1 (green) to mark the
fusome and spectrosomes (green), as well as niche cells and anti-Vasa (red) mark all germ cells including
GSCs. (d) hh-Gal4 UAS-GFP line stained with anti-GFP (green) marks the terminal filament cells and cap cells.
(e) Wild-type ovary stained with anti-Decad (green) and anti-Vasa (red). (f) Wild-type ovary stained with anti-
BamC to and anti-FasIII. (g) ) Wild-type ovary with Gef26 mRNA expression. (h) Wild type 6 days after heat
shock somatic stem cells clones (GFP-green), Dapi marks DNA (red). Dapi marks DNA (blue) in (c, d, f). Scale
bars: 50 µm (c); 20 µm (d); 10 µm (e-h)
2 Materials
2.2 Isolation 1. 3–5-day-old male and female flies of control (Oregon-R) and
of Testis and Ovary transgenic lines.
2. Dissecting solution (Drosophila Ringer’s solution): 130 mM
NaCl, 4.7 mM KCl, 1.9 mM CaCl2, and 10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
pH 6.9. Dissolve 7.5 g NaCl, 0.35 g KCl, 0.21 g CaCl2, and
2.38 g HEPES in approximately 1 l distilled water and stir to
dissolve. Adjust to pH 7.2 with 1 N HCl and make the final
volume of 1 l with distilled water. Store the dissecting solution
in a glass bottle at 4 °C or it can be stored at room temperature
for a short time.
3. Drosophila anesthesia CO2 station.
4. Drosophila CO2 fly pads.
5. Paint brush.
6. Dissecting tweezers.
7. Glass microslides.
8. Plastic dropper.
9. Kimwipes.
10. Dissecting microscope.
11. Ice.
12. 70 % (v/v) ethanol.
13. Pipet (20, 200, 1,000 μl).
14. Pipet tips (200, 1,000 μl).
3 Methods
3.2 Generation 1. CPC clones can be generated using the MARCM system [61].
of Somatic Clones 2. Cross the c587-Gal4.UAS-2XEYFP/FM7; FRT40A-tub-Gal80/Cyo
virgin females with males of FRT40A-w+/Cyo; +/TM3, Sb,
hs-Flp.
3. Heat-shock 3–5-day-old males and females carrying a tub-
Gal80 transgene in trans to the mutant-bearing chromosome
for 1 h at 37 °C for 2 days separated by 8–12 h of interval on
each heat shock.
18 Shree Ram Singh et al.
3.3 Isolation 1. Collect adult males and females after 3–5 days of emergence
of Testis and Ovary under CO2 station.
2. Place a slide under dissecting microscope and put drops of dis-
secting solution on the slide.
3. Take the males and females using tweezers. Put one tweezers
at the thorax region and with other tweezers take out the ter-
minalia and isolate the ovaries and testes.
4. Transfer the dissected testis and ovary into separate tubes con-
taining dissecting solution in ice.
5. For ovary, it is better to dissociate ovaries into ovarioles and
then fragment each ovariole into pieces with fine tungsten nee-
dles before putting in dissecting solution or fixation solution.
13. Using the above protocols, many diverse types of antibodies can
be tested to study male and female GSCs. For references and
details of the antibodies, see refs. [9–60].
3.6 In Situ 1. Probes can be prepared using SP6/T7DIG RNA labeling kit
Hybridization (Roche) following the manufacturer’s instructions.
(See Note 5) 2. Dissect the 3–5-day-old males and females as mentioned in
Subheading 3.3.
3. Fix the tissues for 30 min in 4 % paraformaldehyde in HEPES
buffer at room temperature.
4. Wash the tissues three times for 5 min each in 1× PBX.
5. Incubate the tissues in 50 μg/ml proteinase K for 5 min
(see Note 6). Stop the reaction with 2 mg/ml glycine for 2 min
and wash the tissues two times for 5 min each with 1× PPX.
20 Shree Ram Singh et al.
6. Fix and wash the tissues again if using step 5. If not, wash the
tissues for 10 min in 1:1 ratio of 1× PBX:hybridization
buffer.
7. Pre-hybridize the tissues for about 1 h at 65 °C in a water bath
(for RNA probes) in hybridization buffer. It is advised to pre-
heat the hybridization buffer.
8. Denature the probe by heating at 70 °C for 10 min in a water
bath, and then rapidly cooling in ice.
9. Then heat the hybridization buffer at 65 °C in a water bath,
mix the probe (in a ratio 1:50 probe:hybridization buffer), and
put on tissue.
10. Hybridize overnight at 65 °C in a water bath. Make sure to
have moist chamber where you can place the tube. No need to
shake.
11. Next day, after hybridization, wash the tissues six times for
30 min each in hybridization buffer in a water bath at
65 °C.
12. Then wash for 15 min in 4:1 ratio hybridization buffer:1× PBX
at room temperature.
13. Then wash for 15 min in 3:2 ratio hybridization buffer:1× PBX
at room temperature.
14. Then wash for 15 min in 2:3 ratio hybridization buffer:1× PBX
at room temperature.
15. Then wash for 15 min in 1:4 ratio hybridization buffer:1× PBX
at room temperature.
16. Then wash two times for 15 min each in 1× PBX at room
temperature.
17. For nonfluorescent staining with NBT and X-phosphate, incu-
bate the tissue overnight in a 1:2,000 dilution of alkaline
phosphatase-conjugated anti-DIG antibody in 1× PBX at 4 °C;
then three times, 15 min each, with 1× PBX; and after that,
three times for 15 min each with NMTT. Prepare the color
reaction by adding 4.5 μl NBT and 3.5 μl X-phosphate in 1 ml
NMTT. Incubate the tissue for 10–30 min depending on the
color reaction; once you see the color changing in the tissue by
looking at the dissecting microscope, it is better to stop the
reaction by putting 1× PBX. Put in 50 % glycerol for 1 h and
tissue can be put in 90 % glycerol overnight. It is better to
mount in 90 % glycerol. Image can be taken using the micro-
scope with bright-field and DIC capabilities.
18. For fluorescent in situ hybridization (FISH), we used Fast
Red staining procedure by following the manufacturer’s
instructions. Follow the above method from steps 1 to 17,
and then incubate tissues in 2 % blocking solution. Add pri-
mary antibody anti-DIG, incubate the tissues at 37 °C for
Genetic, Immunofluorescence Labeling, and In Situ Hybridization Techniques… 21
4 Notes
Acknowledgments
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Chapter 3
Abstract
The germaria of the fruit fly Drosophila melanogaster present an excellent model to study germline stem
cell–niche interactions. Two to three adult stem cells are surrounded by a number of somatic cells that
form the niche. Here we describe how Drosophilae germaria can be dissected and specifically immuno-
stained to allow for identification and analysis of both the adult stem cells and their somatic niche cells.
Key words Drosophila, Germarium, Ovary, Adult stem cells, Stem cell niche, Germline, Ovarian
soma, Immunostaining
1 Introduction
Adult stem cells usually reside in the stem cell niche, a unique
physiological microenvironment that helps stem cells to carry on
self-renewing divisions throughout the lifetime of an organism.
The niche includes cellular and noncellular elements that can be
divided into one of the two main mechanistic types—physical con-
tacts and diffusible factors [1]. Close contacts include tight junc-
tions, adherens junctions, gap junctions, the Notch signaling
pathway, the basement membrane, and extracellular matrix pro-
teins. Diffusible factors, which are secreted by niche cells and travel
over varying distances to keep stem cell identity, often affect tran-
scription. Stem cells must be anchored to the niche through cell–
cell interactions so that they will stay both close to niche factors
that specify self-renewal and far from differentiation stimuli.
Presently the existence of a stem cell niche has been demon-
strated for mammalian adult stem cells in the hematopoietic, epi-
dermal, neural, and intestinal systems. However, the stem cell
niches involved in maintenance of adult mammalian tissues and
particularly their role in cancer development remain complex,
poorly defined, and difficult to study in vivo [2].
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_3, © Springer Science+Business Media, LLC 2013
25
26 Annekatrin König and Halyna R. Shcherbata
Fig. 1 Scheme of larval ovary and adult germarium. (a) The primordial germ cells (PGC) that can be identified
by their characteristic spherical spectrosomes (SS spherical skeletal organelles) are intermingled with somatic
cells (IC, intermingled cells). Stacks of the terminal filament (TF) cells have already formed in late L3 larvae.
Cap cells (CpCs) are forming in late L3 larvae through the early pupal stages at the base of TFs. Two popula-
tions of somatic cells (APC, apical cells, BC, basal cells) are also found in the larval ovary. (b) In the adult
ovaries, the individual ovarioles with the germaria are separated by peritoneal sheath. The germline stem cells
(GSCs) are positioned at the anterior of the germarium and directly attached to cap cells. Upon asymmetric
division, the stem cells give rise to another stem cell and a differentiating daughter, the cystoblast (CB). The
cystoblast divides four more times with incomplete cytokinesis, forming the cyst. During that process, the
spherical spectrosomes of the GSCs elongate and branch to form the fusome (Fu). The terminal filament cells
are in close proximity to the cap cells, but have a more oval shape. The GSCs are furthermore in contact with
another type of somatic cells that presents an important component of the niche: the escort cells (EC). Follicle
cells (FC) that are produced by follicle stem cells (FSC) encapsulate the developing egg. Anterior is to the left
Fig. 2 Pre-adult ovary and adult germaria. (a) In the late larval and early pupal ovaries, terminal filament stacks
become visible (outlined in yellow ). The PGCs (arrow ), that can be identified by their spherical SSs, are not
separated yet into individual ovarioles and intermingled with somatic cells. (b) Adult ovaries consist of several
germaria, each containing 2–3 GSCs, that can be not only identified by their characteristic Adducin-marked
SSs but also stained with the stem cell marker pMad. Directly attached to the stem cells are several somatic
cells that are forming the stem cell niche: the CpCs can be marked using LaminC or Engrailed. DE-Cadherin
staining shows the adhesion contacts between the GSCs and CpCs. Furthermore, ECs and CpCs are marked
here with Traffic jam
2 Materials
2.1 Fly Husbandry 1. Standard cornmeal agar food (recipes can be found at http://
fly.bio.indiana.edu/).
2. Yeast paste: Dry yeast should be mixed in 5 % propionic acid
(see Note 1).
Table 1
A subset of antibodies that are useful to study germline stem cell niche interactions is shown
2.5 DNA Staining 1. DAPI solution: Make a 100× DAPI solution (1 mg/ml) and
and Mounting store aliquots at −20 °C. For staining, dilute in PBS
(see Note 10).
2. Glycerol: 70 % Glycerol, 3 % n-propyl gallate (NPG)
(see Note 11).
3. Tungsten needles.
3 Methods
3.1 Dissection All steps are carried out at room temperature unless otherwise
stated. During all incubations and washes, the Eppendorf tubes are
placed on a nutator.
3.1.1 Adult Ovaries 1. Immobilize 5–10 female flies by putting them on an ice block.
2. The ovaries are positioned in the abdomen of the fly and are
simple to find in well-fed individuals (see Note 12). Dissect the
flies in 1× PBS using a stereomicroscope, and hold the fly with
one pair of tweezers at the thorax. Carefully open the cuticle at
the posterior end of the animal with another pair of tweezers.
If necessary, gently push the abdomen to squeeze out the
paired ovaries. Remove all remnants of guts and cuticle and
place the ovaries in an Eppendorf tube using Pasteur pipettes
(see Note 13).
3.1.2 Larval Ovaries 1. Pick up late third instar larvae from the wall of the food vial or
bottle.
2. Select a female larva and hold with a pair of tweezers at the
anterior end.
3. The larval ovaries are located in the fat body. Cut off the larval
head and hold the posterior end of the remaining larval body
with one pair of tweezers. Carefully now invert the larvae by
pulling it over the tweezers with another pair of tweezers.
Remove cuticle and guts and transfer the fat body into an
Eppendorf tube or a 24-well plate (see Note 14).
30 Annekatrin König and Halyna R. Shcherbata
3.1.3 Fixation 1. Add fixing solution and incubate for 10 min. Remove the
fixing solution carefully and wear protective gloves when
handling the fixative.
2. Wash the ovaries three times for 15 min each with PBT
(see Notes 15 and 16).
4 Notes
References
1. Walker MR, Patel KK, Stappenbeck TS (2009) 7. McKearin D, Ohlstein B (1995) A role for the
The stem cell niche. J Pathol 217(2):169–180. Drosophila bag-of-marbles protein in the dif-
doi:10.1002/path.2474 ferentiation of cystoblasts from germline stem
2. Scadden DT (2006) The stem-cell niche as cells. Development 121(9):2937–2947
an entity of action. Nature 441(7097): 8. Song X, Wong MD, Kawase E, Xi R, Ding BC,
1075–1079. doi:10.1038/nature04957 McCarthy JJ, Xie T (2004) Bmp signals from
3. Song X, Zhu CH, Doan C, Xie T (2002) niche cells directly repress transcription of a
Germline stem cells anchored by adherens differentiation-promoting gene, bag of mar-
junctions in the Drosophila ovary niches. bles, in germline stem cells in the Drosophila
Science 296(5574):1855–1857. doi:10.1126/ ovary. Development 131(6):1353–1364.
science.1069871 doi:10.1242/dev.01026
4. Decotto E, Spradling AC (2005) The Drosophila 9. Konig A, Yatsenko AS, Weiss M, Shcherbata
ovarian and testis stem cell niches: similar somatic HR (2011) Ecdysteroids affect Drosophila
stem cells and signals. Dev Cell 9(4):501–510. ovarian stem cell niche formation and early
doi:10.1016/j.devcel.2005.08.012 germline differentiation. EMBO J 30(8):
5. Xie T, Spradling AC (1998) Decapentaplegic 1549–1562. doi:10.1038/emboj.2011.73
is essential for the maintenance and division of 10. Shcherbata HR, Ward EJ, Fischer KA, Yu JY,
germline stem cells in the Drosophila ovary. Reynolds SH, Chen CH, Xu P, Hay BA,
Cell 94(2):251–260 Ruohola-Baker H (2007) Stage-specific differ-
6. Yu JY, Reynolds SH, Hatfield SD, Shcherbata ences in the requirements for germline stem
HR, Fischer KA, Ward EJ, Long D, Ding Y, cell maintenance in the Drosophila ovary. Cell
Ruohola-Baker H (2009) Dicer-1-dependent Stem Cell. 1(6):698–709. doi:10.1016/j.stem.
Dacapo suppression acts downstream of 2007.11.007
Insulin receptor in regulating cell division of 11. Hatfield SD, Shcherbata HR, Fischer KA,
Drosophila germline stem cells. 2009 Nakahara K, Carthew RW, Ruohola-Baker H
136(9):1497–507. doi:10.1242/dev.025999 (2005) Stem cell division is regulated by the
Visualization of Adult Stem Cells Within Their Niches Using the Drosophila Germline… 33
Abstract
Morphometry is a classical quantitative method often used in biology to provide a data basis for functional
interpretations/interactions of a particular organ or system. Herein we took advantage of this valuable
approach to evaluate the spermatogonial stem cell niche using the horse testis and immunocytochemical
localization of GFRA1 [glial cell line-derived neurotrophic factor receptor produced by Sertoli cells)] as an
example. Using the NIH ImageJ free software, we describe in detail all the necessary steps to investigate this
specific and crucial microenvironment. Based on several recently published papers from our research group,
this approach has proved to be fast, simple, and adaptable to a wide range of species and has the potential
to be easily reproducible in different laboratories.
Key words Morphometry, Morphology, Testis, Spermatogenesis, Spermatogonial stem cell, Niche,
GFRA1, ImageJ
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_4, © Springer Science+Business Media, LLC 2013
35
36 Paulo Henrique Almeida Campos-Junior et al.
2 Materials
3 Methods
3.1 Image 1. The images can be acquired using any system for image capture.
Acquisition The optimal resolution for further morphometrical analysis is
2,576 × 1,932 pixels.
2. Each image should be captured with a seminiferous tubule
cross section in a central position in order to observe the
neighboring components such as other tubules or interstitial
elements. In general, we use 200× magnification, but this
choice should be adapted according to the seminiferous tubule
diameter of the particular species investigated.
9. Using the angle tool, join the points 1–0–2 to obtain the angle
of the specific evaluated region (e.g., tubule–tubule area).
10. Just after the angle acquisition, press “ctrl + M” to obtain the
respective value (e.g., 53.096°).
Morphometric Evaluation of the Spermatogonial Stem Cell Distribution… 41
3.3 Data Analysis 1. Calculate the mean cell density per area (degree) in each
region.
2. Apply the adequate statistical tests to compare different
regions.
4 Notes
References
1. Hofmann MC (2008) Gdnf signaling pathways (2010) Spermatogenesis in fish. Gen Comp
within the mammalian spermatogonial stem cell Endocrinol 165(3):390–411
niche. Mol Cell Endocrinol 288:95–103 9. Yoshida S (2012) Elucidating the identity and
2. Nóbrega RH, Greebe CD, van de Kant H, behavior of spermatogenic stem cells in the
Bogerd J, França LR, Schulz RW (2010) mouse testis. Reproduction 144:293–302
Spermatogonial stem cell niche and spermato- 10. Chiarini-Garcia H, Meistrich ML (2008) High
gonial stem cell transplantation in zebrafish. resolution light microscopic characterization of
PLoS One 5:e12808 spermatogonia. Methods Mol Biol 450:95–107
3. Campos-Junior PHA, Costa GMJ, Lacerda 11. Phillips BT, Gassei K, Orwig KE (2010)
SMSN, Rezende-Neto JV, de Paula AM, Spermatogonial stem cell regulation and sper-
Hofmann MC, França LR (2012) The matogenesis. Philos Trans R Soc Lond B Biol
spermatogonial stem cell niche in the Sci 365:1663–1678
collared peccary (Tayassu tajacu). Biol Reprod 12. Huckins C (1971) The spermatogonial stem
86(155):1–10 cell population in adult rats. 3. Evidence for a
4. Costa GMJ, Avelar GF, Rezende-Neto JV, long-cycling population. Cell Tissue Kinet
Campos-Junior PHA, Lacerda SMSN, Andrade 4:335–349
BS, Thomé RG, Hofmann MC, Franca LR 13. Ehmcke J, Schlatt S (2008) Identification and
(2012) Spermatogonial stem cell markers and characterization of spermatogonial subtypes
niche in equids. PLoS One 7:e44091 and their expansion in whole mounts and tis-
5. Lacerda SMSN, Aponte PM, Campos-Junior sue sections from primate testis. Methods Mol
PHA, Costa GMJ, Segatelli TM, Silva MA, Biol 450:109–118
França LR (2012) An overview on spermato- 14. Meng X, Lindahl M, Hyvönen ME, Parvinen
gonial stem cell physiology, niche and trans- M, de Rooij DG, Hess MW, Raatikainen-
plantation. Anim Reprod 9:798–808 Ahokas A, Sainio K, Rauvala H, Lakso M,
6. Oatley JM, Brinster RL (2012) The germline Pichel JG, Westphal H, Saarma M, Sariola H
stem cell niche unit in mammalian testes. (2000) Regulation of cell fate decision of
Physiol Rev 92:577–595 undifferentiated spermatogonia by GDNF.
7. Fawcett DW, Neaves WB, Flores MN (1973) Science 287:1489–1493
Comparative observations on intertubular 15. Leal MC, Cardoso ER, Nóbrega RH, Batlouni
lymphatics and the organization of the intersti- SR, Bogerd J, França LR, Schulz RW (2009)
tial tissue of the mammalian testis. Biol Reprod Histological and stereological evaluation of
9:500–532 zebrafish (Danio rerio) spermatogenesis with
8. Schulz RW, França LR, Lareyre JJ, Le Gac F, an emphasis on spermatogonial generations.
Chiarini-Garcia H, Nóbrega RH, Miura T Biol Reprod 81:177–187
Chapter 5
Abstract
Hematopoietic stem cells (HSC) undergo multilineage differentiation or self-renewal to maintain normal
hematopoiesis and to sustain the size of the HSC pool throughout life. These processes are determined by a
complex interplay of molecular signals between HSC and other cellular components such as osteoblasts
(OB), stromal cells, endothelial cells, and a number of extracellular matrix (ECM) proteins. Through changes
in its physical properties within the bone marrow (BM) microenvironment, collagen, which is one of the
most critical ECM proteins, can modulate HSC function and maintenance of the competence of the hema-
topoietic niche (HN). At present, there is no consensus as to how different cellular elements of the niche
collaborate and interact to promote HSC self-renewal or differentiation to maintain hematopoiesis.
Deciphering these interactions and the impact of mechanical properties of the collagen microstructures
within the HN has critical clinical implications in the areas of stem cell homing, engraftment, and mainte-
nance of HSC function. In this chapter, we describe several of the in vitro methodologies for establishing and
maintaining HSC in vitro including the isolation of OB, stromal cells, and hematopoietic progenitor cells, as
well as the establishment of both two-dimensional (2D) and three-dimensional (3D) coculture systems.
Key words Hematopoietic stem cells, Osteoblasts, Stromal cells, Stem cell niche, Collagen matrix
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_5, © Springer Science+Business Media, LLC 2013
43
44 Brahmananda Reddy Chitteti et al.
2 Materials
4. Sterilized T pins.
5. Sterilized filters (Mesh Screens—297 μm or Sweeny filter).
6. Complete alpha minimum essential medium (αMEM) consist-
ing of αMEM supplemented with 10 % fetal calf serum (FCS),
1 % penicillin/streptomycin (P/S), and 1 % L-glutamine.
7. 1× PBS.
2.2 Stromal Cell 1. 25 or 75 cm2 tissue culture flasks or tissue culture dishes.
Preparation 2. Ficoll purchased as a ready-to-go solution. Make sure that the
bottle of Ficoll is warmed up to room temperature before using.
3. Complete Iscove’s modified Dulbecco’s medium (IMDM)
consisting of IMDM supplemented with 10 % FCS, 1 % P/S,
and 1 % L-glutamine.
4. Heparin medium consisting of Hank’s Balanced Salt Solution
(HBSS) medium supplemented with 1 % P/S and 20 U/mL
heparin.
5. β-Mercaptoethanol prepared at 2 × 10−2 M in complete IMDM
(100×).
6. Methylprednisolone or hydrocortisol prepared at 2 × 10−3 M in
complete IMDM (100×).
2.3 Hematopoietic 1. Ficoll purchased as a ready-to-go solution. Make sure that the
Stem Cells bottle of Ficoll is warmed up to room temperature before using.
2. Complete IMDM consisting of IMDM supplemented with
10 % FCS, 1 % P/S, and 1 % L-glutamine.
3. Heparin medium consisting of HBSS medium supplemented
with 1 % P/S and 20 U/mL heparin.
4. PBS solution supplemented with 1 % FCS.
5. Fluorochrome-labeled primary antibodies for immunostain-
ing. These antibodies may vary in number and specificities
depending on the phenotypic definition of the cells that will be
identified and isolated by flow cytometric cell sorting.
3 Methods
Fig. 1 A representative dot plot showing the 2-day calvarial osteoblast surface phenotype by flow cytometry.
83 % of osteoblasts prepared as described in Subheading 3.1 are lineage (CD45, CD31, and Ter119) negative
and Sca-1 negative. Among these lineage- and Sca1-negative cells, 90.1 % cells are CD51 positive, 95 % cells
are osteopontin positive, and 34 % cells are CD166 (ALCAM) positive
Fig. 2 (a) A representative figure showing osteoblasts that were isolated from the 2-day calvariae of C57Bl/6
pups as described in Subheading 3.1 and were cultured for 4 days. (b) A representative figure of stromal cells
prepared from adult C57Bl/6 mice and cultured for 4 weeks as described in Subheading 3.2. Please note that
stromal cells shown in this figure are not yet at a monolayer stage
2. The next day, add HSC (1,000 LSK cells) to the OB and/or
stromal cell cultures.
3. All cultures are then supplemented with a cocktail of cytokines
containing recombinant murine SCF and IL3 (10 ng/mL),
IGF1 and TPO (20 ng/mL), IL6 and Flt3 (25 ng/mL), and
OPN (50 ng/mL). Maintain cultures for 1 week in medium
consisting of 1:1 mix of IMDM and αMEM supplemented with
10 % FCS, 1 % Pen/Strep, and 1 % L-glutamine. On day 5
replenish the cultures again with the same cytokine mixture.
4. It should be noted that by day 7 cultures containing LSK cells
and cytokines have numerous hematopoietic cells making visu-
alization of OB and/or stromal cells difficult. Importantly, OB
and/or stromal cells should remain healthy throughout the
coculture period (e.g., they are not peeling off).
5. Hematopoietic cells are typically harvested on day 7 and
counted. Fold increase in the number of cells derived from
LSK cells is calculated relative to d0 count of 1,000. At this
time cells can be analyzed by standard in vitro assays such as
phenotyping and colony forming assays, or they can be used in
in vivo transplantation assays [24, 25].
3.5 Preparation of 1. All steps for 3D coculture system preparation should be per-
3D Coculture System formed on ice (4 °C) unless otherwise mentioned.
2. To accurately pipette the viscous collagen solution, a positive
displacement pipette should be used; please see Note 6.
3. Transfer collagen solution into a 15 mL tube and keep it on
ice. The volume of collagen solution is chosen based on the
desired final collagen concentration of the polymerization
reaction or stiffness (G’) of the polymerized matrix. Refer to
Table 1 for polymerization reaction reagents and recipes (reci-
pes are based on collagen polymerization potential).
4. Add sterile 0.01 N HCl to the collagen solution. Invert several
times to mix well.
5. Neutralize collagen solution to achieve neutral pH (7.4) by add-
ing sequentially 10× PBS, sodium hydroxide, and CaCl2. Invert
to mix after the addition of each component. Maintain the
neutralized collagen solution on ice until ready to polymerize.
6. Aliquot cell solutions containing specified number of stromal
cell, OB, and HSC into a 50 ml tube.
7. Centrifuge cell solution at 500 × g for 10 min at 4 °C. Decant
the supernatant. Vortex gently to disrupt the cell pellet.
8. Transfer neutralized collagen solution to cell pellet and mix by
inverting. It is important to achieve a homogenous distribu-
tion of cells within the collagen solution.
52 Brahmananda Reddy Chitteti et al.
Table 1
The amount of collagen and other reagents required to make a particular stiffness (Pa)
three-dimensional scaffolds
3.6 Harvesting 1. Prepare the cell extraction solution (for example, 1 mL per
Cells from 3D 500 μL tissue construct). Filter cell extraction solution through
Coculture System a sterile 0.2 μM syringe filter. Warm solution to 37 °C imme-
diately prior to use.
2. Add 1 mL of cell extraction solution to a 15 mL sterile conical
tube.
3. Using a sterile forceps, transfer tissue construct from well plate
to tube containing cell extraction solution.
4. Shake tubes at 120 rpm, 37 °C, for 20 min. Invert tubes sev-
eral times every 5–10 min.
5. Add an equal volume of complete medium (Subheading 2.3
above) and pipette up and down gently to ensure complete
digestion of the collagen construct.
In Vitro Construction of 2D and 3D Simulations of the Murine Hematopoietic Niche 53
Fig. 4 A representative figure of murine HSC (lineage− Sca1+ cKit+ or LSK cells)
proliferating in 200PA collagen constructs that are prepared as described in
Subheading 3.4
54 Brahmananda Reddy Chitteti et al.
4 Notes
Acknowledgments
References
1. Till JE, McCulloch EA (1961) A direct mea- home of HSC differentiation and mobilization.
surement of the radiation sensitivity of normal Physiology (Bethesda) 20(5):349–356
mouse bone marrow cells. Radiat Res 14: 10. Zhang J, Niu C, Ye L, Huang H, He X, Tong
213–222 WG, Ross J, Haug J, Johnson T, Feng JQ,
2. Abramson S, Miller RG, Phillips RA (1977) Harris S, Wiedemann LM, Mishina Y, Li L
The identification in adult bone marrow of (2003) Identification of the haematopoietic
pluripotent and restricted stem cells of the stem cell niche and control of the niche size.
myeloid and lymphoid systems. J Exp Med Nature 425(6960):836–841
145:1567–1579 11. Arai F, Hirao A, Ohmura M, Sato H, Matsuoka
3. Snodgrass R, Keller G (1987) Clonal fluctua- S, Takubo K, Ito K, Koh GY, Suda T (2004)
tion within the haematopoietic system of mice Tie2/angiopoietin-1 signaling regulates
reconstituted with retrovirus-infected stem hematopoietic stem cell quiescence in the bone
cells. EMBO J 6:3955–3960 marrow niche. Cell 118(2):149–161
4. Van Zant G, Scott-Micus K, Thompson BP, 12. Calvi LM, Adams GB, Weibrecht KW, Weber
Fleischman RA, Perkins S (1992) Stem cell JM, Olson DP, Knight MC, Martin RP,
quiescence/activation is reversible by serial Schipani E, Divieti P, Bringhurst FR, Milner
transplantation and is independent of stromal LA, Kronenberg HM, Scadden DT (2003)
cell genotype in mouse aggregation chimeras. Osteoblastic cells regulate the haematopoietic
Exp Hematol 20:470–475 stem cell niche. Nature 425(6960):841–846
5. Ogawa M (1993) Differentiation and prolif- 13. Heissig B, Hattori K, Dias S, Friedrich M,
eration of hematopoietic stem cells. Blood Ferris B, Hackett NR, Crystal RG, Besmer P,
81(11):2844–2853 Lyden D, Moore MA, Werb Z, Rafii S (2002)
6. Till JE, McCulloch EA (1980) Hemopoietic Recruitment of stem and progenitor cells from
stem cell differentiation. Biochem Biophys the bone marrow niche requires MMP-9
Acta 605:431–443 mediated release of kit-ligand. Cell 109(5):
7. Jones RJ, Celano P, Sharkis SJ, Sensenbrenner 625–637
LL (1989) Two phases of engraftment estab- 14. Wilson A, Oser GM, Jaworski M, Blanco-Bose
lished by serial bone marrow transplantation in WE, Laurenti E, Adolphe C, Marieke A, Essers
mice. Blood 73(2):397–401 H, Macdonald O, Trumpp A (2007) Dormant
8. Schofield R (1978) The relationship between and self-renewing hematopoietic stem cells
the spleen colony-forming cell and the hema- and their niches. Ann N Y Acad Sci 1106:
topoietic stem cell. Blood Cells 4(1–2):7–25 64–75
9. Kopp H-G, Avecilla ST, Hooper AT, Rafii S 15. Weiss L, Sakai H (1984) The hematopoietic
(2005) The bone marrow vascular niche: stroma. Am J Anat 170(3):447–463
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16. Ho AD (2005) Kinetics and symmetry of divi- microscopy of collagen fibrillogenesis and
sions of hematopoietic stem cells. Exp Hematol extracellular matrix assembly in vitro.
33(1):1–8 Biopolymers 54:222–234
17. Sagar BM, Rentala S, Gopal PN, Sharma S, 22. Horowitz MC, Fields A, DeMeo D, Qian HY,
Mukhopadhyay A (2006) Fibronectin and Bothwell AL, Trepman E (1994) Expression
laminin enhance engraftibility of cultured and regulation of Ly-6 differentiation antigens
hematopoietic stem cells. Biochem Biophys by murine osteoblasts. Endocrinology 135(3):
Res Commun 350(4):1000–1005 1032–1043
18. Campbell A, Wicha MS, Long M (1985) 23. Chitteti BR, Cheng YH, Kacena MA, Srour
Extracellular matrix promotes the growth and EF (2013) The hierarchical organization of
differentiation of murine hematopoietic cells osteoblasts reveals the significant role of
in vitro. J Clin Invest 75(6):2085–2090 CD166 in hematopoietic stem cell mainte-
19. Bailey JL, Critser PJ, Whittington C, Kuske nance and function. Bone 54(1):58–67
JL, Yoder MC, Voytik-Harbin SL (2011) 24. Chitteti BR, Cheng YH, Poteat B, Rodriguez-
Collagen oligomers modulate physical and Rodriguez S, Goebel WS, Carlesso N, Kacena
biological properties of three-dimensional self- MA, Srour EF (2010) Impact of interactions
assembled matrices. Biopolymers 95(2): of cellular components of the bone marrow
77–93 microenvironment on hematopoietic stem and
20. Kreger ST, Bell BJ, Bailey J, Stites E, Kuske J, progenitor cell function. Blood 115(16):
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Polymerization and matrix physical properties 25. Cheng YH, Chitteti BR, Streicher DA, Morgan
as important design considerations for soluble JA, Rodriguez-Rodriguez S, Carlesso N, Srour
collagen formulations. Biopolymers 93(8): EF, Kacena MA (2011) Impact of matura-
690–707 tional status on the ability of osteoblasts to
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McCallister ME, Robinson JP, Voytik-Harbin and progenitor cells. J Bone Miner Res 26(5):
SL (2000) Time-lapse confocal reflection 1111–1121
Chapter 6
Abstract
Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells, such as
erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages, and also undergo self-renewal
to sustain hematopoiesis throughout an organism’s lifetime. HSCs emerge and expand during mouse
embryogenesis. HSC regulation is governed by two types of activity: intrinsic activity programmed pri-
marily by cell autonomous gene expression, and extrinsic factors, which originate from the so-called
niche cells surrounding HSCs. Previously, we reported that endothelial niche cells regulate HSC genera-
tion at aorta-gonad-mesonephros region and placenta, and that hepatoblastic niche cells regulate HSC
differentiation in mouse embryonic liver. In the course of those studies, we employed immunohisto-
chemistry, flow cytometry, and the laser capture microdissection system to assess embryonic regulation
of the mouse hematopoietic niche.
Key words Hematopoietic stem cells, Niche cells, Embryo, Visualization, Laser capture microdissection
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_6, © Springer Science+Business Media, LLC 2013
57
58 Daisuke Sugiyama and Tatsuya Sasaki
2 Materials
2.3 Laser Capture All solutions are made RNAse-free by treatment with diethyl
Microdissection pyrocarbonate.
1. Mouse embryos.
2. Primary antibody solution: PBS containing 1 % BSA with goat
anti-mouse Kit (1:500; R&D Systems).
60 Daisuke Sugiyama and Tatsuya Sasaki
3 Methods
3.1 Immunohisto- Carry out all procedures at room temperature unless otherwise
chemistry specified.
1. Dissect out mouse embryos and fix in 2 % paraformaldehyde in
PBS overnight.
2. Equilibrate embryos in 30 % sucrose in PBS overnight.
3. Wash them in PBS.
4. Embed embryos in OCT compound and freeze in liquid
nitrogen.
5. Slice tissues at 20 μm thickness using a cryostat and transfer to
glass slides.
6. Dry sections thoroughly (see Note 3).
7. Wash in PBS 3 times for 10 min each.
8. Block in 1 % BSA in PBS for 1 h.
9. Incubate sections with primary antibody solution at 4 °C
overnight.
10. Wash in PBS 3 times for 30 min each.
11. Incubate with secondary antibody solution at room tempera-
ture for 30 min (see Note 4). After adding secondary antibody
solution to glass slides, carry out all procedures in a darkroom
to preserve fluorescence.
12. Wash samples in PBS 3 times for 30 min each.
13. Mount samples on coverslips using fluorescence mounting
medium. Wait until medium dries thoroughly before analyzing
samples (see Note 5).
14. Evaluate using a confocal microscope (Fig. 1).
Isolation of Embryonic Hematopoietic Niche Cells 61
Fig. 1 Confocal images of hematopoietic organs during embryogenesis. Tissues sliced at 20 µm were stained
with antibodies and observed under confocal microscopy. (a) Hematopoietic cell clusters in the aortic region at
10.5 dpc. CD34 (red), Kit (green), and TOTO-3 (blue). (b) Hematopoietic cell clusters in the placenta at 10.5 dpc.
CD31 (red), c-Kit (green), and TOTO-3 (blue). (c) Hematopoietic progenitor cells in the liver at 12.5 dpc. DLK-1
(red), c-Kit (green), and TOTO-3 (blue). Although cryosections of 7–10 µm thickness have been widely used
previously for immunohistochemistry, sections shown here were of 20 µm and provide clear images of hema-
topoietic cell clusters and surrounding cells
3.2 Flow Cytometry Carry out all procedures at room temperature unless otherwise
specified.
1. Obtain placentas or fetal livers from pregnant mothers. Remove
deciduas and umbilical vessels from placentas.
2. Pass placentas or livers through 21-gauge needles to disrupt
tissue prior to collagenase treatment (see Note 6).
3. Put tissues into collagenase solution and incubate on a shaker
for 30 min at 37 °C (see Note 7). After incubation, pipette tis-
sue up and down very gently approximately 10 times to obtain
a single-cell suspension.
4. Filter suspension through 40-μm nylon cell strainers.
5. Add PBS to the suspension and centrifuge at 900 × g for 5 min.
6. For hemolysis, add lysing buffer to samples and wait for
15 min.
7. Centrifuge cells at 900 × g for 5 min and rinse with PBS.
Resuspend the cell pellet in 100–200 μl PBS.
8. Add all color-conjugated antibodies to the single-cell suspen-
sion. Add 0.3 μl of each antibody per 1.0 × 106 cells.
9. Incubate samples on ice for at least 30 min.
10. Add PI buffer to the suspension to remove dead cells by flow
cytometry.
11. Endothelial cells are defined as CD31+/CD34+/c-Kit−/
Ter119−/CD45−, mesenchymal cells as CD31−/CD34−/c-Kit−/
Ter119−/CD45−, hepatoblasts as CD45−/Ter119−/DLK-1+,
and sinusoid endothelial cells as CD45−/Ter119−/LYVE-1+/
62 Daisuke Sugiyama and Tatsuya Sasaki
c-Kit- c-Kit-Ter119-CD45-
Endothelial
cells
Ter119
SSC
CD31
10.5 dpc
AGM region
Mesenchymal
cells
c-Kit CD45 CD34
c-Kit- c-Kit-Ter119-CD45-
Endothelial
cells
SSC
Ter119
CD31
11.5 dpc
placenta
Mesenchymal
64.4 cells
c-Kit CD45 CD34
DLK-1-CD45-Ter119-
104 104
Sinusoid
103 103 2.17 Endothelial
CD45/Ter119
cells
12.5 dpc
CD31
102 102
fetal liver
1.52 101
101 Hepatoblasts
0 0
0 101 102 103 104 0 101 102 103 104
Dlk-1 Lyve-1
Fig. 2 Sorting of endothelial and mesenchymal cells by flow cytometry. Single-cell suspensions were prepared
from the aorta-gonad-mesonephros (AGM) region at 10.5 dpc (lower), placenta at 11.5 dpc (middle), and fetal
liver at 12.5 dpc (lower). Endothelial cells (CD31+/CD34+/Kit−/Ter119−/CD45−) and mesenchymal cells (CD31−/
CD34−/Kit−/Ter119−/CD45−) were sorted out from the AGM region and placenta, and hepatoblasts (CD45−/
Ter119−/DLK-1+) and sinusoid endothelial cells (CD45−/Ter119−/LYVE-1+/CD31+) were sorted from fetal liver
by flow cytometry
3.3 Laser Capture Carry out all procedures at 4 °C (see Note 8).
Microdissection
1. For this procedure, do not fix embryos and omit the sucrose
System equilibration step.
2. Embed placentas in OCT compound and freeze in liquid
nitrogen.
3. Slice tissues at 20 μm thickness with a cryostat and transfer to
glass slides. Place samples on ice and immediately store at
−80 °C until use.
Isolation of Embryonic Hematopoietic Niche Cells 63
c-Kit
Real-time PCR
LCM
Fig. 3 Laser capture microdissection (LCM) strategy. After staining placenta sections with anti-c-Kit antibody,
the edges of hematopoietic cell clusters expressing c-Kit and prospective niche cells surrounding those cells
are simultaneously marked under a microscope using software equipped with an LCM system. Hematopoietic
cell clusters expressing c-Kit (the center black rings) are dissected and removed and residual niche cells are
dissected out and collected into a tube for PCR analysis
4 Notes
Acknowledgments
References
from pre-circulatory yolk sac, but with low 18. Ema H, Nakauchi H (2000) Expansion of
frequency. Dev Biol 301:53–61 hematopoietic stem cells in the developing
12. Samokhvalov IM, Samokhvalova NI, liver of a mouse embryo. Blood 95:
Nishikawa S (2007) Cell tracing shows the 2284–2288
contribution of the yolk sac to adult haemato- 19. Sugiyama D, Arai K, Tsuji K (2005) Definitive
poiesis. Nature 446:1056–1061 hematopoiesis from acetyl LDL incorporating
13. Mikkola HK, Gekas C, Orkin SH, Dieterlen- endothelial cells in the mouse embryo. Stem
Lievre F (2005) Placenta as a site for hemato- Cells Dev 14:687–696
poietic stem cell development. Exp Hematol 20. Zhang CC, Kaba M, Ge G, Xie K, Tong W,
33:1048–1054 Hug C, Lodish HF (2006) Angiopoietin-like
14. Zeigler BM, Sugiyama D, Chen M, Guo Y, proteins stimulate ex vivo expansion of hema-
Downs KM, Speck NA (2006) The allantois topoietic stem cells. Nat Med 12:240–245
and chorion, when isolated before circulation 21. Sugiyama D, Kulkeaw K, Mizuochi C, Horio
or chorio-allantoic fusion, have hematopoietic Y, Okayama S (2011) Hepatoblasts comprise a
potential. Development 133:4183–4192 niche for fetal liver erythropoiesis through
15. Sasaki T, Mizuochi C, Horio Y, Nakao K, cytokine production. Biochem Biophys Res
Akashi K, Sugiyama D (2010) Regulation of Commun 410:301–306
hematopoietic cell clusters in the placental 22. Sugiyama D, Kulkeaw K, Mizuochi C (2013)
niche through SCF/Kit signaling in embry- TGF-beta-1 up-regulates extra-cellular matrix
onic mouse. Development 137:3941–3952 production in mouse hepatoblasts. Mech Dev
16. Johnson GR, Moore MA (1975) Role of stem 130(2–3):195–206
cell migration in initiation of mouse foetal liver 23. Mizuochi C, Fraser ST, Biasch K, Horio Y,
haemopoiesis. Nature 258:726–728 Kikushige Y, Tani K, Akashi K, Tavian M,
17. Houssaint E (1981) Differentiation of the Sugiyama D (2012) Intra-aortic clusters
mouse hepatic primordium. II. Extrinsic ori- undergo endothelial to hematopoietic pheno-
gin of the haemopoietic cell line. Cell Differ typic transition during early embryogenesis.
10:243–252 PLoS One 7:e35763
Chapter 7
Abstract
The availability of mesenchymal stem cells (MSCs) or skeletal stem cells (SSCs) is vital to many of the tissue
engineering strategies currently being developed for repairing bone and cartilage. One difficulty with using
this cell population is that SSCs represent only a small fraction of the cells available from an individual patient’s
bone marrow sample, typically less than 1 in 10,000. Therefore, methods have been devised to enrich the
proportion of MSCs obtained from a bone marrow sample using hybridoma cell lines to generate antibodies
to cell surface antigens specific for MSCs. Stro-1 is the most widely targeted of these cell surface antigens. The
protocol described overleaf is used to isolate and enrich the Stro-1 positive fraction of cells from a bone mar-
row aspirate to provide a sample enriched for MSCs for use in both in vitro and in vivo studies.
Key words Stro-1, Mesenchymal stem cells, Skeletal stem cells, Bone marrow, Magnetic-activated cell
sorting
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_7, © Springer Science+Business Media, LLC 2013
67
68 Emma L. Williams et al.
Fig. 1 (a) Stro-1 expression by HK cells: Primary antibody—Stro-1 hybridoma supernatant (neat); secondary
antibody—Alexa Fluor 488 (green) goat-anti-mouse IgM (1:50 dilution); nuclei counterstained with DAPI (blue,
1:100 dilution). (b) Negative control showing positive nuclear staining with DAPI only
2 Materials
2.1 Preparation of 1. Add 1 tub of powdered alpha Modified Eagle’s medium (Gibco,
Alpha-MEM Medium UK) to a clean 1 l volumetric flask using a spatula and funnel.
2. Add 1 l of distilled water, followed by 2.2 g of sodium hydro-
gen carbonate.
3. Affix stopper and invert to mix. Remove stopper and using a
magnetic “flea,” mix thoroughly on a bench top stirrer for
approximately 1 h.
4. Filter sterilize the medium into autoclaved glass bottles, using
a bottle-top filter (Nalgene) prior to use.
2.3 Preparation 1. For this step we use a hybridoma cell line, maintained within
of Stro-1 Hybridoma the lab, for which we keep frozen aliquots in liquid nitrogen.
Supernatant 2. Day 1 Wake cells up from liquid nitrogen by adding the con-
(See Notes 1–5) tents of one vial to a T75 containing 20 ml of DMEM
(PAA) + 20 % FCS.
3. Day 2/3 Check the cell growth—if healthy and at high density,
split into two T75s, adding 20 ml DMEM + 10 % FCS to each
flask.
4. Day 4/5 Check cells again—if very dense split again into two
T150s, adding 40–50 ml DMEM + 10 % FCS to each flask.
5. Day 6/7 Once the cells are growing well, switch to using
DMEM + 5 % FCS. Cells will continue to need splitting every
2–3 days to keep them healthy. Additional cells that are not
required for use can be frozen down in FCS + 10 % DMSO and
stored in liquid nitrogen.
6. Once the hybridoma cells have been growing well for about 2
weeks, pellet the cells from 1 confluent T150 and resuspend
the pellet in 10 ml DMEM + 20 % FCS. Split the remaining
T150 into 2× T150 and top up the medium to 50 ml per flask
with DMEM + 5 % FCS.
7. Wet the membrane of a CL flask (Integra Celline Classic 350
bioreactor I90010) by adding 15 ml of plain DMEM to the
medium compartment (green lid). Leave for 5 min.
8. Inoculate the cells suspended in DMEM + 20 % FCS into the
cell compartment (white lid). Remove all air bubbles by pipet-
ting the medium up and down and tipping flask. Replace the
cap tightly.
70 Emma L. Williams et al.
3 Methods
3.1 Bone Marrow 1. Under sterile conditions within a Class II extraction hood,
Isolation transfer the bone marrow sample from the universal tube it was
placed in the operating theatre to a 50 ml Falcon tube (Greiner
Bio-One, UK).
2. Suspend the bone marrow sample in 10 ml of alpha modified
Eagle’s medium (α-MEM—Gibco, UK) and 1 % penicillin/
streptomycin (P/S—diluted from 100× concentrate, PAA,
UK) and shake vigorously.
3. Pipette the bone marrow suspension into a new Falcon tube.
Resuspend the remaining bone marrow sample in a further
10 ml α-MEM + P/S and transfer to the new Falcon tube,
repeating this until the bone fragments are white and clean.
4. Centrifuge the cell suspension at 250 × g for 4 min at room
temperature to obtain a cell pellet.
Stro-1 Based Isolation of MSCs from Adult Human Bone Marrow 71
5. Carefully pour off the supernatant and resuspend the cell pellet
in 10 ml α-MEM + P/S, using the mechanical drawing action
of the pipette to break up the cell pellet.
6. Resuspend the cell pellet in 25 ml α-MEM + P/S and pipette it
through a cell strainer (0.70 μm pore size (Fisher)) into another
Falcon tube (see Note 6).
3.2 Removal of Red 1. Prepare the bone marrow sample as described above.
Blood Cells Using 2. Pipette 20 ml of lymphocyte separation medium (LSM) (PAA)
Lymphocyte into a fresh Falcon tube (see Note 7). Using a 3 ml pastette,
Separation Medium add the cell suspension very carefully onto the LSM, holding
the pastette close to the side of the tube and just below the
meniscus of the LSM to add the cell suspension as a steady
stream and form a uniform layer.
3. Centrifuge the LSM/cell suspension at 800 × g for 40 min at
18 °C, with the brakes off (see Note 8).
4. Remove the Falcon tube from the centrifuge. There should be
a layer of medium at the top, bone marrow mononuclear cells
between this and the LSM, and the blood cells at the bottom
of the tube.
5. Using a pastette, carefully remove the cell layer, followed by
the medium into a new Falcon tube and top up to 50 ml with
α-MEM + P/S.
6. Centrifuge twice at 240 × g for 10 min at 4 °C to remove all
traces of LSM.
3.3 MACS Stro-1 1. After the final centrifugation step of the mononuclear cell sep-
Isolation aration described above, pour off the supernatant and resus-
pend the pellet in 2 ml of blocking buffer (see Subheading 2.2).
2. Place in the fridge for 30 min.
3. Thaw the Stro-1 hybridoma supernatant and keep in the fridge
ready for use.
4. Remove the cell suspension from the fridge and add 8 ml of
chilled MACS buffer, to give a total volume of 10 ml (see
Subheading 2.4). Centrifuge at 300 × g for 5 min.
5. Pour off the supernatant and resuspend the cells in 1 ml undi-
luted Stro-1 hybridoma supernatant. Incubate in the fridge for
30 min, mixing regularly.
6. Centrifuge the cell suspension at 300 × g for 5 min. Pour off
the supernatant and wash the cells in 20 ml of chilled MACS
buffer, centrifuging at 300 × g for 5 min, for a total of three
washes.
7. Do a cell count and resuspend in 80 μl MACS buffer per 1 × 107
cells.
72 Emma L. Williams et al.
4 Notes
1. When culturing Stro hybridoma cells, check the cells every day
to assess growth. Timescales given are approximate and have
to be adjusted according to how the cells look.
2. Avoid spinning down Stro hybridoma cells as much as possible
as they do not tolerate this very well.
3. Do not use penicillin/streptomycin in the cell cultures.
4. Healthy Stro hybridoma cells look like round shiny spheres
and grow well at high density.
5. The color of the medium is a good indicator of cell growth
(red—good, orange—need splitting, yellow—dead cells).
6. When pipetting the cell suspension through the cell strainer,
gently scrape the bottom of the filter to prevent it from clog-
ging up.
Stro-1 Based Isolation of MSCs from Adult Human Bone Marrow 73
References
1. Kolf CM, Cho E, Tuan RS (2007) Mesenchymal antibody, STRO-1. Blood 78(1):55–62, Epub
stromal cells. Biology of adult mesenchymal 1991/07/01
stem cells: regulation of niche, self-renewal and 3. Stewart K, Walsh S, Screen J, Jefferiss CM,
differentiation. Arthritis Res Ther 9(1):204, Chainey J, Jordan GR et al (1999) Further char-
Epub 2007/02/24 acterization of cells expressing STRO-1 in cul-
2. Simmons PJ, Torok-Storb B (1991) tures of adult human bone marrow stromal cells.
Identification of stromal cell precursors in J Bone Miner Res 14(8):1345–1356, Epub
human bone marrow by a novel monoclonal 1999/08/24
Chapter 8
Abstract
Marrow stromal cells (MSCs) are relatively rare cells difficult to visualize in marrow biopsies or detect in
aspirated marrow. Under specific conditions MSC can be expanded in vitro and the population can give
rise to several mesenchymal lineages. “MSC” also refers to mesenchymal stem cells which implies that all
cells in the population are multipotent. It is generally agreed that while there may be a few multipotent
stem cells in an MSC population the majority are not stem cells. In either case MSCs do not produce
hematopoietic cells. Although MSCs have been isolated and characterized from several tissues, bone mar-
row is their most common source for research and clinical use. Primary MSC populations can be derived
from bone marrow mononuclear cells with relative ease, but it is important to recognize the cellular het-
erogeneity within a culture and how this may vary from donor to donor. In this chapter, we describe
methodology to derive primary MSCs from bone marrow screens, an otherwise discarded by-product of
bone marrow harvests used for clinical transplantation. We also describe some useful techniques to charac-
terize and manipulate MSCs—both primary and immortalized cell lines.
Key words Marrow stromal cells (MSCs), Bone marrow screen, Reverse-transfection, FACS,
AutoMACS, CD146, siRNA, miRNA, Long-term culture (LTC)
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_8, © Springer Science+Business Media, LLC 2013
75
76 Aravind Ramakrishnan et al.
1.2 MSC: Misleading Most of the initial interest in these cells after their initial descrip-
Misnomer tion by Dexter centered around the mechanistic basis of their
interaction between hematopoietic cells and how they support
hematopoiesis [1, 20]. The mid-1990s, however, witnessed a sig-
nificant interest in stromal cells, which came to be denoted inap-
propriately as mesenchymal stem cells (MSCs), for a wide spectrum
of clinical uses ranging from regeneration of damaged tissues like
heart and liver to immune modulation of allogeneic graft versus
Primary Marrow-Derived Stromal Cells: Isolation and Manipulation 77
1.2.1 Defining MSC MSCs are typically defined as cells that possess the ability to form
colony-forming unit-fibroblast (CFU-F) and differentiate into
multiple mesenchymal cell types (including osteoblasts, chondro-
cytes, adipocytes, and bone marrow stromal cells) under appropri-
ate in vitro culture conditions [39, 40]. Although it is generally
accepted that these mesenchymal lineages likely originate from
common precursors (much in the same way that a single self-
renewing hematopoietic stem cell can self-renew and give rise to
mature hematopoietic lineages), unequivocal evidence for such a
single common precursor that repopulate all mesenchymal lineages
has been lacking [38]. This is due to both the obvious difficulty in
depleting all mesenchymal cells from a host and the difficulty in
reliably transplanting a single stromal cell into such a depleted
host. Although only a small portion of the stromal cells from fresh
bone marrow isolates or early bone marrow cultures would be con-
sidered true MSCs, the term is often inappropriately applied to all
cells in a marrow-derived stromal culture.
Given the heterogeneity of the stromal population, several
groups have attempted to enrich for the CFU-F population using
cell surface markers. The first effort reported was the generation of
the murine monoclonal antibody STRO-1, an IgM antibody spe-
cific for an undefined polysaccharide motif. STRO-1 binds to
approximately 10 % of bone marrow mononuclear cells (BMMNCs),
most of which are nucleated erythroid cells [41]. When nucleated
78 Aravind Ramakrishnan et al.
1.3 MSC Cultures Although culture conditions can be varied to promote stromal
growth over hematopoietic growth resulting in further enrichment
of fibroblast-like cells, they nevertheless remain highly heteroge-
neous and contaminated with cells of hematopoietic origin, most
commonly macrophages. These cultures are typically referred to as
MSC cultures. Setting up primary MSC cultures is relatively
straightforward, but one must expect wide variation among donors
and between cultures from the same donor. Differences in growth
kinetics and relative proportions of different cell types are com-
mon, as is the ability to support different hematopoietic stages and
lineages. MSC quality is also influenced by viral infections like
cytomegaloviruses (CMV), and technical issues which are often
unavoidable, including a delay in processing, or a significant con-
tamination by erythroid cells necessitating hypotonic hemolysis
of samples [49]. Even adjusting for these factors, the phenotypic
and functional characteristics of these cells are difficult to predict.
Cloned stromal cell lines have circumvented many of these
problems; however, results from cell lines must be reproduced in
primary cells to be considered valid. While cell lines are readily
available, descriptions of current techniques to isolate primary
cells and manipulate them in vitro are not as available.
Most tissues that have discernible connective tissue components
have mesenchymal progenitors that can be grown in cultures that
resemble MSC [38]. Bone marrow has remained the most com-
monly used and best studied source of MSCs due to relative ease of
access by bone marrow aspiration. Although normal bone marrow
aspirates can be obtained by volunteer donations or remunerated
study participants, this is often problematic for obvious reasons.
Although relatively safe, bone marrow aspirates are painful and inva-
sive and major complications like severe hemorrhage from a severed
aberrant blood vessel or damage to internal organs have occurred.
Primary Marrow-Derived Stromal Cells: Isolation and Manipulation 79
2 Materials
2.1 IRB Approval As with any other work involving biological materials, appropriate
and Personnel Training training of personnel, and up-to-date institutional approval of
protocols, is essential. Institutional Review Board (IRB) approval
has to be in place before the work commences. Depending on the
particular institution, the investigators may be eligible for expedited
review and human subject exclusion given that the screens may be
provided after removing personal identifiers and that the material is
otherwise discarded. Proper training and certification of personnel
in handling of biohazardous materials are similarly critical given that
the marrow screens could be a source of infection although donors
are typically tested extensively prior to marrow harvest (see Note 1).
2.2 Cell and Tissue Proper cell and tissue culture techniques and equipment are of
Culture Setup critical importance in any tissue culture methodology, but espe-
cially so for primary MSC cultures given that they could be con-
taminated at multiple steps of their setup and maintenance, and the
relatively long periods (weeks to months) that they may need to be
maintained. Most laboratories which perform tissue culture are set
up with biosafety laminar flow cabinets (or TC hoods), incubators
that can maintain temperature, CO2 content and humidity (TC
incubator), benchtop centrifuges to spin down cells, vacuum traps,
water baths, standard inverted microscopes (for cell counting and
monitoring of cell growth), etc. The setup and maintenance of
these are hence not discussed in detail here.
2.3 Bone Bone marrow screens are filters used to remove particulate materials
Marrow Screens including marrow spicules, fat globules, and small clots from the
bone marrow aspirated from donors for the purpose of transplanta-
tion (Fig. 1). They can be procured from the cell processing or
80 Aravind Ramakrishnan et al.
Fig. 1 Schematic of bone marrow harvest and screens. The bone marrow aspi-
rate collected from donors is hooked up typically to two filters called screens
connected in series (500 μM first followed by 200 μM) to filter out particulate
matter including spicules, clots, and tissue fragments. The filtrate is collected for
infusion (processed marrow). The marrow screens are then made available to
research laboratories with the entry port and exit connected to each other
Fig. 2 Extraction of bone marrow mononuclear cells (BMMNC) from marrow screens. (a) Screens are typically
provided with the two filters (500 and 200 μM) connected in series as a sterile loop. (b) Carefully disengage
the entrance port of the 500 μM filter from the exit port of the 200 μM filter. (c) Pour any liquid contents left in
the screen to a 50 cc conical tube. (d) Inject 25 cc of PBS–EDTA into the entrance port of the 500 μM filter and
collect the run through carefully. (e) Try to dislodge any particulate material in the filters into the PBS–EDTA
solution. (f) Approximately 50 cc of bone marrow particulate material is eluted into a 50 cc conical tube.
(g) The cell pellet after the first wash is rich with erythroid cells which need to be mostly removed before
plating in culture. (h) Hemolysis of collected cells in hemolysis buffer at 37 °C. (i) Post-hemolysis and a further
wash in PBS–EDTA, cell pellet is pale and has little visible erythroid contamination
2.4 Human Stromal Various groups have reported on immortalized stromal cell lines,
Cell Lines typically generated using retroviral vectors. Our group has previ-
ously reported on the use of human stromal cell lines isolated after
immortalization with retrovirally expressed human papilloma virus
(HPV) E6/E7 proteins; two cell lines termed HS5 and HS27a
have been used to represent functionally distinct stromal niches
that exist within the larger context of the adult vertebrate marrow
microenvironment [50]. HS5 represents a stromal phenotype that
secretes large quantities of cytokines, thus driving differentiation of
HSPC to mature myeloid lineages. HS27a in contrast produces
high levels of HSPC niche-associated genes (such as CXCL12,
Jagged1, and Angiopoietin1) that help maintain HSPC in their
primitive undifferentiated states (see Note 3).
2.5 Flow-Cytometry Flow-based sorting as described here for CD146 expression is typi-
and Sorting of Stromal cally available in large research centers where well-trained staffs per-
Cells Based on CD146 form the sorting using shared instruments (see Note 4). For magnetic
Expression: Flow bead sorting for enrichment of stromal precursors by removal of
Sorters and MACS CD14- and CD45-positive cells, we have utilized proprietary
Sorters magnet-conjugated antibodies from Miltenyi Biotec (see Note 5).
2.6 Medium for MSC Human MSCs can be grown in a variety of medium formulations
Growth and as long as they are replete with serum. These include alpha minimal
LTC Growth essential medium, Dulbecco’s modified Eagle’s medium (DMEM),
and RPMI-1640. DMEM commonly comes in low-glucose or
high-glucose variants and low-glucose formulations are routinely
used by most groups for propagating MSC. Although the other
base medium formulations have differences in components, in our
anecdotal experience, they can be used interchangeably as long as
they are serum supplemented. Fetal bovine serum (FBS) supple-
mented at 10–20 % final volume is considered essential for good
proliferation of all fibroblast cultures including bone marrow-
derived stromal cells. FBS is known to have high concentrations of
several fibroblast mitogens (from the PDGF and FGF family) (see
Note 6). Serum-free formulations with defined chemical additives
such as cytokines and small molecules, as have been developed for
hematopoietic colony growth, would be an important technical
advancement for the field; however at this time FBS remains a
necessary supplement for MSC cultures. Addition of antibiotics
such as penicillin–streptomycin and antifungal agents such as
amphotericin B should be considered, the latter especially in cli-
mates where mold contamination is known to occur in cultures
that have to be maintained for several weeks.
Medium for primary LTC is typically composed of Iscove’s
medium supplemented with screened horse serum (HS) in addition
to FBS and several other additives (see Note 7). Although the human
stromal cell lines Hs27a and Hs5 were initiated in LTC medium
they can be grown in RPMI-1640 supplemented with 10 % FBS.
Primary Marrow-Derived Stromal Cells: Isolation and Manipulation 83
2.7.2 Bovine Serum Dissolve 100 g fraction V BSA in 1 l of deionized water. Filter
Albumin, 10 % Solution sterilize using a low-protein-binding 0.45-μm filter. Store in
50–100 ml aliquots at −20 °C.
2.7.4 LTC Medium 62.5 ml prescreened FBS (screened to support LTC growth) to a
final concentration of 12.5 %.
62.5 ml prescreened HS (screened to support LTC growth) to a
final concentration of 12.5 %.
5 ml of 100× Penicillin–streptomycin.
5 ml of 100× Amphotericin B (optional).
5 ml of L-glutamine.
5 ml of Pyruvate.
250 ml of 2× Isocove’s modified Dulbecco’s medium (IMDM)
prepared from powder (GIBCO).
500 μl of 103 M hydrocortisone stock (final concentration 106 M).
Distilled tissue culture-grade water (endotoxin free)—add to total
of 500 ml.
Mix all components and filter sterilize with 0.45 μm filter and
store at 4 °C for up to 3 months. They can also be frozen at −20 °C
for future use if larger volumes are made.
2.7.6 Solutions
10× PBS
and Buffers
NaCl 800 g
KCl 20 g
Na2HPO4 ⋅2H2O 144 g
KH2PO4 24 g
2.7.12 Other Solutions Trypsin–EDTA (0.25 % or 0.05 % trypsin and 1 mM EDTA) avail-
and Buffers able from various vendors.
Phosphate-buffered saline (PBS).
PBS–EDTA (addition of EDTA to 2 mM final concentration).
Ficoll solution (1.073 or 1.077 g/ml) from various vendors.
3 Methods
3.3 Ficoll Density Density gradient separation based on the polysaccharide Ficoll to
Gradient Separation to separate whole blood or bone marrow to different components is
Yield BONE MARROW a useful technique for isolation of MSCs and stromal precursors
Mononuclear Cells since they are contained in the mononuclear fraction at the inter-
phase between Ficoll and diluted bone marrow. Layering of blood
or marrow on Ficoll is a delicate technique and it is recommended
that it is learned from those proficient with the technique and prac-
ticed on more easily obtainable samples like peripheral blood
before attempting on limited resources like bone marrow aspirate.
Ficoll density (g/cm3) 1.077 is classically used for BMMNC prepa-
ration from human bone marrow (see Note 10). Also important to
remember is that the gradient separation is not absolute and does
result in the loss of BMMNC (including the stromal precursors) to
other fractions. Hence if the sample is limited (such as part of a
clinical procurement) and to be used for direct plating for MSC
cultures, it is recommended that one consider direct hemolysis
instead of Ficoll gradient separation.
1. Move Ficoll from 4° fridge to TC hood at least 30 min before
separation since Ficoll density is sensitive to temperature. Do
not warm in a 37 °C water bath. To expedite equilibration to
room temperature, one can aliquot the Ficoll to the Falcon
tubes about 30 min before separation (step 3 of this protocol).
2. Dilute bone marrow with three volumes of medium HBSS.
Diluting the blood or the BM increases the yield of mononu-
clear cells and decreases the hang-up of RBCs.
88 Aravind Ramakrishnan et al.
Fig. 3 Extraction of bone marrow mononuclear cells (BMMNC) by Ficoll gradient separation. (a) 3 ml of Ficoll
at room temperature is aliquoted into 15 cc Falcon tubes. (b–d) 6–8 mf of dilute bone marrow is carefully
layered on the Ficoll layer taking care not to mix the two layers up. (e) After centrifugation at 400 × g for 30 min
at room temperature, different layers become apparent. The mononuclear cells are trapped in the layer
between Ficoll and serum. (f–j) With a sterile glass pipetted (stuffed with cotton at proximal end for seal), the
mononuclear layer is carefully collected minimizing suction of both the Ficoll layer between and the serum
layer above
Fig. 4 Primary long-term cultures (LTCs or Dexter cultures). (a) Cartoon depicting
vertical layer of an LTC with stromal and hematopoietic elements. (b) Phase con-
trast micrograph of an LTC. Long arrows in both panels depict the cobblestone
areas (CSAs) comprising primitive hematopoietic precursors trapped within the
stromal layers and appear as “phase-dark” cells resembling a cobblestone.
Mature myeloid cells are released into the supernatant when the more primitive
precursor cells in the CSAs divide and mature (short arrow in both panels ) and
appear as “phase-light cells” in phase contrast micrographs
Fig. 5 Morphology of MSC cultures. (a) A typical CFU-F that arises out of a single stromal precursor after 5–7
days of culture. (b) Confluent cultures that develop after 2 weeks or more of culture
3.5 Setting Up and Protocol for human MSC (hMSC) cultures is similar to that of LTCs,
Passaging of Human except that the medium used is DMEM replete with 10 % FCS.
MSC Cultures
1. Plate cells in densities as described above for LTCs.
2. Fibroblast-appearing MSCs should be evident 24–48 h after
plating although the preponderance of non-adherent hemato-
poietic cells might make initial visualization difficult.
3. About 48 h after plating, remove supernatant cells by suction-
ing (with sterile precautions) in the tissue culture hood. The
cultures may be rinsed with HBSS if the hematopoietic cells
form clumps. Refeed with equal amount of fresh medium.
4. CFU-Fs become rapidly evident in the next 3–5 days (Fig. 5a)
and cultures become confluent in about 2 weeks from when
they were plated (Fig. 5b).
5. Since primary cells have contact inhibition, MSCs could be
maintained for several weeks after reaching confluence with-
out splitting cultures; but to expand their numbers, the MSC
cultures can be split at a 1:3 ratio after they reach 75–80 %
confluence.
6. To split, rinse cultures 2–3 times with PBS–EDTA (half the
volume of tissue culture medium) after the medium is suc-
tioned off. Then add trypsin–EDTA (1 ml of 0.25 % or 4 ml of
0.05 % trypsin) and return to 37 °C.
7. After 5 min, determine how dislodgeable the cells are in
response to initial trypsin–EDTA treatment. If needed, the ini-
tial trypsin–EDTA can be suctioned off, an equal volume
added, and the plate returned to 37 °C (this will allow for the
initial trypsin to presumably loosen up the extracellular matrix
and the second trypsin to loosen the cell-to-cell adhesions).
8. After another 5 min, add 2 ml of FBS (or bovine calf serum
which is adequate to inactive trypsin but is much cheaper
92 Aravind Ramakrishnan et al.
than FBS) to inactivate the trypsin. Scrape the cells off the plate
with a sterile cell scraper and transfer cells to a 50 cc conical.
9. Wash plate with 5–10 ml of PBS–EDTA to collect cells that
may have been left behind.
10. Spin cells at 400 × g force for 5 min. Remove supernatant by
suctioning.
11. Resuspend pellet by “racking.” Add requisite amount of medium
(threefold if splitting 1–3) and plate to adherent plastic plates.
12. MSCs will maintain exponential growth typically for at least
4–5 passages and slow down after that. The characteristics of
MSCs will become more homogeneous after each passage and
closer to fibroblasts derived from any other tissue site after a
few passages.
preserving viability. Since these cells are typically larger, the use of
a larger nozzle (100 or 130 μm) for the sorting machine would be
ideal and most modern flow-sorters are equipped to do this. While
preparing the cells, it is important to avoid using trypsin to dis-
lodge the cells since CD146 is known to digest the CD146 epitope
(see Note 11). Finally, viability of cells after sorting is typically
about 50 % at best (again likely reflecting the non-physiological
nature of single-cell capillary flow for these cells) and if these cells
were to be replated for culture, the loss of viability needs to be
considered.
1. Wash cultures initially with PBS–EDTA 3 times. Typically ten
to twenty 100 mm dishes are used for one sorting experiment.
2. Add enough citrate saline solution to cover the bottom culture
(for 100 mm plates, 5 ml of solution). Return plates to 37 °C
incubator for 5 min.
3. Scrape cells off the plate with a sterile cell scraper. Move the
cells to a 50 ml conical. Wash the plate with an additional 5 ml
of PBS–EDTA to collect any remaining cells and add to the
conical tube.
4. Spin cells down at 400 × g force for 5 min. Remove supernatant.
5. Gently resuspend cells by “racking” the pellet. Add 5 ml of
PBS–EDTA and mix thoroughly with the pipetman several
times.
6. Wash a second time with PBS–EDTA and resuspend cells as
described above.
7. Determine if cells are significantly clumped together (primary
MSC cultures are often clumped together) and you may opt to
repeat the PBS–EDTA resuspension step once more. If not,
filter through a sterile 100 μm filter and proceed to counting
and antibody labeling.
8. Determine viable cell number with a hemocytometer count.
9. Resuspend up to ten million cells in a clear 5 ml Falcon tube
(#2058) in sterile FACS buffer in a total volume not to exceed
1 ml (see Note 12).
10. Add 10 μl of anti-CD146 antibody per one million cells.
Incubate at 4 °C for 30 min.
11. Wash three times by adding 4 ml of FACS buffer, spinning at
800 × g force for 5 min, and resuspending the cell pellet after
each wash.
12. After final wash, filter cells once more through a 100 μm filter
and resuspend in a final cell concentration of no more than one
million cells per ml (to prevent cellular clumping).
13. Perform FACS-based sorting of cells on sorting machine, pref-
erably with a 100 or a 130 μm nozzle. Ensure that the cells are
Primary Marrow-Derived Stromal Cells: Isolation and Manipulation 95
3.7 Isolation of MSC An alternate method to enrich for MSCs is by depleting the bone
by Depletion of CD45 marrow of hematopoietic cells. Most mature cells of hematopoietic
and CD14 origin are CD45 (pan leucocyte antigen) positive; some monocytes
and macrophages may not be removed by CD45 depletion alone
and depleting CD14-positive cells in addition further enriches
for stromal precursors. As in the CD146 protocol above
(Subheading 3.6), enriching for adherent cells only by plating cells
in plastic adherent plates for 24 h can reduce the total reagents that
would need to be used if unselected fresh BMMNC is used.
Availability of magnetic conjugated antibodies for CD45 and
CD14 makes this protocol easy to perform with magnetic separa-
tors such as Auto-MACS (described earlier) which we have typi-
cally utilized for this purpose.
1. Isolate BMMNC as per Ficoll gradient separation protocol
above.
2. Plate BMMNC at a density of 50 × 106 cells per 100 mm plate
in MSC medium (DMEM with 10 % FCS).
3. Next day, wash off the non-adherent cells 3 times with HBSS.
4. Add 5 ml of 0.25 % trypsin–EDTA and incubate at 37 °C for
5 min to remove adherent cells. Visualize cells under microscope
to ensure adequate trypsinization. Dislodge with cell scraper if
needed.
5. Rinse flask with 5 ml medium to inactivate trypsin and collect
cells in 15 ml conical.
6. Spin cells down at 1,200 rpm for 5 min.
7. Resuspend cells in AutoMACS buffer (80 μl for up to 107
cells). Add 20 μl each of CD45 and CD14 microbeads.
8. Incubate on ice for 20 min.
9. Wash with 4 ml of AutoMACS buffer after incubation.
10. Perform AutoMACS with “Deplete” protocol, once only.
11. Spin down the negative fraction at 400 × g and resuspend in
MSC medium.
12. Plate in 100 mm dishes or T75 flasks with 0.1–0.5 × 106 cells
per flask; if the yield is low, plate at lower densities as well.
13. Isolated fibroblast colonies should be visible starting days 2–3.
To remove all the non-stromal components, change medium
every 2–3 days for the first 2 weeks as described for MSC
cultures.
96 Aravind Ramakrishnan et al.
3.8 Stromal Stromal cell lines HS5 and HS27are stable and well-characterized
Cell Lines lines that can be used to approximate various tissue microenviron-
ments. They are easy to culture and can be manipulated with rela-
tive ease (at least when compared to primary MSC).
1. Pre-warm medium (RPMI-1640 supplemented with 10 %
FBS) in a 37 °C water bath. Aliquot 45 ml of medium to a
50 cc conical tube.
2. Thaw cryopreserved cell vial (typically containing 5 × 106 cells)
in a 37 °C water bath. As with other cryopreserved cells, the
rule is to “freeze slowly and thaw quickly” to avoid cryogenic
damage to cells.
3. Remove vials to tissue culture hood once the frozen medium is
loosening from the side of the vial. Open vial, and add about
1 ml of pre-warmed medium to vial to complete the thaw.
4. Add the thawed cell–medium mixture to the 50 cc conical tube
containing warm medium. Mix well and spin down cells at
400 × g for 5 min.
5. Suction off supernatant and plate cells to one T75 flask (or
100 mm plate) with 10 ml medium. Transfer to incubator.
6. Determine viability of cells the next day—all viable cells should
be attached and exhibiting fibroblast-like pleomorphic mor-
phology. Cells can be split in about 48 h (1:3) if they are nearly
confluent.
7. Cells can be maintained by replacing medium without splitting
for up to a week after attaining full confluence.
8. To freeze stromal cell lines, use RPMI-1640 supplemented
with 30 % FBS. Resuspend about 5 × 106 cells per ml of this
medium and aliquot to cryovials. Add tissue culture-grade
DMSO to a final volume of approximately 10 % (100 ml to a
1 ml suspension). Move to cryo-freezing containers (such as
“Mr. Frosty” that allows for graded temperature drop) and
place in −80 °C freezer. Move frozen vials to liquid nitrogen in
24–48 h for long-term storage.
3.9 Reverse While stromal cells are not particularly difficult to grow and propa-
Transfection of gate, they can be challenging to transfect transiently with plasmids
Stromal Cell Line with or small RNAs. When working with siRNAs to inhibit specific tran-
siRNA/ miRNA scripts or miRNA, low-efficiency transfection (less than 25 %) is
almost guaranteed to result in a failed experiment, since the down-
stream tests of changes in RNA/protein levels or function are
likely not be reflected in those cells which were not transfected.
Commercially available lipid preparations such as lipofectamine
2000 from Invitrogen have been successfully adapted to the high-
efficiency transfection of several adherent cell types (such as HEK-
293Ts, HeLa). Typical protocols for liposomal transfections for
Primary Marrow-Derived Stromal Cells: Isolation and Manipulation 97
cells add the lipid–RNA mix to already adherent cells. Stromal cells
are typically difficult to transfect with this methodology presum-
ably due to extracellular matrix that is laid down immediately by
freshly plated cells that make lipid-based transfection difficult. One
way around this difficulty is by “reverse transfection.” In reverse
transfection cells are added to the medium that already has the
lipid–RNA mixes; higher efficiency likely results from the cells
being exposed to liposomal mixture before they adhere and secrete
matrix proteins [48]. We use siRNA or miRNA at 5–10 nmol final
concentration, but can be optimized based on the knockdown
achieved. The following are for a 12-well plate, but can be scaled
up or down as needed. All reagents need to be brought to room
temperature before beginning the protocol (see Note 13).
1. In a 5 ml clear sterile Falcon tube (#2058), mix 100 μl
OptiMEM with 2 μl lipofectamine 2000.
2. In a separate tube, mix 100 μl OptiMEM with 0.625 μl of
5 nmol miRNA/siRNA.
3. Let these mixtures stand for 5 min, and then gently mix
together. Keep for 10–15 min.
4. In the interim, plate 500 μl of medium without antibiotics
(usually RPMI with 10 % FCS) in a 12-well plate. Return to
TC incubator at 37 °C for 10–15 min.
5. Add 200 μl of the lipofectamine–RNA–OptiMEM mixture to
the 12-well plate slowly.
6. Return to incubator for another 10–15 min, total of about
30 min.
7. Suspend stromal cells (80,000–100,000) in 500 μl of antibiotic-
free medium (RPMI with 10 % FCS).
8. Add the cell suspension drop by drop to the well containing
the lipid mixture/medium. Gently tilt spread evenly (avoid cir-
cular swirling as this will allow for cells to congregate at the
center of each plate). Return to incubator.
Change medium (RPMI-1640 with 10 % FBS and antibiot-
ics) after 6 h or overnight incubation.
9. We typically can get transfect up to 80 % of the cells with the
above protocol (when using anti-GFP siRNA or plasmid for
controls, Fig. 7).
3.10 Concluding Mesenchymal stromal cells (MSC) can be isolated from bone mar-
Remarks and row with relative ease. Bone marrow screens which are typically
Summary discarded by-products of bone marrow harvests are an excellent
source of marrow cells and should be considered by investigators
with access to large clinical centers. The foremost consideration
while working with MSCs is to realize the wide variability of almost
all biological characteristics amongst MSCs established from vari-
ous donors. This is partly due to the complex nature of the culture
98 Aravind Ramakrishnan et al.
Fig. 7 Reverse transfection of stromal cell lines. (a) Stromal cell line HS27a stably expressing green fluorescent
protein (GFP) was transfected with control scrambled siRNA and visualized by inverted fluorescent microscopy
after 48 h. (b) Hs27a-GFP cell lines transfected with anti-GFP siRNA showing marked reduction in GFP expres-
sion of most cells
4 Notes
3. Both Hs27a and Hs5 are available from American Type Culture
Collection (ATCC, Manassas, VA) or can be directly obtained
from Beverly Torok-Storb laboratory in Seattle, WA.
4. We have used the FACS Aria sorter (Beckton, Dickinson and
Company, or BD) for our studies but multicolor sorters from
other manufacturers such as Beckman Coulter should work
just as well for these applications.
5. Several varieties of instrumentation are available for separation
of cells. The AutoMACS automated system is particularly use-
ful for larger cell numbers and can sort cells maintaining asep-
tic status and it is the system we use routinely. This instrument
is also typically available through flow-cytometry core facilities.
The protocol could be optimized for use with other magnetic
sorters with relative ease.
6. Since FBS is a complex biological product mass-produced as a
side-product from slaughterhouses, biological variability from
batch to batch is inevitable and it is recommended to screen lots
of FBS for performance in the particular biological assay of inter-
est. Commercial suppliers often provide small (~50 ml) aliquots
of FBS for testing to individual laboratories and hold those lots
for several weeks to allow for testing. Batches screened by the
commercial suppliers (deemed appropriate for MSC growth) are
also available directly albeit at typically much higher costs.
7. The horse serum needs to be specifically screened for the
ability to support LTCs and not MSCs.
8. Avoid delays in anticoagulant-free state given that marrow cell
pellets have powerful pro-coagulants activated and could result
in clots and clumps.
9. With complete or near-complete hemolysis, the supernatant
should be wine red in color and the cell pellet is pale or pinkish
but not frank red. If hemolysis is inadequate steps 1–3 can be
repeated 1–2 times (further hemolysis is unlikely to be helpful)
keeping in mind not to allow exposure to hemolytic buffer for
more than 5 min each time.
10. Recently, reports have indicated that Ficoll at 1.073 may be
more suitable for MSC progenitor isolation although this
observation has not been rigorously validated.
11. Protocols that do not use trypsin tend to leave primary MSCs
in a clumped state, so extra precaution needs to be taken to get
the cells into single-cell suspension.
12. Given the extreme stickiness of primary stromal cells, it would
be ideal to limit total cells in the approximately five million cells.
13. Also note that transfection needs to be performed in antibiotic-
free medium as the presence of antibiotics will reduce transfec-
tion efficiency and cell viability.
100 Aravind Ramakrishnan et al.
Acknowledgments
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Chapter 9
Abstract
Extracellular matrix can regulate multipotent mesenchymal stromal cells (MMSC) differentiation, with
potential applications for tissue engineering. A relief of mineralized bone takes essential effect on MMSC
fate. Nevertheless, delicate structure and a hierarchy of niches for stromal stem cells and its quantitative
parameters are not practically known. Here, we describe the protocol to define the basic approach provid-
ing a guiding for in vitro identification of quantitative features of artificial calcium phosphate niches which
promotes osteogenic differentiation and maturation of stromal stem cell.
Key words Prenatal stromal cells of human lung, Calcium phosphate surface, Short-term culture,
Artificial osteogenic niches, Alkaline phosphatase, Acid phosphatase, Osteocalcin,
Immunocytochemistry, Light microscopy, Scanning electron microscopy
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_9, © Springer Science+Business Media, LLC 2013
103
104 Igor A. Khlusov et al.
2 Materials
3. Acetone (С3H6O).
4. Alcohol (ethyl hydroxide, С2H5OH; 30°): Mix 31 mL of 96°
ethanol with 69 mL water.
5. Alcohol (ethyl hydroxide, С2H5OH; 50°): Mix 52 mL of 96°
ethanol with 48 mL water.
6. Alcohol (ethyl hydroxide, С2H5OH; 70°): Mix 73 mL of 96°
ethanol with 27 mL water.
7. Alcohol (ethyl hydroxide, С2H5OH; 90°): Mix 94 mL of 96°
ethanol with 6 mL water.
8. Alcohol (ethyl hydroxide, С2H5OH; 100°): Add 96° ethanol
2–3 times to heat-treated copper sulfate (CuSO4) crystals (see
Note 13).
9. 24-Well plastic plate.
10. Fume hood.
11. Phillips S 515 or Zeiss EVO 50 XVP scanning electron micro-
scopes (SEM).
3 Methods
3.1 Short-Term 1. Prior to use, sterilize the samples of CP-coated titanium plates
Cultivation with by dry-heart manner (Binder FD 53, Germany) for 60 min at
Composite Samples 160 °C.
2. Place one CP-coated titanium specimen in each plastic well
(1.86-cm2 area) of 24-well plate (Orange Scientific, Belgium).
Use plastic wells without composite samples as control of cell
growth (see Note 14).
3. Prepare HLPSC suspension in osteogenic medium with a con-
centration of 3 × 104 viable karyocytes/mL. Estimate cell via-
bility by means of 0.4 % trypan blue staining (see Note 15).
4. Add cell suspension in a volume of 1 ml per well of plastic
plates with or without CP-coated titanium specimens. Use
plastic wells with cultural medium probes along to estimate
initial levels of molecular metabolites (see Note 14).
5. Incubate the cell culture for 4 days in a humidified atmosphere
of 95 % air and 5 % CO2 at 37 °C (see Note 16).
6. Collect the suspensions of nonadherent cells from wells into
SCT.
7. Centrifugate SCT at 500 × g for 15 min.
8. Collect the supernatants from SCT and freeze immediately
0.9 mL aliquots at −20 °C before immunoenzyme and bio-
chemical tests.
108 Igor A. Khlusov et al.
3.2 Cells Fixation 1. Remove composite samples from plastic wells. Dry the samples
and plastic plates with adherent cells in the air for 24 h.
2. Fix composite samples and plastic plates with adherent cells in
formalin vapors for 30 s to use the (immuno)cytochemical
staining probes (see Note 18).
3. Fix other composite samples with adherent cells in glutaric
dialdehyde solution for at least 24 h at 4 °C to use SEM.
4. Wash with water for 1 min and air-dry.
5. Store fixed samples at 4 °C.
3.3 Cytochemical 1. Place composite samples with adherent cells to pure plastic
Staining for ALP by wells of a 24-well plate (see Note 14).
Diazocoupling 2. Use plastic wells with adherent cells after removing composite
Technique samples as control of ALP staining.
3. Add 0.5 mL of SWS into each well of a 24-well plate.
4. Incubate in dark at 37 °C for 45 min.
5. Aspirate SWS and rinse 3 × 1 min in distilled water.
6. Air-dry composite samples and 24-well plate.
7. ALP staining criteria (Fig. 1): blue sites of cytoplasmic enzy-
matic activity (see Note 19). Positive staining control: neutro-
cytes of human blood.
3.4 Cytochemical 1. Place composite samples with adherent cells to pure plastic
Staining for ACP by wells of a 24-well plate (see Note 14).
Diazocoupling 2. Use plastic wells with adherent cells after removing composite
Technique samples as control of ALP staining.
3. Add 0.5 mL of SWS for each well of a 24-well plate.
4. Incubate in dark at 37 °C for 2 h.
5. Make the procedures as in previous Subheading 3.3.
6. ACP staining criteria (Fig. 2): pink sites of cytoplasmic enzy-
matic activity (see Note 20). Positive staining control: neutro-
cytes of human blood.
Artificial Osteogenic Microterritories for Stromal Stem Cells 109
Fig. 1 Fourth day culture of prenatal stromal cells of human lung (HLPSC) on
plastic surface of 24-well plate. Cells with alkaline phosphatase (ALP) under-
staining are marked by black arrows. Magnification, 350×
Fig. 4 ALP stained HLPSC on calcium phosphate surface of composite samples. (a) Blue cell sites stained with
fast blue PP salt. (b) Cell site with garnet color stained with fast garnet Gbc salt. ALP-positive cells are marked
by black arrows. Magnification, 500×
3.7 Scanning 1. Place composite samples with adherent cells in pure 24-well
Electron Microscopy plastic plates (see Note 26).
2. Add 1 mL of 1 % OTS for 30 min.
3. Wash the samples 2 × 5 min with 1 mL of PBS.
4. Dehydrate the samples through a graded series of alcohols
(30°; 50°; 70°; 90°; 100°) for 15 min with each alcohol
concentration.
5. Rinse the samples 2 × 15 min with acetone (see Note 27) and
air-dry.
6. Store the samples at 4 °C in dark place before SEM.
7. Examine the samples in Phillips S 515 or Zeiss EVO 50 XVP
SEM at an accelerating voltage of 15 kV.
8. Result: (1) spreaded cells with rounded shape (Fig. 5) or fibro-
blast-like cells on the surface of samples (Fig. 6); (2) three-
dimensional (3D) shape of osteoblast-like cells in the sockets
(Fig. 7) (see Note 28).
4 Notes
Fig. 5 Scanning electron microscopy (SEM) image of rounded HLPSC on calcium phosphate surface of com-
posite samples. Cell is marked by an arrow. Magnification, 5,000×
Fig. 6 SEM image of fibroblast-like HLPSC on calcium phosphate surface of composite samples. Cell is marked
by black arrows. Magnification, 5,000×
Artificial Osteogenic Microterritories for Stromal Stem Cells 113
350
300
Square(S) of ALP staining, µm2 r = 0,939, p = 0,00001
250
200
150
100
50
-50
-100 0 100 200 300 400 500 600 700 800
S of sockets, µm2
Fig. 9 A regression curve of the squares of ALP-stained sites in HLPSC cells and surrounding sockets of rough
calcium phosphate surface
Fig. 10 SEM image of rough calcium phosphate surface. Spherolites with pores
are presented. Magnification, 1,250×
Acknowledgments
The authors are deeply indebted to: professor Yu.P. Sharkeev and
E.V. Legostaeva Ph.D. (Institute of Strength Physics and Materials
Science, SB of RAS, Tomsk, Russia) for designing and digital imag-
ing of titanium specimens with calcium phosphate coating; K.V.
Zaitsev Ph.D. (Stem Cells Bank Ltd., Tomsk, Russia) for cell cul-
ture provision; Joint Use Center for Materials Science (Tomsk
State University, Tomsk, Russia) for microscopic equipment use.
This work was supported by the Federal Goal Program of
Russian Federation (grant No 8036).
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Chapter 10
Abstract
Mature megakaryocytes (MM) can be up to 65 μM in diameter and due to their size, viable and pure MM
populations have been difficult to isolate in large numbers. Here in, we report a fluorescence activated cell
sorting (FACS) method by which viable and pure populations of 8 N, 16 N, 32 N, and 64 N MM can be
isolated from murine bone marrow (BM).
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_10, © Springer Science+Business Media, LLC 2013
121
122 Shen Y. Heazlewood et al.
antibody and the DNA binding dye Hoechst 33342, viable, pure
8 N, 16 N, 32 N, and 64 N MM can be sorted from murine BM.
Lineage depleted central BM cells were sorted at ~5,000 events per
second using a 100 μM nozzle set at 20 psi. MM function was
unaffected, MM isolated in this manner are able to form pro-
platelet extensions whilst in culture.
2 Materials
2.1 Isolation 1. Adult C57Bl/6J (Ly5.2), GFP, or RFP mice, 6–8 week old
of Bone Marrow (see Note 1).
2. Sterile #11 surgical blade and #3 handle.
3. Phosphate buffered saline (PBS): pH 7.2, 310 mOsm
(see Note 2) supplemented with 2 % Se (Serum): Defined
bovine calf serum, iron supplemented.
4. 1 ml syringes attached to 21-gauge and 23-gauge needles to
flush central marrow from bones.
5. 50 ml conical tubes for collection of BM.
6. 100 μm nylon cell strainers.
7. Hemocytometer and microscope equipped with phase contrast
or an automated cell counter. We use the Sysmex KX-21N
(Japan).
3 Methods
3.1 Isolation 1. Sacrifice three mice by cervical dislocation. Dissect and clean
of Bone Marrow femurs, tibias, and iliac crests (see Note 5).
2. Using a 1 ml syringe containing PBS-2 % Se attached to a
21-gauge needle, repeatedly flush out the central marrow into
a 50 ml centrifuge tube containing 40 ml of PBS-2 % Se by
inserting the needle in turn into each epiphysis of the femoral
124 Shen Y. Heazlewood et al.
shaft and the knee epiphysis of the tibia. To flush the iliac crest,
insert a 1 ml syringe fitted with a 23 gauge needle and flush the
BM into the same 50 ml centrifuge tube. Wash the cells by
centrifuging at 300 × g for 5 min at 4 °C (see Note 6).
3. Decant supernatant and resuspend the cell pellet in 10 ml
PBS-2 % Se.
4. Filter the cell suspension through a 100 μm nylon cell strainer
into a fresh 50 ml conical tube (see Note 7).
5. Dilute cells to 40 ml with PBS-2 % Se and perform a cell count.
3.2.3 Immunomagnetic 1. Add 0.25 ml of the washed Dynabeads to the cell suspension.
Separation 2. Incubate for 5 min at 4 °C with gentle tilting and rotation.
3. Place tube in the magnet for 2 min.
4. Transfer supernatant containing unbound (lineage negative)
cells to a fresh 5 ml polypropylene collection tube.
Isolating Viable High Ploidy Megakaryocytes 125
3.3.2 Gating Strategies The set up of any flow cytometer, including controls for compen-
for the Isolation of MM sation, is essential for the identification and accurate sorting of spe-
cific subsets of cells. Aliquots of the following cell samples are
required for selection of instrument settings and fluorescence
compensation.
1. Unstained BM cells for setting scatter profiles and gains for
background fluorescence.
126 Shen Y. Heazlewood et al.
Table 1
Flow cytometry instrumentation setup
3.3.3 Cell Collection Cells are sorted and collected at 4 °C into 1.7 ml microtubes
containing 0.5 ml of PBS-2 % Se (see Note 22). Reanalysis of the
sorted populations is not routinely performed due to the low cell
numbers recovered. Instead, the purity of the sorted populations is
assessed using a hemocytometer and microscope equipped with
phase contrast and fluorescence (see Notes 23–27).
4 Notes
Fig. 2 FACS isolation of viable, pure MM sorted on CD41 expression. (a) Enrichment gate for SSChighCD41bright
MM (R1). (b) Final sort gate for SSChighCD41bright MM post-enrichment (R2). (c) Phase contrast light microscopy
image of FACS sorted SSChighCD41bright MM (20×). (d) FACS sorted MM labeled with CD41 FITC (green), DNA is
shown in blue (40×)
Table 2
Sequential gating strategy and region (R) definitions for sorting MM based on ploidy
Fig. 3 Isolation of viable and pure high ploidy MM. (a) All cells are processed through the FSChighSSChigh gate
(R1), during a two-way enrichment sort of 8 N + 16 N MM one-way (R2) and 32 N + 64 N the other way (R3).
To ensure that adequate events can be visualized and the gates are set accurately, R2 and R3 gates are set on
an image that is not gated through R1. (b) All cells are repassed through R1, during the subsequent purity
recovery sort of 8 N MM one-way (R4) and 16 N MM one-way (R5). (c) All cells are repassed through R1, during
the subsequent purity recovery sort of 32 N MM one-way (R6) and 64 N MM one-way (R7). (d) Phase contrast
light microscopy images of sorted high ploidy MM. Hoechst stained DNA is shown in blue (40×)
Isolating Viable High Ploidy Megakaryocytes 129
onto the sorter and to enrich for MM, these mature lineage
cell types are depleted using the Dynabeads system. The num-
ber of Dynabeads used was optimized based on depletion effi-
ciency and experimental cost.
10. Beads are washed to remove the sodium azide in the bead stor-
age buffer.
11. This step is to ensure the removal of any residual Dynabeads
from the cell suspension.
12. During our experiments, we have found the PE conjugation of
CD41 is much brighter than FITC-CD41 and therefore we
use the two antibodies at different concentrations. Due to
both the size of MM and their high expression of CD41, when
sorting, a logarithmic scale for flurochrome detection is used
with decreased FITC or PE voltage settings. The FITC or PE
voltage is set based on CD41 positive MM being set at the
third decade.
13. We also tested the DNA binding dye Nuclear Red (Draq 5)
which does not require a UV laser. The ploidy populations are
not as distinct as those seen with Hoechst 33342 and due to
the cost of the reagent; we do not use Nuclear Red routinely.
However, if no UV laser is available, this reagent can be substi-
tuted for Hoechst 33342.
14. In order to reduce the incidence of nozzle clogs during sort-
ing, sort as soon as possible after labeling and filter the sample
immediately prior to sorting.
15. Although some FACS machines are able to sort “four-ways,”
due to the large size of the target cells and possible accuracy
issues, we only ever sort MM “two-ways.”
16. When we first attempted to sort MM, our samples were occa-
sionally contaminated by unwanted hemopoietic cells that
would proliferate in culture. Although in theory only large
(Forward Scatter; FSChigh) cells of high granularity (SSChigh)
should be sorted, because CD41 is also expressed on hemopoi-
etic stem and progenitor cells, these cells could be sorted if
they remained as cell clumps or were attached to MM. To opti-
mize purity, we tested a number of before sorting and during
sorting strategies (see Notes 17–19).
17. Prior to sorting, we performed a Nycoprep gradient pre-
lineage depletion. However, we determined that this addi-
tional step reduced the number of MM by 82.7 %.
18. We also tested a red cell lysis with NH4Cl prior to the lineage
depletion step. We demonstrated that this additional step
reduced our MM population by 47.1 %.
19. To maximize MM purity, our lineage depleted central BM
sample was first sorted through the “enrich” mode of the
Isolating Viable High Ploidy Megakaryocytes 131
Acknowledgments
The authors thank Dani Cardozo for assistance with animal work,
Michael Reitsma and Andrew Fryga for intellectual input and flow
cytometric support. In addition, we also thank Kathryn Flanagan
and Karen Clarke for flow cytometric support.
Isolating Viable High Ploidy Megakaryocytes 133
References
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19(4):250–5 Botrocetin agglutination of rat megakaryocytes:
2. Heazlewood SY, Neaves RJ, Williams B, a rapid method for megakaryocyte isolation.
Haylock DN, Adams TE, Nilsson SK (in press) Exp Hematol 20:1085–1089
Stem Cell Research 12. Hussein K (2011) Gene expression profiling in
3. Levine RF, Hazzard KC, Lamberg JD (1982) laser-microdissected bone marrow megakaryo-
The significance of megakaryocyte size. Blood cytes. Methods Mol Biol 755:429–439
60:1122–1131 13. Mazharian A (2012) Assessment of mega-
4. Nakeff A, Maat B (1974) Separation of mega- karyocyte migration and chemotaxis. Methods
karyocytes from mouse bone marrow by veloc- Mol Biol 788:275–288
ity sedimentation. Blood 43:591–595 14. Tolhurst G, Carter RN, Miller N, Mahaut-Smith
5. Levine RF, Fedorko ME (1976) Isolation of MP (2012) Purification of native bone marrow
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achieved with a two-step separation technique. try. In: Macey MG (ed) Flow cytometry:
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misnomer? Blood 60:213–219 the interactions between haemopoietic stem
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Chapter 11
Abstract
The infusion of different substances into the left ventricle of the brain allows substances to reach the
subventricular zone, one of the neural stem cell niches in the adult brain. Implantation of an osmotic
minipump delivers proteins, virus and drugs directly into the lateral ventricle to act locally. Here we
describe this method consisting on a cannula implanted into the lateral ventricle and linked to an osmotic
minipump via catheter. The cannula is fixed over the brain and the minipump is placed subcutaneously.
This system can be maintained from days up to several weeks and ensures constant and regular delivery of
the desired biological product.
Key words Brain, Minipump, Skull, Cannula, Stereotaxic frame, Substances administration, Neural
stem cell niche
1 Introduction
There are two neural stem cell niches in the adult mammalian
brain: the subventricular zone (SVZ) and the dentate gyrus in the
hippocampus (DG) [1, 2]. Located on the lateral wall of the lateral
ventricles, the SVZ is composed of different cell populations,
including a monolayer of ependymal cells that lines the ventricle,
NSCs, transit-amplifying cells, neural progenitors (neuroblasts),
astrocytes, and a dense network of blood vessels. The stem cell
niches contain several cell types that produce a range of factors to
control how fast the stem cells divide and the type of cells to which
they give rise.
The brain is not an easily accessible organ to inject substances,
because it is surrounded and protected by the skull. To access the
SVZ, niche it is necessary to get through the skull after calculating
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_11, © Springer Science+Business Media, LLC 2013
135
136 María Victoria Gómez Gaviro et al.
a b cannula
z
SVZ
y
cb ob
Lateral
ventricles
Fig. 1 Schematic of the brain coordinates to localize different regions and the mouse LV. (a) “y” indicates
rostro-caudal orientation, “z” dorsoventral, and “x” the lateral coordinates. Ob olfactory bulb; Cb, cerebellum;
SVZ, subventricular zone. (b) Schematic of a brain coronal section with the lateral ventricles and SVZ localiza-
tion; it shows how the cannula penetrates into the LV
2 Materials
2.1 The Osmotic 1. The system is provided with the flow moderator (Alzet
Minipump minipumps 1007D, flow rate 0.5 μl/h). This minipump delivers
substances for 7 days.
2. Brain infusion kit 3 (Alzet): includes the brain infusion can-
nulae, vinyl catheters tubes, and depth-adjustment spacers.
Cannulae in kit number 3 are 3 mm long, so, the cannula pen-
etrates 3 mm under the skull, where the lateral ventricles are.
3. Water bath 37°.
4. NaCl (0.9 %) (Sigma).
5. Forceps, scissors, insulin syringe, and insulin needle.
3 Methods
3.1 Preparation of A range of sizes, flow rates and durations are available. The
the Osmotic Minipump minipump should be chosen and according to the needs of the
experiment (the flow rate and volume). Proliferation studies in
the mouse SVZ can be done with Alzet 1007D or Alzet 1003D
minipumps [3].
Prepare the system the day before the surgery using sterile
conditions (in a hood or other sterile cabinet and gloves should
be used).
Load the osmotic minipump (Alzet minipumps 1007D, flow
rate 0.5 μl/h) with the substance avoiding making bubbles.
Incorporate the needle into the minipump with an insulin syringe
(see Note 1). Afterwards, connect the minipump with the flow
moderator.
138 María Victoria Gómez Gaviro et al.
Flow moderator
catheter
cannula
minipump
salt sleeve
Semi-permeable
membrane
Impermeable
membrane
Suspension, protein…
Fig. 2 Components of the minipump system. The osmotic minipump is composed of the flow moderator and
the minipump itself. The cannula and the catheter must be linked first and the catheter is then connected to
the minipump
3.2 Surgery Prepare the surgical area: stereotaxic frame, mask, vaporizer, O2,
sevoflurane, buprenorphine, fentanyl, surgical instruments.
The mouse will be anesthetized using general anesthesia by
inhalation.
Induction anesthesia should be done using an induction cham-
ber, with 2 % O2 and 6 % sevoflurane. When the animal is asleep
and has no reflex (check the pedal reflex) decrease the dose of
Delivery Substances with the Osmotic Minipump 139
anesthetic to 2–3 % and the O2 to 1–1.5 %. Take the animal out the
box and place it to the stereotactic frame. Put the mask on the
mouth/nose of the animal.
Administer 0.2 μg/ml/g of the analgesic fentanyl (or equiva-
lent) with an intraperitoneal (i.p.) injection. Fix the tail and the
legs to the surface. Spread ethanol 70 % over the head skin. Cut the
skin over the skull and open a hole with a scalpel number 15 on top
of the head (between the eyes and over them) and expose the skull
area (see Note 4). Clean up the surface of the skull to localize the
bregma. Crack the skull softly with a scalpel to remove the menin-
ges and visualize the bregma easily.
With the stereotactic frame measure 0.0 mm relative to bregma,
1.2 mm lateral, and 3.5 mm deep to inject the substance into the
SVZ. Alternatively, locate the position relative to bregma −2.0 mm
posterior, −1.95 mm lateral, and −1.9 mm ventral to the pial sur-
face to inject the substance in the hippocampus (in case of injec-
tions into the DG). Make a small hole with a drill in the area where
the coordinates indicate. This hole will allow you to open enough
space subcutaneously with the forceps to introduce the minipump
in the intercapsular region, without the need to open a new hole
on the back. Put a little amount of glue between the cannula and
the skull, in order to fix it to the skull. Close the skin over the can-
nula and put glue over the skin (see Note 5). Inject Buprenorphine
i.p. Decrease the anesthetic concentration but maintain the oxygen
for post-operative care.
Once the mouse is awake, the osmotic minipump will deliver
the substance directly into the ventricle or the DG homogenously
during a long period of time.
4 Notes
1. This step must be done very carefully and slowly. The mini-
pump will be full when a small drop appears over the top of the
minipump.
2. In the case of an adult MF1 mouse, the length of the catheter
should be around 2 cm. This must be long enough to avoid
the movement of the minipump in the interacapsular space.
3. The minipump should be manipulated with hands as little as
possible; it is advisable to use forceps to handle it.
4. Hair on the top of the brain can be removed with a shaver or
with scissors.
5. If the hole is small it is more convenient to close it with surgical
glue instead of sutures; it is quicker.
140 María Victoria Gómez Gaviro et al.
Acknowledgments
References
1. Doetsch F (2003) A niche for adult neural stem 6. Xia HJ, Suda S et al (2011) ACE2-mediated
cells. Curr Op Genet Dev 13:534–550 reduction of oxidative stress in the central nervous
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and their niches. Science 311:1880–1885 nomic function. PLoS One 6(7):U548–U558
3. Gómez-Gaviro MV, Scott C et al (2012) 7. Bedard AM, Maheux J et al (2011) Continuous,
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Chapter 12
Abstract
The subependymal zone (SEZ), frequently named as adult subventricular zone (SVZ), is a niche of adult
neural stem and progenitor cells that lines a large extension of the lateral ventricles of the brain. The majority
of the studies do not analyze the SEZ throughout its entire extension. Instead, studies of cell populations
within the SEZ typically focus their analysis on a narrow space between specific bregma coordinates that
provides a perspective of only a small portion of the SEZ. We have previously proposed a standard division
for the SEZ at the anterior–posterior and dorsal–ventral axes based on external brain anatomical hallmarks
(Falcao et al., PLoS One 7:e38647, 2012). Herein, we describe in detail the procedure and a stereological
approach that can be used to obtain an unbiased estimation of the SEZ cell proliferation under physiologi-
cal and pathological conditions. This approach takes into consideration clear SEZ anatomical divisions,
both on the anterior–posterior and dorsal–ventral axes, which will standardize future studies on the SEZ.
Key words Subependymal zone, Adult subventricular zone, Neural progenitor cells, Stereology,
Proliferation, Topography
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_12, © Springer Science+Business Media, LLC 2013
141
142 Ana Mendanha Falcão et al.
2 Materials
2.1 BrdU Preparation The BrdU dose commonly used to assess proliferation in the SEZ,
for Proliferation both for mice and rat is 50 mg/kg. In order to inject twice the
Assessment volume equivalent to the body weight of the rat prepare a solution
of 25 mg/mL of BrdU (Sigma, St. Louis, MO, USA) dissolved in
sterile saline (0.9 % w/v NaCl). For a volume of 10 mL weight
0.25 g of BrdU in an analytical balance and add it to 10 mL of
Unbiased Stereological SEZ Proliferation Analysis 143
3 Methods
3.2 Brain Freezing 1. Upon transcardiac perfusion with saline, collect the brain and
Procedure and Slices place it in a rectangular mold embedded in tissue tek O.C.T.
Collection compound (Thermo Scientific) that provides an appropriate
matrix for cryostat sectioning at −20 °C (see Note 7).
2. Snap frozen the brain by immersing the mold in a recipient
with isopentane and then into liquid nitrogen for a couple of
minutes until it is frozen (see Note 8).
3. Section the brain in a cryostat and collect the sections to Super
Frost plus slides (Menzel-Glazer from Thermo Scientific).
Make 20 μm coronal sections and start collecting all slices as
soon as the ventricle begins. Stop collecting brain sections
when you see large ventral ventricles at the level of the hippo-
campus. The bregma coordinates for the beginning and end of
the SEZ are the following: rat, bregma coordinates 2.28 to
−3.60 mm; mice, 1.18 to −2.06 mm (see Note 9).
4. The methodology to collect the sections is the following: make
series of 8 slides and collect consecutive sections to consecutive
slides, i.e., if you have series of 8 slides, in one slide each brain
section will be 160 μm distant from the subsequent (Fig. 1).
Following this methodology you will obtain slides with repre-
sentative sections at a defined constant distance from each
other (stereological requirement for proliferation assessment).
The sections collected to glass slides should be stored in slide
boxes and frozen at −20 °C (see Note 10).
A A A A A A A A
S1 1 S1 2 S1 3 S1 4 S1 5 S1 6 S1 7 S1 8
+ + + + + + +
1 2 3 4 5 6 7 8
9* 10 11 12 13 14 15 16
17* 18 19 20 21 22 23 24
25* 26 27 28 29 30 31 32
A A A A A A A A
S2 1 S2 2 S2 3 S2 4 S2 5 S2 6 S2 7 S2 8
33* 34 35 36 37 38 39 40
n* n+1"
n+8*
Sn
Fig. 1 Schematic representation of methodology used to collect brain sections. Sections are represented in
numbers (from 1 to 40 and then the following consecutive numbers) and are collected following the number
order represented in the figure; thus, 1 is the first brain section collected and 40 represents the 40th brain
section sliced. The distance between consecutive brain sections collected in the same slide (marked with an *
in slide 1 of series 1) is 160 μm (20 μm × 8); marked with a + in slides 1–8 of series 1 are slices collected
consecutively. Because they contain contiguous brain sections, the first slide (1) and the second slide (2) of the
first (S1) series are basically identical. To select representative sections of the SEZ choose the necessary series
of the same slide number (Sn = series n (n≥1) of the slide n (n1≤n≤8))
3.4 Proliferation Sampling methodology: The most important rule in this part is to
Assessment perform a systematic uniform sampling, i.e., to analyze sections at
Throughout the SEZ constant distance intervals, for instance 160 μm or 320 μm (or
even more or less). Using the methodology described above to col-
lect sections in the cryostat, if the analysis is performed for every
section in one slide the SEZ brain sections will be separated by
160 μm intervals; if the analysis is performed for every other sec-
tion in a slide, the brain sections will have 320 μm intervals between
them (see Note 23).
Random selection of first section: the first section to be analyzed is
the one displaying a well-defined juxtaposed ependymal layer
(Fig. 2) (see Note 24).
Microscope and software: To estimate the cell proliferation rates
throughout the SEZ use, for example, the Visiopharm Integrator
system (VIS) software in an Olympus BX51 microscope (Olympus,
Hamburg, Germany) or similar software. By using this software
you can draw the areas of interest and count within these areas the
nuclei stained for BrdU or Ki67 (in brown) with a count tool.
Delimitate the SEZ areas at low magnification (40×) and perform
the counting of BrdU positive cells within this defined areas at
high magnification (400×) (see Note 25).
Identification of the different SEZ divisions and regions: The coronal
sections collected in the cryostat comprise SEZ between bregma
Unbiased Stereological SEZ Proliferation Analysis 147
Fig. 2 Detail of the anterior subependymal zone displaying the ependymal layer
juxtaposed (arrows) and with BrdU positive cells stained in brown
cc
DG
LV
aca ac
A I P PP
Bregma
A I P PP
Table 1
Anterior–posterior axis anatomical references for the mouse and rat SEZ divisions
Bregma Bregma
coordinates coordinates
SEZ mouse (mm) rat (mm) Anatomical references
Anterior [1.18; 0.74] [2.28; 1.44] From the beginning to the end of the genu
of the corpus callosum
Intermediate [0.74; −0.14] [1.44; −0.12] From the end of genu of the corpus callosum
to the decussation of anterior commissure
Posterior [−0.14; −0.94] [−0.12; −1.72] From the decussation of anterior commissure
to the beginning of the hippocampus
Post posterior [−0.94; −1.94] [−1.70; −3.60] From the beginning of the hippocampus to
the fusion of the dorsal and ventral parts
of the lateral ventricle
Bregma coordinates are according to Paxinos and Franklin [15] for mice and Paxinos and Watson [14] for rat
analyzed for one animal. Repeat this procedure for all animals.
Group animals into different experimental conditions and calculate
the mean proliferation rate for the group by averaging the prolif-
eration rates of animals within the same group. If, within the inter-
mediate SEZ there is the need to distinguish between regions, i.e.,
RMS, dorsal, ventral and dorsolateral, proceed as mentioned
above. Estimate the BrdU positive cells for each region and the
correspondent areas for every section, average rates obtained for
each section to obtain the proliferation rate of one animal. Repeat
this procedure for all animals and estimate the mean proliferation
rate of a determined region for the group. The same rationale is
applied to estimate every specific division and/or region of the
SEZ (see Note 27).
Statistical analysis: Data can be presented as the mean (±SEM) and
analyzed with any statistical package software such as GraphPad
PRISM 5 software (GraphPad Software Inc., San Diego, CA). The
analysis consists of one-way analysis of variance (ANOVA) with
Bonferroni multiple comparison post-test analysis for single-factor
multiple group comparisons to determine differences between
three or more groups.
4 Notes
14. This step allows the linearization of the DNA strands were the
BrdU is inserted. If antigen retrieval is not performed prior to
this step, 30 min in HCl is not enough to detect BrdU stain-
ing; instead 1 h in HCl will work, however it may result in
nuclear damage, which will difficult the analysis under the
microscope. Use fresh HCl 2 M and always wear gloves.
15. This step is required to avoid nonspecific staining when devel-
oping the immunohistochemistry, because external horserad-
ish peroxidase (HPR) coupled to streptavidin is added to the
tissue and will bind to the biotinylated secondary antibody.
16. Verify if (a) the chamber is humid so your antibody solution
won’t evaporate and (b) the slides are not leaning and there-
fore the antibody is equally distributed.
17. This antibody can be reused once.
18. Streptavidin can be reused once.
19. Observing in the microscope while development occurs will
allow determining the time necessary to see strong brown
staining without background, it may vary between 2 and
10 min.
20. Hematoxylin diluted 4× provides a weaker staining and makes
it easier to observe the BrdU nuclear brown staining.
21. The slides can be kept in xylene for some minutes until sec-
tions are covered with a coverslip.
22. Be careful not to introduce air bubbles between coverslip and
sections.
23. Notice that shorter distance intervals will provide you more
accurate estimation but will increase the time you will spend
on the analysis. Intervals of 160 μm or 320 μm between ana-
lyzed sections typically provide accurate estimations for SEZ
proliferation analysis.
24. The first section analyzed must be assigned randomly (stereol-
ogy principle). In fact the process is already random because it
is not known which slide has the first section comprising the
beginning of the SEZ. This would only be possible if every
section collected would be stained prior to the selection of
slides. If you perfuse animals with PFA (and not only saline as
described herein) the first section is likely to have the ependy-
mal layer not juxtaposed but instead a slightly opened ventricle
can be observed. Furthermore, the areas estimated for the
SEZ will be inferior due to the shrinkage of the brain caused
by PFA perfusion and by the histological procedures.
25. Alternatively, if you perform all the protocol with fluorescence
immunohistochemistry you can do the same analysis by taking
images, in a fluorescence microscope or confocal microscope,
of the entire SEZ and then estimate the areas and the cell
152 Ana Mendanha Falcão et al.
References
Abstract
Embryonic stem cells (ESC) are totipotent, self-renewing, and clonogenic, having potential to differenti-
ate into a wide variety of cell types. Due to regenerative capability, it has tremendous potential for treating
myocardial infarction (death of myocardial tissue) and type 1 diabetes (death of pancreatic beta cells).
Understanding the components regulating ESC differentiation is the key to unlock the regenerative
potential of ESC-based therapies. Both the stiffness of extracellular matrix (ECM) and surrounding niche/
microenvironment play pivotal roles in ESC differentiation. Matrix metalloproteinase-9 (MMP9) induces
fibrosis that causes stiffness of the ECM and impairs differentiation of cardiac stem cells into cardiomyo-
cytes. Here, we describe the method of ESC culture and differentiation, and the expression of MMP9 and
its inhibitor, tissue inhibitor of metalloproteinase-4 (TIMP4) in differentiating ESC.
Key words Stem cell, MMP9, TIMP-4, Differentiation, Extracellular matrix, Cardiomyocytes
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_13, © Springer Science+Business Media, LLC 2013
153
154 Paras Kumar Mishra et al.
2 Materials
Use all the materials in sterile conditions. The ESC and mouse
embryonic fibroblast (MEF) cells should be stored in a liquid
nitrogen tank, whereas the culture medium should be stored
at 4 °C.
2.3 MEF Culture 1. The MEF culture medium is similar to the ESC culture medium
Medium Components except the concentration of FBS is 10 %.
2.4 MEF Inactivation 1. MEFs are frozen in MEF culture medium + 10 % DMSO at
5 × 106/cryovial and stored in liquid nitrogen. MEFs can be
inactivated by γ-irradiation (5,000 rads) in a cell irradiator
packed with dry ice to prevent thawing during irradiation.
They are used as a feeder layer for ESCs (see Note 3).
3 Methods
3.1 MEF Culture 1. Thaw frozen MEFs quickly in a water bath maintained at 37 °C
for 90 s and dilute vial contents with MEFs medium in a 15 ml
conical tube and spin at 270 × g for 5 min (see Note 3).
2. One thawed cryovial of 5 × 106 MEFs is enough for 1 confluent
10 cm plate.
3. Culture γ-irradiated MEFs on a sterile tissue culture plate
coated with 0.1 % gelatin in ultrapure water under a biosafety
cabinet and place in a cell culture incubator maintained at
37 °C with 5 % CO2 (see Note 4).
4. Remove gelatin at the time of ESC plating (see Note 5).
5. To plate MEFs, resuspend the pelleted MEFs in 10 ml MEF
medium, distribute into the gelatin coated plate, and place in
incubator for at least 24 h to allow MEFs to become confluent
before adding ESCs.
156 Paras Kumar Mishra et al.
Fig. 1 The different stages of embryoid body (EB). (a) One day after plating of embryonic stem cell (ESC) on
mouse embryonic fibroblasts (MEFs) shown in low magnification (10×). The round shape EB is shown by
arrows. (b) The 3 days EB shown in high magnification (20×). (c) The MEF free undifferentiated floating EB.
(d) Differentiated and attached EB
3.2 ESC Culture 1. Twenty-four hours after plating MEFs, remove the medium.
Wash 1× with DPBS. Add 10 ml ESC medium with LIF. Place
back into the incubator for 1 h.
2. Thaw frozen ESCs quickly in a water bath maintained at 37 °C
for 90 s and dilute cryovial contents with ESC medium into a
15 ml conical tube (see Note 3).
3. Spin the tube at 270 × g for 5 min.
4. Remove the supernatant by vacuum using sterilized pipette.
5. Add 5 ml of fresh warm ESC medium with LIF and resuspend
the pellet gently (see Note 6).
6. Distribute this 5 ml of ESC suspension to the 10 cm plate
containing the MEFs and place back into the incubator
(see Note 7).
7. The plated ESCs exhibit a small round morphology. They
attach to the MEFs and proliferate forming oval shaped colo-
nies (Fig. 1a).
Cardiac Stem Cell Niche, MMP9, and Culture and Differentiation… 157
3.3 ESC 1. Use ESC medium without LIF for differentiation of ESC (ESC
Differentiation differentiation medium).
2. To remove feeder cells from ESC, coat a 10 cm plate with
0.1 % gelatin for 30–45 min.
3. Remove gelatin at the time of ESC plating (see Note 5).
4. Trypsinized ESCs with MEFs with 5 ml of TrypLE Express
and disaggregate it with a fire polished pipette.
5. Transfer the disaggregated ESC into a 15 ml tube.
6. Add 8 ml of ESC medium to the 5 ml of trypsinized cells and
mix it by pipetting up and down.
7. Centrifuge the cell containing medium at 270 × g for 5 min.
8. Remove the supernatant by vacuum using sterilized Pasteur
pipette.
9. Resuspend the pellet with 10 ml of fresh medium.
10. Transfer the medium into 0.1 % gelatin coated plate and incu-
bate it for 1 h (see Note 9).
11. After 1 h, transfer the medium from 10 cm gelatin coated plate
to a 15 ml tube.
12. Centrifuged the tube at 270 × g for 5 min.
13. Remove the supernatant and resuspend the pellet in 1 ml of
ESC medium.
14. Count the ESC number (see Note 10).
15. Dilute ESCs in a manner that 540,000 ESCs are suspended in
36 ml of ESC medium without LIF (15,000 cells/ml). This
volume is good for two 6-well plates at 3 ml of cell suspension/
well of the plate (see Note 11).
16. The plated cells are not disturbed for 48 h. Differentiating
ESCs will begin to agglomerate into free-floating EBs.
158 Paras Kumar Mishra et al.
Fig. 2 Different regions of differentiated ESC. (a) From central (black spot, arrow ) to the remote (distant from
the black spot ) of the differentiated EB at low magnification (10×). (b) The high magnification (20×) view of
the area of differentiated ESC, where contractile cardiomyocytes are observed
Fig. 3 Expression of MMP9 in differentiating ESC. (a) The expression of MMP9 (green color ) in differentiating
area, the central region of EB. The blue color is DAPI, which stains nucleus. (b) The expression of MMP9 in the
terminally differentiated region (remote from the center of EB). (c) The expression of MMP9 in undifferentiated
EB. Scale bar is 50 µm
Fig. 4 The expression of TIMP4 (green color) in differentiating region of EB. The left panel show DAPI (blue) that
stains nucleus, the middle panel show TIMP4 staining (green) and the right panel show merged imaged with
blue and green. Scale bar is 50 µm
160 Paras Kumar Mishra et al.
Fig. 5 The differentiated EBs are treated with 5 and 25 mM of D-glucose for 24 h and stained with MMP9
(green) and DAPI (blue). (a) The left panel control (5 mM) group show less expression of MMP9. (b) The right
two panels (b) (i), and (b) (ii) show robust expression of MMP9 (green). Scale bar is 50 µm
4 Notes
Acknowledgments
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Chapter 14
Abstract
Study of stem cell phenotype and functions requires their proper isolation. Stem cells isolated from skeletal
muscle are a useful tool to explore molecular pathways involved in the regulation of myogenesis. Among
progenitor cells, a subset of cells, called reserve cells, has been identified, in vitro, in myogenic cell cultures.
This subset of cells remains undifferentiated while the main population of progenitor cells commits to
terminal myogenic differentiation. When replated, these reserve cells grow as new colonies of progenitors.
At the time of differentiation, they reform both differentiated myotubes and undifferentiated reserve cells.
Here, we present a protocol to obtain and further isolate reserve cells from both human and murine
myogenic cell cultures, together with techniques to analyze their cell cycle status.
Key words Skeletal muscle, Stem cells, Satellite cells, Myogenic progenitor cells, Myogenesis, Human,
Mouse, Cell cycle, Nocodazole synchronization, Reserve cells
1 Introduction
Skeletal muscle stem cells, also called satellite cells, reside under the
basal lamina of the myofibers and are normally quiescent. Following
damage to the myofiber, these stem cells proliferate and differenti-
ate to form new myofibers, and self-renew to reconstitute the res-
ervoir of quiescent satellite cells [1–3]. Satellite cells may be
isolated from both human and murine muscle, and give rise to
myogenic precursor cells in vitro. Myogenic cell cultures have been
used for a long time to parallel in vivo experiments and to under-
stand cellular and molecular mechanisms of myogenesis [4, 5].
A subpopulation of quiescent, noncycling, undifferentiated cells has
been identified in myogenic precursor cell cultures. When replated,
these cells, named “reserve cells” (RCs), give rise to cultures that
eventually form both differentiated myotubes and new RCs
[6–11]. RCs, that are in vitro capable of both self-renewal and
myogenic differentiation may be related to satellite cells described
in vivo, responsible for skeletal muscle growth and regeneration.
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_14, © Springer Science+Business Media, LLC 2013
165
166 Rana Abou-Khalil et al.
2 Materials
3 Methods
3.2 Human Myogenic 1. Plate human primary skeletal myogenic cells at a concentration
Cell Synchronization of 3,000 cells/cm2 in 100 mm petri dish in 10 ml of growth
medium.
2. Add 100 μg/ml of bFGF at a final concentration of 10 ng/ml
and grow cells for 4 days at 37 °C.
3. Add Nocodazole to the culture at a final concentration of
1 μg/ml. Incubate for 16 h at 37 °C (see Notes 11 and 12).
4. Remove medium, wash cells twice with PBS (1×) and add
10 ml of fresh growth medium.
5. Assess cell synchronization by flow cytometry using BrdU
staining to monitor actively cycling cells, as described by APC
BrdU flow kit catalog provided by supplier. Briefly, myogenic
cells are incubated with BrdU at a final concentration of 10 μM
in growth medium for 16 h. Cells are subsequently fixed and
treated for staining with anti-BrdU as recommended by man-
ual supplier. BrDU stained cells are analyzed by flow cytometry
(Fig. 1) (see Note 13).
Fig. 1 Synchronization of human myogenic cells. G2/M cell cycle arrest following
Nocodazole treatment was confirmed using APC BrdU Flow kit, by measuring
cell-incorporated BrdU (anti-BrdU antibody) and total DNA content (7-AAD). Cells
incubated with only bFGF (a) present normal cell cycle profile with cells in G1/G0
(R4), S (R2 and R3), and G2/M (R5) phase (the small cell population in the bottom
left represent necrotic/apoptotic cells). Rare cells are observed in R3 region as
primary human myogenic cells exhibit a long cell cycle (more than the incubation
time with BrdU (16 h)). When cells are treated with Nocodazole (b), the majority
of myogenic cells are observed in G2/M phase (R5), confirming their synchroni-
zation and G2/M arrest after Nocodazole treatment
Skeletal Muscle Reserve Cells: Isolation and Cell Cycle Analysis 171
3.4 Isolation Obtaining Satellite cells. We use two different approaches, previ-
of Murine RCs ously described, to isolate pure satellite cells:
(A) Cultures of isolated myofibers prepared from EDL and Soleus
muscles [19]. Single myofibers are separated from intact mus-
cles following collagenase digestion and mechanical triturat-
ing. Single myofibers are plated on Matrigel, and proliferating
satellite cell progenies migrate out from the host myofiber after
2 days of plating.
(B) Cultures of FACS-sorted satellite cells prepared from limb
muscles [20]. Satellite cells are isolated from hindlimb muscles
following collagenase–dispase digestion. Then, satellite cells
can be isolated based on negative selection for CD45, CD31,
and Sca1, and positive selection for CD34 and α7-integrin.
1. Coat petri dishes with Matrigel. Thaw an aliquot by placing it
on ice for at least 30 min to allow the Matrigel to completely
liquefy. Use a chilled glass pipette to draw up the diluted Matrigel
solution and coat the dishes (1 ml per 60 mm petri dish).
Fig. 2 Evaluation of the quiescence of human myogenic cells with Hoechst and
Pyronin Y. Non-treated human myogenic cells show cells present in all phases of
the cell cycle (b) while the pyronin Y labelling is lost after RNAse treatment (a).
When Ang1, a promotor of RC quiescence, is added, the number of cells in G0 is
increased (c) while when bFGF, a promotor of cell cycle, is added, the number of
cells in G1 is increased (d)
Skeletal Muscle Reserve Cells: Isolation and Cell Cycle Analysis 173
Let the solution in the dish, on ice, for 2–3 min, and then use
the same chilled pipette as before to remove the Matrigel solu-
tion and place it back in the aliquot tube that was kept on ice.
This will create a thin Matrigel coating at the bottom of the
dishes.
2. Plate cells in the Matrigel-coated culture dishes in 2 ml of
growth medium. When using 35-mm dishes: if starting from
FACS-sorted cells, plate 2 × 104 cells, if starting from isolated
myofibers, plate 50 fibers.
3. Grow cells for 9 days at 37 °C and replace growth medium
every 3 days (see Notes 21–23).
4. At day 9, cells should be at 80 % of confluent density (see Note
24), remove growth medium, wash twice with PBS (1×), and
add 2 ml of differentiation medium.
5. Incubate with differentiation medium for 6 days at 37 °C
(see Note 25).
6. Remove differentiation medium and wash cells three times
with PBS (1×).
7. Trypsinate (0.1 ml/cm2 of Trypsin EDTA 0.25 %) for 3–5 min
at 37 °C. Monitor cell detachment under microscope
(see Note 26).
8. Transfer detached cells into a 50 ml conical centrifuge tube
containing 10 ml of growth medium, rinse with 5 ml of growth
medium to inactivate trypsin activity.
9. Place a 40 μm cell strainer on top of 50 ml conical centrifuge
tube and transfer collected cells through the filter. Wash filter
with 10 ml of growth medium.
10. Centrifuge cells that have passed through the filter (RCs) for
10 min at 300 × g at room temperature.
11. Discard supernatant and resuspend RC cell pellet with 10 ml
of growth medium.
12. RCs may be used for molecular analysis or may be plated to
give rise to new colonies.
13. Myotubes collected in the top of the filter may be recovered for
molecular analyses. Place the filter containing the myotubes in a
Petri dish, add some medium and collect the cells in the filter.
4 Notes
Fig. 4 Culture of murine cells showing differentiated myotubes and side reserve
cells. After differentiation step, murine cell cultures are labelled for the transcrip-
tion factor Pax7 that marks quiescent and proliferating myogenic cells and for
Myosin Heavy Chain (MyHC) that is expressed by fullly differentiated myogenic
cells. Pax7pos MHCneg cells are located beside Pax7neg MHCpos large myotubes
Acknowledgments
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71:1466–1470 (2004) Cytometry of the cell cycle: cycling
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down-regulation of MyoD and Myf-5 generates Satellite cells attract monocytes and use macro-
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retinoblastoma-like protein p130 is involved in 20. Pasut A, Oleynik P, Rudnicki MA (2012)
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(1998) The muscle regulatory factors MyoD 22. Zammit PS, Relaix F, Nagata Y et al (2006)
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Chapter 15
Abstract
Skeletal muscle tissue has a remarkable capability of regenerating in pathological conditions or after injury.
The principal muscle stem cells, satellite cells, are responsible for this prompt and efficient process.
Normally quiescent in their niches underneath the basal lamina of each muscle fiber, satellite cells become
activated to repair or form new fibers. Ideally, healthy donor stem cells could be transplanted to regenerate
the skeletal muscle tissue to repair a genetic defect. However, to be efficient, cell grafting requires modula-
tion of the host muscle environment to allow homing of, and regeneration by, donor satellite cells. Here,
we provide methods to modulate the host mouse muscle environment in order to destroy or preserve the
muscle niche before transplanting donor satellite cells. We also describe methods to investigate donor-
derived muscle regeneration and self-renewal.
Key words Stem cells, Satellite cells, Skeletal muscle, Muscle regeneration, Differentiation,
Self-renewal
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_15, © Springer Science+Business Media, LLC 2013
179
180 Luisa Boldrin and Jennifer E. Morgan
Fig. 1 Histology of transverse sections of murine wild type muscle, stained with Hematoxylin and Eosin. In
uninjured muscle, myofibers are mature, with nuclei in a peripheral position. Three days after myotoxin
(notexin) injection, muscle fibers are destroyed. Four days after injury, myotubes begin to form. Five days after
injury, newly formed, small, basophilic, centrally nucleated fibers are present. Scale bar = 100 μm
2 Materials
2.1 Host and Donor Breeding of mice and experimental procedures were carried out in
Mouse Strains the Biological Services Unit of University College London,
Institute of Child Health, in accordance with the Animals (Scientific
Procedures) Act 1986. Experiments were carried out under Home
Office licence.
2.1.1 Host Mouse Strain The mdx mouse, which is the genetic and biochemical homologue
of human Duchenne Muscular Dystrophy [8], was crossed to a
nude (Foxn1–/–) background to generate dystrophin-deficient,
immunodeficient mdx nu/nu mice [9]. This is a valuable mouse
model for cell transplantation, as the lack of T cells prevents immu-
nological rejection of donor cells. Furthermore, in mdx nu/nu
mice aging occurs prematurely, making them a good model to
study an advanced stage of dystrophy [10]. In transplantation
experiments, as extensive degeneration and regeneration occurs in
mdx at 3 weeks of age, this is an optimal time to graft donor cells.
2.1.2 Donor Mouse To investigate the contribution of donor cells to muscle regenera-
Strains tion, we isolate donor cells from the 3F-nlacZ-2E transgenic
mouse, where all the myonuclei that express myosin light chain-3F
also express β-Gal [11] (Fig. 2). Satellite cells in these mice are
β-Gal negative [10, 11] (Fig. 2).
To investigate if grafted donor cells give rise to satellite cells,
we use the Myf5nlacZ/+ mouse to isolate donor cells. In this mouse,
the majority of satellite cells expresses, β-Gal [12, 13]. β-Gal
expression in nuclei underneath the myofiber basal lamina marks
donor-derived satellite cells (Fig. 2). However, it should be taken
into account when performing analyses that newly regenerated
myonuclei retain Myf5nlacZ expression for a short time [14, 15].
To investigate integration of donor cells in muscle, inside and/
or outside the myofibers, we use the β-actin-Cre:R26NZG mouse
(obtained from crossing a homozygote male β-actin-Cre (FVB/
N-Tg(ACTB-cre)2Mrt/J)—a kind gift from Massimo Signore,
UCL—with an homozygote female R26NZG (Gt(ROSA)26Sortm1(CAG-
lacZ,-EGFP)Glh
) (The Jackson Laboratory, USA)). Nuclei underneath
the basal lamina of myofibers expressing β-Gal can be either myo-
nuclei or satellite cells. If found outside the myofiber basal lamina,
they can be myoblasts or other cell types (Fig. 2).
Fig. 2 Single fibers isolated from different donor mouse models. In the 3F-nlacZ-2E myofiber, only the myonu-
clei are X-gal stained. The X-gal negative, DAPI positive nucleus on the fiber (arrow) is a satellite cell. In the
Myf5nlacZ/+ myofiber, only satellite cells are X-gal positive; note that a strong X-gal staining can quench DAPI
signal (arrow). In the β-actin-Cre:R26NZG myofiber, all the nuclei are X-gal positive. Size bar = 10 μm
2.6 Detection of 1. Gum Tragacanth (Sigma G-1128) and cork disks to mount
Regenerated Muscle harvested muscles (as detailed in ref. [16]).
Fibers of Donor Origin 2. Isopentane and liquid nitrogen.
2.6.1 Muscle Freezing
3 Methods
Myotoxin Injury 1. Inject the myotoxin percutaneously into the TA of the anes-
thetized mouse in the volume and concentration described in
Subheading 2.2.2.
3.2 Preparation of a 1. After pooling all fibers in a single dish with plating medium,
Pure Population of release satellite cells by physical trituration for 5 min with a
Donor Mouse Satellite 19G needle mounted on a 1 ml syringe. Pass cell suspension
Cells through 40 μm cell filter to remove hypercontracted fibers.
Counting of stripped satellite cells is very difficult and ambigu-
ous due to the large amount of debris sized similarly as the very
small freshly-isolated satellite cells. As the number of satellite
cells per fiber is known [15], counting the number of fibers
before stripping allows the expected number of satellite cells to
be estimated [4, 10, 18].
2. If it is necessary to reduce the volume of medium containing
stripped satellite cells, perform two rounds of centrifugation:
the first at 240 × g for 15 min, to collect bigger cells, the sec-
ond at 600 × g for 20 min at 4 °C to collect the smaller cells (see
Note 3). Collect both pellets and mix them in the desired
volume of medium.
186 Luisa Boldrin and Jennifer E. Morgan
3.3.2 Test of Donor- 1. Three weeks after cell grafting (see Note 6), anesthetize mice
Derived Satellite Cell and inject notexin into TA muscle(s) as described in
Functionality Subheadings 3.1.2 and 3.1.2.1.
Fig. 3 Detection of donor-derived muscle regeneration. (a) When 3F-nlacZ-2E satellite cells are grafted in mdx
nu/nu mice, their contribution to muscle regeneration is demonstrated by the presence of X-gal positive myo-
nuclei inside fibers that express dystrophin (shown by immunostaining in a serial section). (b) Grafting of
β-actin-Cre:R26NZG satellite cells allows us to determine whether satellite cells give rise to cells other than
satellite cells or myofibers. (c) Grafting of satellite cells isolated from Myf5nlacZ/+ myofiber allows the detection
of donor-derived satellite cells on myofibers (5× magnification). Identification of donor-derived satellite cells
can be performed on transverse sections: these are cells that are X-gal positive and are at the myofiber periph-
ery (arrows), underneath the basal lamina (stained by laminin antibody). Nuclei counterstained by DAPI. Size
bar = 50 μm
Fig. 4 Test of functionality of donor-derived satellite cells. In a grafted mdx nu/nu muscle that has been left for
3–4 weeks to allow complete regeneration, myofibers of both host and donor origin are destroyed by notexin
injection. This promotes satellite cell-mediated regeneration. (a) Schematic representation of muscle sections
1 week after notexin injection: fibers that have not been destroyed by notexin are of larger diameter with nuclei
in a peripheral position (in grey ) and may be of either host (dystrophin negative) or donor (dystrophin positive
in red ) origin. Fibers that are regenerating 7 days after notexin injury are small, centrally-nucleated and
strongly express neonatal myosin (green ); these may be either of host (dystrophin negative) or donor (dystro-
phin positive in red ) origin. (b) Newly regenerated donor-derived myofibers in a grafted and notexin-injured
host muscle immunostained with neonatal myosin (green ) and dystrophin (red ) antibodies. Nuclei counter-
stained by DAPI. Size bar = 50 μm
4 Notes
1. Time and dose of irradiation are critical variables for cell graft-
ing [4, 17]. A radiation dose higher than 18 Gy can also pro-
mote donor cell engraftment if host satellite cells are
incapacitated but not totally depleted, thus preserving a func-
tional host niche. Three days after 25 Gy irradiation of host
muscle, nearly all host satellite cells are depleted, whilst on the
day of radiation at least some host satellite cells remain in their
niches. In keeping with the necessity of preserving the host
muscle satellite cell niche, 25 Gy radiation promotes donor-
derived muscle regeneration only if applied on the day of,
rather than 3 days before, grafting. Similarly, using 18 Gy irra-
diation, a comparable amount of donor-derived muscle is
formed if irradiation is applied up to 3 days but not 4 weeks
before cell grafting, when nearly all host satellite cells are
depleted [4].
2. As recovery is always quicker in the case of inhalable anesthe-
sia, this is to be preferred to injectable anesthesia whenever
possible.
3. It is preferable to use a refrigerated centrifuge at 4 °C. This
helps to maintain cell viability.
4. Keep cells on ice after isolation until injection.
5. In an in vivo experiment, the best control is the muscle of the
contralateral leg (either untreated or treated with medium alone).
6. It is necessary to wait a sufficient time for muscle regeneration
to have occurred, before analysis or performing a further
procedure—usually 3–4 weeks.
7. Fixation can be compromised if pH of PFA is not adjusted
to 7.5.
8. Dytrophin production in host fibers regenerated by donor cells
is segmental: only fragments of fibers where donor nuclei are
integrated express dystrophin protein [14].
190 Luisa Boldrin and Jennifer E. Morgan
Acknowledgments
The authors thank Miss Rowan Asfahani for the pictures presented
in Fig. 1. This work was supported by Muscular Dystrophy
Campaign (grant code RA3/776) and Wellcome Trust University
Award (grant code 08241/Z/07/Z).
References
1. Charge SB, Rudnicki MA (2004) Cellular and regulatory sequences confer regionalized car-
molecular regulation of muscle regeneration. diac and skeletal muscle expression in trans-
Physiol Rev 84(1):209–238 genic mice. J Cell Biol 129(2):383–396
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fibers. J Biophys Biochem Cytol 9:493–495 Arnold H, Buckingham M (1996) Gene
3. Boldrin L, Muntoni F, Morgan JE (2010) Are targeting the myf-5 locus with nlacZ reveals
human and mouse satellite cells really the expression of this myogenic factor in mature
same? J Histochem Cytochem 58(11):941– skeletal muscle fibres as well as early embryonic
955. doi:10.1369/jhc.2010.956201 muscle. Dev Dyn 206(3):291–300
4. Boldrin L, Neal A, Zammit PS, Muntoni F, 13. Beauchamp JR, Heslop L, Yu DS, Tajbakhsh
Morgan JE (2012) Donor satellite cell engraft- S, Kelly RG, Wernig A, Buckingham ME,
ment is significantly augmented when the host Partridge TA, Zammit PS (2000) Expression
niche is preserved and endogenous satellite of CD34 and Myf5 defines the majority of qui-
cells are incapacitated. Stem Cells 30(9):1971– escent adult skeletal muscle satellite cells.
1984. doi:10.1002/stem.1158 J Cell Biol 151(6):1221–1234
5. Harris JB (2003) Myotoxic phospholipases A2 14. Blaveri K, Heslop L, Yu DS, Rosenblatt JD,
and the regeneration of skeletal muscles. Gross JG, Partridge TA, Morgan JE (1999)
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6. McGeachie JK, Grounds MD, Partridge TA, studies on isolated fibers. Dev Dyn
Morgan JE (1993) Age-related changes in rep- 216(3):244–256
lication of myogenic cells in mdx mice: quanti- 15. Collins CA, Olsen I, Zammit PS, Heslop L,
tative autoradiographic studies. J Neurol Sci Petrie A, Partridge TA, Morgan JE (2005)
119(2):169–179 Stem cell function, self-renewal, and behav-
7. Emery AE (2002) The muscular dystrophies. ioral heterogeneity of cells from the adult mus-
Lancet 359(9307):687–695. doi:S0140- cle satellite cell niche. Cell 122(2):289–301
6736(02)07815-7, [pii] 10.1016/ 16. Collins CA, Zammit PS (2009) Isolation
S0140-6736(02)07815-7 and grafting of single muscle fibres.
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Gomeza V, Foster K, Muntoni F, Wells DJ, doi:10.1007/978-1-59745-060-7_20
Dickson G (2011) Chronic systemic therapy 17. Gross JG, Bou-Gharios G, Morgan JE (1999)
with low-dose morpholino oligomers amelio- Potentiation of myoblast transplantation by
rates the pathology and normalizes locomotor host muscle irradiation is dependent on the
behavior in mdx mice. Mol Ther 19(2):345– rate of radiation delivery. Cell Tissue Res
354. doi:10.1038/mt.2010.261 298(2):371–375
9. Partridge TA, Morgan JE, Coulton GR, 18. Neal A, Boldrin L, Morgan JE (2012) The satel-
Hoffman EP, Kunkel LM (1989) Conversion lite cell in male and female, developing and adult
of mdx myofibres from dystrophin-negative to mouse muscle: distinct stem cells for growth and
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Nature 337(6203):176–179 doi:10.1371/journal.pone.0037950
10. Boldrin L, Zammit PS, Muntoni F, Morgan JE 19. Hoffman EP, Morgan JE, Watkins SC,
(2009) Mature adult dystrophic mouse muscle Partridge TA (1990) Somatic reversion/sup-
environment does not impede efficient pression of the mouse mdx phenotype in vivo.
engrafted satellite cell regeneration and self- J Neurol Sci 99(1):9–25
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doi:10.1002/stem.162 sor cells injected into irradiated mdx mouse
11. Kelly R, Alonso S, Tajbakhsh S, Cossu G, muscle persist after serial injury. Muscle Nerve
Buckingham M (1995) Myosin light chain 3F 22(2):174–185
Chapter 16
Abstract
Amniotic fluid-derived stem (AFS) cells have been described as an appealing source of stem cells because
of their (1) fetal, non-embryonic origin, (2) easy access during pregnancy overcoming the ethical issues
related both to the use of human embryonic cells and to the postnatal tissue biopsy with donor site mor-
bidity, and (3) their undemanding ability to be expanded. We and others have demonstrated the broad
differentiation potential and here we describe the established protocol we developed to obtain c-Kit+
human AFS cells, starting from second trimester amniocentesis samples.
Key words Human stem cells, Amniotic fluid stem cells, Magnetic selection, c-Kit, Fetal cells, CD117
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_16, © Springer Science+Business Media, LLC 2013
191
192 Michela Pozzobon et al.
CD117+ selection
Fig. 1 Schematic representation of the procedure to obtain and expand CD117 positive cells from fresh amni-
otic fluid samples. After the first centrifugation you can use a fraction of the cell pellet to analyze by flow
cytometry the level of markers present in the total population before culture and selection
adipose, bone, nerve [4], muscular [5, 6], vascular, and hematopoietic
tissues [7]. This selection allows the purification of a more homog-
enous population of cells from the amniotic fluid, which is usually
populated by several cell types [8]. Considering the low number of
CD117 positive cells in the amniotic fluid (range 1–5 %) we devel-
oped an accurate step-by-step methodology for their selection
which take in account both their low number and the limited
amount of fluid obtained at amniocentesis (see Fig. 1).
2 Materials
2.1 Culture Medium 1. Prepare the pre-selection medium: using a sterile syringe
and Plastic Ware plugged to a needle, inject 10 mL of sterile distilled water to
the Chang Medium C Lyophilized Supplement (catalog num-
ber T101-019) (IrvineScientific, Santa Ana, CA, USA) and let
equilibrate until no powder is visible inside the vial.
2. Transfer reconstituted Chang Medium C Supplement in the
bottle of Chang Medium B Basal (catalog number T101-019)
(IrvineScientific, Santa Ana, CA, USA) making a 100 mL final
volume of medium called “Chang B + C.” This medium should
be completed with your antibiotics (Penicillin and Streptomycin,
see Note 1) and also the L-Glutamine (2 mM final) (catalog
number 25030) (Gibco-BRL, Bethesda, MD, USA) at the
moment of use.
3. Expansion medium: MEM Alpha (catalog number A10490-01)
(Gibco-BRL, Bethesda, MD, USA) containing 20 % Chang
B + C, 15 % Fetal Bovine Serum (FBS, catalog number 10106-
169) (Gibco-BRL, Bethesda, MD, USA), antibiotics, and
L-Glutamine (2 mM final) (see Note 2).
Isolation of c-Kit+ Human Amniotic Fluid Stem Cells from Second Trimester 193
2.3 Flow Cytometry 1. Tubes (or other supports) suitable for your cytometer.
Analysis 2. Antibodies anti-human antigens: CD29 FITC (catalog num-
ber 0791), HLA-DR PE (catalog number 0464), CD105 PE
(catalog number A07414), HLA-ABC FITC (catalog number
IM1838) (Beckman Coulter, Brea, CA, USA), CD44 FITC
(catalog number 560977), 7-aminoactinomycin D (7AAD)
(BD Biosciences, San José, CA, USA), CD73 PE (catalog
number 344004), CD90 FITC (catalog number 328018)
(BioLegend, San Diego, CA, USA).
194 Michela Pozzobon et al.
3 Methods
3.1 Fresh Sample: 1. Centrifuge amniotic fluid sample at 300 × g for 5 min. Remove
Sample Seeding the supernatant making sure not to disturb the cell pellet.
2. Resuspend cells in 3 mL of Chang B + C medium and seed over
a glass coverslip (see Note 4) covering the inside of a
35 mm × 10 mm petri dish.
3. Check daily the cell morphology and change medium after 5–7
days of culture (see Note 5).
4. Once clusters of cells with mesenchymal-like morphology start
to grow (see Note 6 and Fig. 2), immune-selection and subse-
quent expansion should be performed [9].
5. Before proceeding to the next step you should prepare the
Chang Complete medium.
Fig. 2 Morphological aspect of unselected (a, b) and CD117 selected amniotic fluid stem cells (c, d). Images
were taken with phase contrast microscope (scale bars = 100 µm). (a) Morphology of a sample presenting an
epithelial and overconfluent appearance, such samples usually do not give a lot of success for selecting CD117
positive cells. (b) Aspect of a heterogeneous sample with mesenchymal-like cells and also some big-rounded
cells (floating or adherent): choose this type of sample for cells selection. (c) The few cells obtained after
immune-selection and (d) the proliferation of those cells with conservation of the mesenchymal-like morphol-
ogy after culture in expansion medium
Isolation of c-Kit+ Human Amniotic Fluid Stem Cells from Second Trimester 195
3.2 Immune- 1. Take out the supernatant from the petri dish and wash twice
Selection with PBS. Add 500 μL of Trypsin and incubate for 3–5 min at
37 °C. Check at the microscope the detachment of the cells
(see Note 7).
2. Collect cell suspension and add at least 2 mL of α-MEM con-
taining 25 % of FBS. Centrifuge cells at 300 × g for 5 min.
Remove the supernatant making sure not to disturb the cell
pellet and resuspend in 300 μL of buffer.
3. Add 100 μL of FcR Blocking Reagent and 100 μL of CD117
MicroBeads, mix well, and incubate for 15–20 min at 4 °C.
4. Add 2 mL of buffer to the cell suspension and centrifuge at
300 × g for 10 min. Remove the supernatant making sure not
to disturb the cell pellet and resuspend in 500 μL of buffer.
5. Place a MS Column on the Separator attached to the MultiStand
and wash it once with 500 μL of buffer, place a 15 mL tube
under the tip of the column to collect the flow-through (the
negative fraction can be retrieved centrifuging this tube if
needed).
6. Once the first wash has passed through the column put your
cell suspension onto the column.
7. Wash the column three times with 500 μL of buffer.
8. Remove the column from the magnetic support and place it on
a 15 mL tube for collection: add 1 mL of buffer onto the col-
umn and using the plunger immediately flush out the content.
9. Repeat steps 5–8 another time to reduce contamination by
unwanted cells (use one new column).
3.3 Cells Expansion 1. Add 2 mL of buffer to the cell suspension from Subheading 3.2,
step 8 and centrifuge at 300 × g for 10 min. Remove the super-
natant making sure not to disturb the cell pellet and resuspend
in an appropriate volume of expansion medium (see Note 8).
2. We recommend to start expanding selected cells in a well of a
24-multiwell plate and then—before confluence—split them in
other appropriate surfaces to maintain subconfluence (<70–
80 %) (see Note 9). Usually we put around 2 × 104 cells in
100 mm × 20 mm dishes and 1 × 105 cells in 150 mm × 25 mm
dishes [10].
3. Freezing of cells should be performed in FBS (or Horse Serum)
containing 5–10 % DMSO and no less than 5 × 105 cells should
be frozen in a cryotube (expand cells until passage 3 in a
150 mm × 25 mm dish before obtaining enough cells to freeze).
In our experience you can thaw 5 × 105 cells in a 150 mm × 25 mm
dish (thawing in a smaller dish can bring cells directly to con-
fluence) [11].
196 Michela Pozzobon et al.
4 Notes
CD105
CD29
CD90
CD73
BEFORE SELECTION
HLA-ABC
% of total
HLA-DR
CD44
CD105
CD29
CD90
CD73
AFTER SELECTION
HLA-ABC
% of total
HLA-DR
CD44
CD117
Fig. 3 Representative cytograms of the fresh and expanded (passage 4) cells analyzed for the surface markers
CD29, CD44, CD90, CD73, CD105, HLA-ABC, HLA-DR, and CD117. CD117 surface expression generally dimin-
ishes after expansion; on the contrary, after culture mesenchymal stem cell markers are up-regulated. HLA-
ABC marker in culture is homogeneously expressed, whereas HLA-DR remains negative both in fresh and
expanded CD117 positive cells [14]
References
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(2010) On the origin of amniotic stem cells: of 25(1):100–106
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(2007) Molecular and proteomic characteriza- stem cells. Biol Reprod 74(3):545–551
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from amniotic fluid: comparison to bone mar- (2012) Immunosuppressive properties of
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Chapter 17
Abstract
It is widely accepted that mammalian stem cells reside in a specialized cellular and a cellular microenvironment
called the niche. The niche contrary to other tissues is characterized by a low partial Oxygen pressure
(ppO2). This microenvironment protects stem cells from deleterious effects of O2 on proteins and DNA,
through the production of reactive oxygen species (ROS). In addition there is now solid evidence that this
physiological hypoxia helps stem cells maintaining their major characteristics: multipotency and ability to
differentiate and migrate from the niche to specialized tissues in order to fulfill the needs of the organism.
Immuno Histological techniques can stain stem cells in situ by specific Abs (such as against CD34 and
CD45 for Hematopoietic Stem Cells HSC). However, a universal marker of hypoxia is Hypoxia-Inducible
Factor-1, HIF-1, which is stabilized by low ppO2 and acts as a transcription factor to regulate a vast array
of genes downstream. HIF-1, together with pimonidazole, a chemical compound interacting with proteins
that are reduced in a hypoxic environment, are bona fide markers of the stem cell niche.
Key words Stem cell niche, Multipotency, Hypoxia, Pimonidazole, Regenerative medicine
1 Introduction
1.1 General Oxygen is a critical component of the stem cell niche which can be
Introduction defined as a specialized microenvironment made of cellular and a
cellular components that integrate systemic and local cues to regulate
the biology of stem cells [1]. As stem cells are vital to the organism,
from a finalistic point of view it is understood that the niche is dedi-
cated to preserving the functions of stem cells [2]. This is achieved
by avoiding metabolic stresses such as those generated by free
oxygen radicals/reactive oxygen species ROS. Furthermore it helps
maintaining long term survival in quiescent state, multipotency,
proliferation and migration capacities of stem cells.
In mammalian tissues, the partial pressure of Oxygen, ppO2
drops progressively after it enters the lungs, from 21 to less than
9 % in most tissues [3] and as low as 2 % in the most hypoxic tissues
and therefore constitute “physiologic normoxia” [4]. The cur-
rently best known niche is the hematopoietic stem cell HSC niche
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_17, © Springer Science+Business Media, LLC 2013
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200 Ali Dalloul
1.2 Biological High ppO2 can be detrimental to stem cells, conversely, hypoxia is
Functions of Hypoxia protective by lowering the local production of ROS. ROS production
is however finely tuned and has also protective effects.
ROS are potentially harmful to stem cells. These anions (O2–)
and hydrogen superoxide H2O2 are detrimental for the lifespan of
HSC, partly through the stimulation of the p38MAPK pathway
[15]. ROS affects genetic stability and their deleterious effects
include DNA damage, aneuploidy, and telomere shortening, all of
which can be prevented in hypoxia [16].
Maintaining quiescence and multipotency is influenced by the
regulation of response to ROS. The FoxO genes [17, 18] are critical
in regulating this response, indeed mice with conditional deletion
of Foxo in HSC, show an increase in cell cycling and apoptosis in
parallel to an increase in ROS species [19]. Critical enzymes for
the production of ROS are NADP oxidase isoforms Nox. Nox are
transmembrane molecules that function as oxygen sensors; they
are produced mainly by differentiated myeloid cells [20] and to a
lesser extent by progenitor cells [21]. Interestingly, in response to
hypoxia, Nox2 induces the production of Matrix Metalloproteinases
MMP, which in turn enhances the migration of stem cells [22].
Hypoxia and Visualization of the Stem Cell Niche 201
2 Materials
+
I Notch
glucose
Survival, multipotency
Lactic acid
LDH
VEGF, CX3CR1, CXCR4, CXCR7
pyruvate
PDK1 PDH
Mitochondrial
respiration
GT, LDH, PDK1
Fig. 1 Molecular action of Hypoxia-activates HIF-1. Under low ppO2, Prolyl Hydroxylase PH, is not activated and
does not tag HIF-1a on pralines for ubiquitination and degradation. HIF-1a dimerizes with HIF-1b to translocate
into the nucleus and interacts with hypoxia-responsive element HRE on the promoters of various genes.
Transcription of chemokine receptor genes to CXCL-12/SDF-1 and CX3CL1/fractalkine, VEGF, genes involved
in multipotency, and genes involved in the inhibition of mitochondrial respiration is activated. HIF-1 interacts
with Notch-ligand(L) to activate the cleavage of Notch. HIF-1 interacts thereafter with intracellular Notch to
activate stemness genes. HIF-1 activates the transcription of glucose transporters, GT, Lactate dehydrogenase
LDH, and Pyruvate kinase dehydrogenase-( PDK-)1. This allows lactic acid to be produced from pyruvate, while
inhibiting pyruvate dehydrogenase and downstream, tricarboxylic (Krebs) cycle, electron transport, ATP and
ROS production. As a result, mitochondrial respiration is inhibited
3 Methods
4 Notes
References
1. Scadden DT (2006) The stem cell niche as an 9. Harrison JS, Rameshvar P, Chang V et al
identity of action. Nature 441:1075 (2002) Oxygen saturation in the bone marrow
2. Brahimi-Horn MC, Pouyssegur J (2007) of healthy volunteers. Blood 99:394
Oxygen, a source of life and stress. FEBS Lett 10. Matsumoto A, Matsumoto S, Sowers AL et al
581:3582–3591 (2005) Absolute oxygen tension (p(O(2)) in
3. Eliasson P, Johnsson JI (2010) The hemato- murine fatty and muscle tissue as determined
poietic stem cell niche: low in oxygen but a by EPR. Magn Reson Med 54:1530–1535
nice place to be. J Cell Physiol 222:17–22 11. Mendez Ferrer S, Michurina TV, Ferraro F
4. Simon MC, Keith B (2008) The role of oxygen et al (2010) Mesenchymal and hematopoietic
availability in embryonic development and stem cells form a unique bone marrow niche.
stem cell function. Nat Rev Mol Cell Biol Nature 466:829–834
9:285–296 12. Basciano L, Nemos C, Foliguet B et al (2011)
5. Wilson A, Trumpp A (2006) Bone-marrow Long term culture of mesenchymal stem cells
haematopoietic-stem-cell niches. Nat Rev in hypoxia promotes a genetic program main-
Immunol 6:93–106 taining their undifferentiated and multipotent
6. Yin T, Li L (2006) The stem cell niches in status. BMC Biol 12:12–24
bone. J Clin Invest 116:1195–1201 13. Pistoia V, Raffaghello L (2010) Potential of
7. van Tavazoie M, der Velken L, Silva-Vargas V mesenchymal stem cells for the treatment of
et al (2008) A specialized vascular niche for autoimmune disorders. Expert Rev Clin
adult neural stem cells. Cell Stem Cell Immunol 6:211–218
3:279–288 14. Rodesch F, Simon P, Jauniaux E (1992)
8. Li Z, Bao S, Wu Q et al (2009) Hypoxia- Oxygen measurements in endometrial and
inducible factors regulate tumorogenic capacity trophoblastic tissues during early pregnancy.
of glioma stem cells. Cancer Cell 15:501–513 Obstet Gynecol 80:283–285
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15. Ito K, Hirao K, Arai K et al (2006) Reactive 23. Vassilopoulos G, Wang PR, Russel DW (2003)
oxygen species act through p38MAPK to limit Trans-planted bone marrow regenerate liver
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Nature Med 12:446–451 24. Hung SC, Pochampally RR, Hsu SC, Sanchez
16. Estrada JC, Albo C, Benguria A et al (2012) C, Chen SC, Spees J, Prockop DJ (2007)
Culture of human mesenchymal stem cells at Short term exposure of multipotent stromal
low oxygen tension improves growth and cells to low owygen increases their expression of
genetix stability by activating glycolysis. Cell CX3CR1 and CXCR7 and their engraftment
Death Differ 19:743–755 in vivo. PLoS One 2:e416
17. Thotova ZR, Kollipara BJ, Huntly BH et al 25. Weidemann A, Johnson RS (2008) Biology of
(2007) FoxOs are critical mediators of hema- HIF-1 alpha. Cell Death Differ 15:621–627
topoietic stem cell resistance to physiologic 26. Wang Y, Wan C, Deng L et al (2007) The
oxidative stress. Cell 128:325–339 hypoxia-inducible factor 1 alpha pathway
18. Miyamoto K, Araki KY, Naka K et al (2007) couples angiogenesis to osteogenesis during
Foxo3a is essential for the maintenance of the skeletal development. J Clin Invest 117:
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1:101–112 27. Kim JW, Tshernyshyov I, Semenza GL, Dang
19. Son BR, Marquez-Curtis LA, Kurcia M et al CV (2006) HIF-1-mediated expression of
(2006) Migration of bone marrow and cord pyruvate dehydrogenase kinase: a metabolic
blood mesenchymal stem cells in vitro is regu- switch required for cellular adaptation to
lated by stromal-derived factor-1-CXCR4 and hypoxia. Cell Metab 3:177–185
hepatocyte growth factor c-met axes and 28. Gustafsson MV, Zheng X, Pereira T et al
involves matrix metalloproteinases. Stem Cells (2005) Hypoxia requires notch signaling to
24:1254–1264 maintain the undifferentiated cell state. Dev
20. Urao N, Inomata H, Razvi M et al (2008) Cell 9:617–628
Role of nox2-based NADPH oxidase in bone 29. Lendahl U, Zimmertman LB, McKay MD
marrow and progenitor cell function involved (1990) CNS stem cells express a new class of
in neovascularisation induced by hinlimb isch- intermediate filament proteins. Cell 60:
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progenitor cells express multiple isoforms of results in hypoxia with increased hypoxia-
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reactive oxygen species. Biochem Biophys Res lar endothelial factor A in bone marrow. Stem
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Chapter 18
Abstract
The presence of disseminated tumor cells (DTCs) in the bone marrow is associated with poor prognosis of
cancer patients. However, little is known about the biology of DTCs due to lack of relevant animal models.
Here, we describe the methods for detecting and isolating human DTCs from the murine bone marrow
niche by PCR using human Alu sequences and by fluorescence-activated cell sorting and immunohistochem-
istry using anti-HLA antibody. These strategies could be useful for exploring the biology of DTCs.
Key words Disseminated tumor cells, Bone marrow niche, ALU, Human leukocyte antigens,
PCR, Flow cytometry, Animal model, Prostate cancer, Immunohistochemistry
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_18, © Springer Science+Business Media, LLC 2013
207
208 Yusuke Shiozawa et al.
Tumor Inoculation
Identify PCa Cells by PCR Isolate PCa Cells by FACS Detect PCa Cells by IHC
with ALU sequence with Anti-HLA-ABC Antibodies with Anti-HLA-ABC Antibodies
Fig. 1 Schematic experimental model of human disseminated tumor cells detection in murine marrow. Human
cancer cells are inoculated into immunodeficient animals (e.g., intracardiac injection, intravenous injection,
subcutaneous implantation, intratibial injection). At the termination of the experiment, bone tissues are
harvested. Thereafter, human disseminated tumor cells in the murine tissues are identified by polymerase
chain reaction (PCR), fluorescence-activated cell sorting (FACS), and immunohistochemistry (IHC)
2 Materials
2.1 DTC Detection 1. DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA, USA).
by PCR 2. ABI PRISM™ 96-Well Optical Reaction Plate with Barcode
(code 128) (Applied Biosystems, Foster City, CA, USA).
3. MicroAmp Optical Adhesive Film Kit (Applied Biosystems).
4. TaqMan® Universal PCR Master Mix (Applied Biosystems).
5. RNase-Free Water (QIAGEN).
6. TaqMan® Gene Expression Assay (murine β-actin) (Applied
Biosystems).
7. ALU Primer Mix (Applied Biosystems): 5 μl of TaqMan®
TAMRA™ Probe (100 μM) (5′-FAM-ATT AGC CGG GCG
TGG TGG CG-TAMRA-3′), 10 μl of ALU forward primer
(50 μM) (5′-CAT GGT GAA ACC CCG TCT CTA-3′), 10 μl
of reverse primer (50 μM) (5′-GCC TCA GCC TCC CGA
GTA G-3′), 75 μl of RNase-free water (see Note 1).
8. ABI PRISM® 7700 Sequence Detection System (Applied
Biosystems).
3 Methods
3.1 DTC Detection 1. Create a standard curve comparing total mouse cells to ß-actin
by PCR cycles: Count and separate 1 × 107 mouse cells into one tube,
dilute 1:10 to make a total of eight standards ranging from
1 × 107 cells to 1 cell.
2. Extract genomic DNA using the DNeasy Blood & Tissue kit.
3. Plate samples in a PCR plate: 15 μl TaqMan Master Mix;
12.5 μl H2O; 1.5 μl mouse actin primer; 1 μl DNA sample.
4. Run the 2nd step PCR reaction for 40 cycles (95 °C for 15 s
and 60 °C 1 min) after an initial single cycle of 50 °C for 2 min
and 95 °C for 10 min using an ABI PRISM 7700 instrument.
5. Note the threshold cycle.
6. Make the graph using Excel by plotting cell number on the
x-axis and cycle number on the y-axis on a scatter plot, and
then add a trendline (see Note 3).
Detection of Disseminated Tumor Cells 211
Fig. 2 Representative bone marrow histology following establishment of disseminated tumor cells. Intracardiac
injection of PCa cells (PC3) into severe combined immunodeficiency (SCID) mice was performed. Twenty-four
hours later, the long bones were harvested and human disseminated tumor cells in murine marrows were
visualized by immunofluorescent imaging with anti-HLA-ABC antibodies. Vehicle injection (saline) served as a
negative control. Original magnification 60× Zoom2. Bar = 20 µm
4 Notes
Acknowledgments
References
1. Eyles J, Puaux AL, Wang X et al (2010) Tumor 6. Taichman RS, Cooper C, Keller ET et al
cells disseminate early, but immunosurveillance (2002) Use of the stromal cell-derived fac-
limits metastatic outgrowth, in a mouse model tor-1/CXCR4 pathway in prostate cancer
of melanoma. J Clin Invest 120:2030–2039 metastasis to bone. Cancer Res 62:
2. Pantel K, Alix-Panabieres C, Riethdorf S 1832–1837
(2009) Cancer micrometastases. Nat Rev Clin 7. Muller A, Homey B, Soto H et al (2001)
Oncol 6:339–351 Involvement of chemokine receptors in breast
3. Morgan TM, Lange PH, Porter MP et al cancer metastasis. Nature 410:50–56
(2009) Disseminated tumor cells in prostate 8. Taichman RS (2005) Blood and bone: two
cancer patients after radical prostatectomy and tissues whose fates are intertwined to create
without evidence of disease predicts biochemi- the hematopoietic stem-cell niche. Blood 105:
cal recurrence. Clin Cancer Res 15:677–683 2631–2639
4. Nash KT, Phadke PA, Navenot JM et al (2007) 9. Shiozawa Y, Pedersen EA, Havens AM et al
Requirement of KISS1 secretion for multiple (2011) Human prostate cancer metastases target
organ metastasis suppression and maintenance the hematopoietic stem cell niche to establish
of tumor dormancy. J Natl Cancer Inst footholds in mouse bone marrow. J Clin Invest
99:309–321 121:1298–1312
5. Holmgren L, O’Reilly MS, Folkman J (1995) 10. Havens AM, Pedersen EA, Shiozawa Y et al
Dormancy of micrometastases: balanced prolif- (2008) An in vivo mouse model for human
eration and apoptosis in the presence of angio- prostate cancer metastasis. Neoplasia 10:
genesis suppression. Nat Med 1:149–153 371–380
Chapter 19
Abstract
Mounting evidences indicate that leukemic cells in patients with acute myeloid leukemia (AML) are
derived from leukemia stem cells (LSC). In analogy to normal hematopoietic stem cells (HSC), LSC
remain mostly dormant and are hence resistant to conventional chemotherapy. Residual, physiological
HSC exist alongside with LSC, with heterogeneous dominance of LSC over HSC in individual patients.
We have devised a flow cytometric method for the identification and separation of these two stem cell
populations based on surface antigen markers such as CD34, CD38, lineage aberrant markers, and alde-
hyde dehydrogenase (ALDH) enzyme activity.
Key words Acute myeloid leukemia, Leukemia stem cells, Hematopoietic stem cells, Flow cytometry,
FACS, Aldehyde dehydrogenase, Aberrant markers
Abbreviations
ALDH Aldehyde dehydrogenase
AM Aberrant marker
AML Acute myeloid leukemia
APC Allophycocyanin
FSC Forward scatter
FACS Fluorescence-activated cell sorting
FITC Fluorescein
HSC Hematopoietic stem cell
LSC Leukemia stem cells
MNC Mononuclear cells
NOD/SCIDNOD/SCID Non-Obese Diabetic/Severe Combined Immune Deficiency
NSG NOD/SCID/interleukin 2 receptor gammanull
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_19, © Springer Science+Business Media, LLC 2013
217
218 Van T. Hoang et al.
PE Phycoerythrin
PI Propidium iodide
SSC Side scatter
1 Introduction
Most authors reported that HSC and LSC share the same
CD34+CD38− phenotype. Thus in patients with AML, this subset
consists of a mixture of both of normal HSC and LSC compart-
ments. Other authors have suggested that several surface markers
were differentially expressed on LSC as compared to HSC. These
included CD90 [11], CD123 [12], aberrant markers (AM) [13],
CD96 [14], CD47 [15] and Tim3 [16]. With the exception of
CD90, which is expressed at a higher level in HSC than in LSC,
all the other markers have been reported to be up-regulated in LSC.
AM can be lymphoid lineage markers, e.g., CD2, CD5, CD7,
CD19, CD22 and CD56, or differentiated myeloid marker, e.g.,
CD11b, which are absent in normal HSC, but are aberrantly
expressed in LSC and immature leukemic blasts. They have been
used for detection of minimal residual disease in the routine diag-
nostics and in combination with CD34 and CD38 for an enrich-
ment of LSC and HSC [13]. The disadvantages of the technique are
that it requires an individual strategy for each patient, a large panel
of antibodies and the quality of the enrichment might vary between
different markers. Other candidates for the separation of HSC and
LSC are the recently published markers CD47 and Tim3. However,
CD47 was expressed unexpectedly high in all hematopoietic cells
including HSC in healthy bone marrow and Tim3 was upregulated
only in some AML samples in our analysis.
In this protocol, we describe a method using the enzyme alde-
hyde dehydrogenase 1 (ALDH) as a marker for the detection and
separation of HSC and LSC from the same patient with AML at
the time of diagnosis as well as in follow-up studies. ALDH belongs
to the ALDH super-family, which is expressed in many tissues and
is responsible for the oxidation of aldehydes to corresponding
carboxylic acids. These enzymes are involved in many metabolic
processes, e.g., alcohols, amino acids, vitamins, steroids, and lipids,
as well as in the detoxification of drug-induced aldehyde substrates
leading to resistance of cells [17]. Moreover, ALDH has been
shown to be expressed at a high level by primitive stem and pro-
genitor cells in the bone marrow [18, 19], brain [20], breast [21],
and in several cancers such as AML [19, 22, 23], breast [21], colon
[24] and prostate cancer [25]. In the bone marrow of both healthy
donors and patients with AML, cells with high activity of ALDH
(ALDHbright) contained significantly higher proportions of long-
term colony initiating cells and engrafted better in mouse models
than their counterparts [19, 22, 23]. However, its expression in
AML is more heterogeneous. In the majority of patients, ALDHbright
cells are very rare. These cells have been demonstrated to contain
residual normal HSC and could be distinguished from CD34+CD38−
LSC that exhibited lower level of ALDH (ALDHdim) [26]. In this
protocol, we demonstrate the identification of the ALDHbright cells
in AML patients and the analysis of their co-expression of AM,
220 Van T. Hoang et al.
2 Materials
2.1 Patient Samples Bone marrow aspirates of patients with AML are collected in hepa-
rin. Samples should be stored at 4 °C and processed as soon as
possible. Frozen mononuclear cells can also be used.
2.2 Materials Biocoll separation solution (1.077 density, Biochrom AG, Berlin).
for Gradient PBS (PAA Laboratories GmbH, Cölbe).
Separation of
EDTA (PAA Laboratories GmbH, Cölbe).
Mononuclear
Cell (MNC) FBS (PAA Laboratories GmbH, Cölbe).
Cell preparation buffer: PBS added with 1 % FBS and 2 mM EDTA.
Store the buffer at 4 °C.
Türk’s solution (Merck, Damstadt).
Or Trypan blue (FulkaChemi AG, Taufkirchen).
Hemocytometer (Neubauer counting chamber).
BD Pharm Lyse™ reagent (BD Bioscience, Heidelberg).
2.3 Materials and FACS snap-cap tubes (12 × 75 mm, polystyrene round-bottom
Antibodies for Flow tubes) (BD Bioscience, Heidelberg).
Cytometry Aldefluor kit (Stem Cell Technologies, Vancouver, BC,
Canada).
Antibodies:
CD38 Phycoerythrin (PE) (clone HB7, BD Bioscience, Heidelberg).
CD7 PE (BD Bioscience, Heidelberg).
CD11b PE (BD Bioscience, Heidelberg).
CD15 PE (BD Bioscience, Heidelberg).
CD19 PE (clone SJ25C1, BD Bioscience, Heidelberg).
CD56 PE (clone MY31, BD Bioscience, Heidelberg).
CD34 Allophycocyanin (APC) (clone 8G12, BD Bioscience,
Heidelberg).
CD45 APC-H7 (clone 2D1, BD Pharmingen, Heidelberg).
Propidium iodide (PI, BD Bioscience, Heidelberg) in order to
exclude dead cells.
Separation of Normal Hematopoietic and Leukemia Stem Cells in AML 221
2.4 Flow Cytometry A FACS sorter that is suitable for Fluorescein (FITC), PE, PI, APC
and APC-H7. The immunophenotyping can be done using a
FACS analyzer. We used a FACScan (BD Bioscience, Heidelberg),
supplemented with a Rainbow laser (Cytek Flow Cytometry
Products, California, USA), which supports the measurement of
APC and APC-H7 fluorochromes, and a FACSAria II sorter (BD
Bioscience, Heidelberg).
3 Methods
3.1 Patient Samples Bone marrow aspirates should be obtained from patients diag-
nosed with AML after informed consent and with the approval of
local ethics committees. Immunophenotyping and other diagnos-
tic analysis can be carried out in the routine diagnostics for the
expression of AM such as CD2, CD7, CD11b, CD15, CD19,
CD22, and CD56 on blasts and CD34+ cells. Candidates for AM
were chosen for individual patient if they are expressed on more
than 50 % of CD34+ cells or more than 50 % of blasts (see Note 1).
3.2 Isolation of MNC 1. Mix 5–20 ml of bone marrow aspirates with PBS to a total
by Ficoll Gradient volume of 35 ml in a 50 ml conical tube.
Centrifugation 2. Place 15 ml of Biocoll separation solution into another 50 ml
conical tube.
3. Carefully overlay the Biocoll separation solution with the bone
marrow/PBS mixture.
4. Centrifuge the tube at 950 × g for 20 min at room temperature
without brake.
5. Carefully transfer the mononuclear cell layer at the interphase to a
new 50 ml conical tube. These cells contain mainly lymphocytes,
monocytes, thrombocytes, blood stem and progenitor cells.
6. Fill the tube with cell preparation buffer, centrifuge at 500 × g
for 5 min with brake, and remove the supernatant.
7. Repeat step 6.
8. Mix a small amount of cells with Türk’s solution and count
them with the Neubauer chamber. White blood cells should be
stained in blue in the Türk’s solution (see Note 2).
3.3 Erythrocyte Lysis After density gradient centrifugation, if the cell preparation still
contains a high number of erythrocytes, an erythrocyte lysis step
should be performed. An erythrocyte to leukocyte ratio higher
than 2:1 might disturb the ALDH staining.
1. Suspend the cell in 500 μl cell preparation buffer.
2. Add 5 ml of 1× BD Pharm Lyse™ reagent to the cell suspension.
3. Incubate 3 min at room temperature.
222 Van T. Hoang et al.
3.4 ALDH Staining Aldefluor substrate is activated, aliquoted, and stored at −20 °C
according to the manufacturer’s instructions. The following proto-
col is adapted from the manufacturer’s instructions for staining a
large number of cells.
1. Adjust sample to a concentration of 1 × 106 to 1 × 107 cells/ml
with Aldefluor assay buffer (see Note 3).
2. Label four FACS tubes for the unstained control (autofluores-
cence), the DEAB control, the ALDH compensation control
and the test tube.
3. Place 100 μl of cells in the unstained control and the rest in the
test tube.
4. Add 2 μl DEAB into the DEAB control tube (see Note 4).
5. Add 5 μl of Aldefluor substrate per 1 ml reaction to the test
tube, mix well, and immediately transfer 100 μl of this mixture
into the DEAB control tube (see Note 5).
6. Incubate the DEAB control and the test tube in water bad at
37 °C for 30 min.
7. Place 100 μl of cells from the test tube in the ALDH compen-
sation control tube.
8. Add 200 μl cold Aldefluor assay buffer to the DEAB and
ALDH control tubes to wash the cells.
9. Centrifuge all the tubes at 500 × g for 5 min and remove
supernatant.
10. Resuspend cells in the control tubes with 200 μl Aldefluor
assay buffer and place on ice or at 4 °C in the dark until FACS
measurement.
11. Cells in the test tube are resuspended in 50 μl Aldefluor assay
buffer and stained with surface markers follows.
3.5 Staining with The emission spectrum of the Aldefluor fluorescent product overlaps
Surface Markers with that of FITC and is compatible with antibodies conjugated to
other fluorochromes. Compensation controls have to be prepared
using cells stained with single fluorochromes, their isotype controls
and/or fluorescence minus one fluorochrome.
1. Label and place 105 cells into each compensation control tube.
2. Wash cells with cell preparation buffer (500 × g, 5 min).
3. Remove supernatant and suspend cells in 50 μl cell preparation
buffer.
4. Add appropriate amount of antibodies to the tubes.
Separation of Normal Hematopoietic and Leukemia Stem Cells in AML 223
Table 1
The excitation and emission of the fluorochromes
Sort sample:
ALDH CD38 CD34 CD45
3.6 Instrument The excitation and emission of the fluorochromes are indicated
Setting and Data in the Table 1 (see Note 6). The instrument setting can vary
Acquisition depending on the flow cytometry system that is provided in your
department. We use the FACScan analyzer and the FACSAriaII
sorter with the CellQuest and FACSDiva software provided by the
manufacturer.
1. Prepare an acquisition template (mask) (Fig. 1).
(a) Create a forward scatter (FSC) vs. side scatter (SSC) dot
plot and a region R1 that encompass MNC (Fig. 1a).
(b) Create a FSC vs. PI dot plot gated on R1 and a region R2
displaying PI negative cells that means living cells (Fig. 1b).
224 Van T. Hoang et al.
Fig. 1 Data acquisition and gating strategies for ALDHbright, ALDHdim and surface marker expressing cells.
The mononucleated cells (MNC) are gated in the region R1 (a). Dead cells are excluded by PI. The R2 gate
includes living cells from R1 (b) and these are displayed in pink in the following plots. DEAB, an ALDH inhibitor,
is used as a control of the ALDH staining (c). ALDH level in MNC is observed in a dot plot (d) and in a density
plot (e). Gate R3 marks ALDHbright cells (R3), which have a high ALDH level and low SSC. They are absent in
the DEAB control tube. ALDHdim cells show an intermediate level of ALDH and are gated in the region R4. The
ALDHbright cells display low FSC and low SSC (f). Expression of the aberrant marker CD56 and the stem/
progenitor cell marker CD34 is demonstrated in (g) and (h), in which the positive cell fractions are gated as R5
(g) and R6 (h). R7 is the gate of blasts, which are characterized by CD45dimSSClow (i). Spectral overlaps were
compensated as demonstrated in k–m
(c) Create a SSC vs. FITC (or FL-1) dot plot gated on R2 for
the detection of ALDH (Fig. 1c, d, see Note 7).
(d) Create dot plots of FSC vs. PE (or FL-2), APC (or FL-4),
and APC-H7 (or FL-5) vs. SSC gated on R2 for the detec-
tion of appropriated fluorochromes (Fig. 1g–i).
(e) Create dot plots for compensation of spectral overlap
(Fig. 1k–m).
2. Acquisition of data.
(a) Place the non-stained tube in the cytometer, adjust FSC,
SSC voltages and gain to center the MNC within the plot
and the R1 gate (Fig. 1a).
Separation of Normal Hematopoietic and Leukemia Stem Cells in AML 225
Fig. 2 Analysis of the aberrant markers (AM) expression in AML blasts and in the ALDHbright cell population.
The ALDHbright cells are gated in R3 with a frequency of 0.53 %. These cells express a very low level of AM
compared to the leukemic blasts suggesting their normal origin
3.7 Data Analysis 1. Add regions R3 and R4 that gate on the ALDHbright and
ALDHdim cells in the ALDH versus SSC plot, respectively.
ALDHbright cells (R3) are characterized as a population with
high ALDH level and low SSC and they are not observed in
the DEAB control tube. ALDHdim cells have an immediate
level of ALDH and can be distinguished from ALDHneg cells,
which are mostly erythrocytes (Fig. 1c–e, see Note 8).
2. Check the FSC/SSC pattern of the ALDHbright cells. They
should have low FSC and low SSC indicating their small size
and low granularity (Fig. 1f).
3. Create a region R5 of AM-expressing cells in the FL-2 vs. FSC
plot (Fig. 1g). Display the frequency of R5 within R2.
4. Create a region R6 of CD34+ cells in the FL-2 vs. FSC plot
(Fig. 1h). Display the frequency of R6 within R2.
5. Create a region R7 of CD45dimSSClow leukemic blasts in the
CD45 vs. SSC plot (Fig. 1i).
6. Display blasts (R7) and ALDHbright cells (R3) in CD34 vs. AM
dot plots and add the same quadrant gate in all plots to sepa-
rate the populations based on CD34 and AM expression
(Fig. 2). Show statistics of the quadrant gate for each plot.
226 Van T. Hoang et al.
Fig. 3 Separation of HSC and LSC based on ALDH level within the CD34+CD38− population. The ALDHbright and
ALDHdim cell populations are identified as R3 and R4 as mentioned before (a and b). CD34+CD38− cells are
marked by the gate R10 (c). Their ALDH levels are demonstrated with the percentages of CD34+CD38−ALDHdim
LSC and CD34+CD38−ALDHbright HSC (d). In the sample 1, CD34 is expressed in a large part of MNC and
CD34+CD38− cells consist mostly of LSC with a minority of residual HSC. The sample 2 is a typical example for
the CD34− AML group, in which leukemic cells have been shown to reside within the CD34− population,
whereas CD34+ cells define normal HSC and their progenitors. In accordance with this observation, the
CD34+CD38− cells of our sample expressed highly ALDH and were found exclusively within the R3 gate and
represent normal HSC
3.8 Sorting of HSC 1. Repeat the instrument settings for ALDH, CD38 PE, CD34
and LSC in ALDH− AML APC, and CD45 APC-H7 as described for the ALDH, AM,
Samples by FACS CD34, and CD45 test tube.
2. ALDHbright and ALDHdim cells are gated in the region R3 and
R4 as described before (Fig. 3a, b).
3. Create a CD34 vs. CD38 dot plot. Add a region R10 of
CD34+CD38−/dim cells (Fig. 3c).
4. Display R10 in an ALDH vs. SSC dot plot. Copy region R3
and R4 to this plot to gate CD34+CD38−ALDHbright and
CD34+CD38−ALDHdim cell population (Fig. 3d).
5. Sort CD34+CD38−ALDHbright HSC and CD34+CD38−
ALDHdim LSC in sterile FACS tubes containing IMDM
Separation of Normal Hematopoietic and Leukemia Stem Cells in AML 227
Fig. 4 FISH analysis for CD34+ALDHbright cells (a) and MNC (b) of an AML sample with a PML/RARA t(15;17)
(q24;q21) specific DNA probe (Kreatech Diagnostics) for detection of a deleted chromosome 17. Nuclei are
stained with DAPI in blue. The probes of chromosome 15 and 17 are labeled in green and red, respectively.
One red signal in the nucleus indicates a deletion of the chromosome 17. CD34+ALDHbright cells representing
normal HSC in this case are free of this deletion (a), which is detected in MNC that mainly consist of leukemic
blasts (b)
4 Notes
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Chapter 20
Abstract
Orthotopic transplantation of tumor tissue into recipient mice has long been established to study the
role of the microenvironment in tumorigenesis and metastasis. Many of these transplantation assays
involve the surgical implantation of an undissociated piece of tumor tissue. However, dissociation of
tumor tissue into a single cell suspension prior to orthotopic transplantation enables the injection of fewer
cell numbers, the selection of tumor-initiating populations by specific purification using antibody staining
and fluorescence-activated cell sorting, and the analysis of tumor-forming efficiency.
In this chapter, we provide a method to perform serial transplantation of tumor cells into their
niche of origin. Visualization of the location of transplanted tumor cells is essential to confirm the success
of the transplant as well as the viability of transplanted cells. We also describe an optimized immunofluo-
rescence protocol to visualize tumor cells shortly after transplantation. This serial transplantation protocol
allows for an experimental tumorigenesis assay to more closely mimic spontaneous tumor formation and is
applicable to many microenvironments.
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_20, © Springer Science+Business Media, LLC 2013
231
232 Heather A. McCauley and Géraldine Guasch
canal and the simple epithelia of the rectum. This anorectal transition
zone is susceptible to squamous cell carcinoma (SCC) formation in
mice and humans associated with loss of TGFβ signaling [7–9].
Mice, in which the gene encoding the transforming growth factor-β
receptor II (TGFβRII) has been conditionally targeted in K14+
cells, develop spontaneous SCC in their anorectal region, while
TGFβRII deficient backskin appears morphologically normal [7].
Orthotopic transplantation is defined by the injection of
tumor cells or the transplantation of tumor tissue into anatomi-
cally appropriate sites, such as their microenvironment of origin.
This has the advantage of creating physiologically relevant pri-
mary tumors that can lead to spontaneous metastases in various
distant sites in contrast to induced metastasis formed after inject-
ing tumor cells in the blood circulation [10]. Placing cells into
their original niche improves their survival and proliferation, as
foreign microenvironments may not support tumor growth in the
same way and can cause misleading results [11]. Many of the pro-
tocols designed for surgical orthotopic transplantation involve
transplanting fragments of tumor, approximately 1 mm3, directly
into a recipient mouse without first dissociating the tumor into
a single-cell suspension [10]. However, dissociating a tumor into
a single-cell suspension prior to orthotopic transplantation is a criti-
cal step if the goal of the experiment is to identify a tumor-initiat-
ing population of cells [12].
Microenvironmental influence has been shown to affect cell
type differentiation. For example, hair follicle bulge stem cells
cultured on extracellular matrix from the corneal limbus and com-
bined with conditioned medium from limbal cells results in the
differentiation of those cells into corneal-like epithelium [13].
This microenvironmental reprogramming is also illustrated with
thymic epithelial cells that function as epidermal and hair follicle
stem cells when exposed to the microenvironment of the skin [14].
Therefore, orthotopic transplantation is crucial for the successful
outcome of tumorigenesis experiments if the goal is to create
tumors that mimic the original tumor.
A single-cell suspension of tumor cells can be sorted by flow
cytometry to dissociate distinct cell populations within a tumor.
This is accomplished by staining with fluorescent antibodies that
recognize cell surface proteins known to be expressed on cells that
display tumor initiating properties. Suspending cells in Matrigel, a
matrix of basement membrane proteins, ensures that the trans-
planted cells do not diffuse away from the surgical location and has
been shown to improve tumor formation [15].
Fluorescent labeling of tumor cells prior to orthotopic trans-
plantation is essential to control the accuracy of surgical technique,
to monitor the precise site of injection, and to visualize interactions
Serial Orthotopic Transplantation of Epithelial Tumors in Single-Cell Suspension 233
2 Materials
2.1 Cell Culture 1. Tumor cell line, fluorescently labeled (we use the
TGFβRII Flox/Flox ×K14Cre × Rosa-Flox-Stop-Flox-EYFP +
anorectal SCC cell line established in our laboratory).
2. Tissue culture dish, 100 × 20 mm (BD Falcon).
3. Epithelial cell culture medium with 0.05 mM Ca2+ [19] (add
83.5 μl of 0.05 M CaCl2 stock per 200 ml of medium).
4. Trypsin-EDTA, 0.25 % (Gibco).
5. Versene solution, sterile (dissolve 0.04 g EDTA disodium salt
(Sigma) in 20 ml 10× PBS) + 0.4 % glucose solution, sterile
(make a 25 % stock by dissolving 12.5 g glucose (Sigma) in
50 ml deionized water).
6. 1× Phosphate-buffered saline, sterile (1× PBS).
7. 15 ml conical tubes (BD Falcon).
8. Bright-Line Hemacytometer, 0.100 mm deep (Hausser
Scientific).
9. Trypan Blue Solution, 0.4 % (Sigma).
10. Centrifuge.
11. Sterile eppendorf tubes.
2.3 Mice Female nude mice, approximately 6–8 weeks old (at the beginning
of the experiment), were used for these experiments. We maintain
a homozygous Nu/Nu strain in the laboratory, which can be
Serial Orthotopic Transplantation of Epithelial Tumors in Single-Cell Suspension 235
purchased from Charles River (see Note 1). Mice are housed in a
barrier facility using ventilated racks and automated watering that
provides a sterile environment for preventing infections. The air
supply for the barrier facility is class 100 HEPA filtered air. All
equipment, food and bedding are autoclaved prior to entry into
the barrier facility through a double door, floor loading autoclave.
Mouse cages are changed using a clean workstation and sterile for-
ceps. The forceps are sterilized, using a glass bead sterilizer,
between cages. Every month, a sentinel is evaluated for serology,
parasitology, and pathology. The barrier veterinary technicians
only work in clean areas and change into scrubs before donning a
sterile gown, mask, shoe covers, bonnet, and gloves. Passage
through an air shower is required to gain access to the barrier facil-
ity. The barrier facility is “clean,” with exception for Helicobacter
spp. and Murine Norovirus (MNV). It is negative for internal and
external parasites.
3 Methods
3.1.2 Day 2 1. Aliquot 500 μl Matrigel in cold eppendorf tubes using a P1000
pipettor and cold tips. These 500 μl aliquots can be stored at
−20 °C until needed to avoid repeated thawing and freezing.
Prepare 30 % Matrigel by diluting Matrigel in cold epithelial
cell culture medium without calcium or serum. Calculate
150 μl 30 % Matrigel per injection. Keep 30 % Matrigel on ice
at all times.
2. Remove cells from the incubator and ensure that cells look
healthy and are approximately 80 % confluent.
3. Under a sterile hood, aspirate medium and wash cells with ster-
ile 1× PBS.
4. Combine equal volumes of 0.25 % Trypsin-EDTA and Versene
solution with 0.04 % glucose. Add 2 ml of this mixture to each
10 cm plate of cells. Incubate 5–8 min at 37 °C.
5. Vigorously pipette all around the plate with a P1000 pipettor
to detach cells and add to 5 ml epithelial cell culture medium
containing 0.05 mM of calcium in a 15 ml conical tube.
6. Centrifuge for 5 min at 450 rcf at room temperature.
7. Carefully aspirate supernatant. To avoid clumping of cells, gen-
tly tap the bottom of the 15 ml tube before adding 2 ml of
epithelial cell culture medium containing 0.05 mM calcium to
the pellet. Resuspend the cells by pipetting two to three times
with a 5 ml pipette to obtain a single cell suspension. Note that
this step is important to have an accurate count in the next step.
8. Using a hemacytometer and trypan blue solution, count the
number of viable cells in the suspension.
9. Transfer 100,000 viable cells per injection to an ice cold eppen-
dorf tube. Centrifuge cells in the eppendorf tube for 5 min at
450 rcf at 4 °C.
10. Carefully aspirate supernatant.
11. Resuspend cells in 150 μl 30 % Matrigel per injection using
a P200 pipettor and cold tips and keep on ice. If possible,
prepare enough cells with 30 % Matrigel for one more injec-
tion than planned to avoid loss of cells in the dead space of the
syringe used during the injection (see Note 2).
Fig. 1 The surgical method of cell transplantation into the anorectal transition zone. (a) Surgical set-up within
a class 100 HEPA filtered mass clean air portable system (single asterisk) includes a surgical table (double
asterisk) and an anesthesia delivery system (triple asterisk) to minimize complications from transplantation.
(b) Mice can be placed into a drop-box receiving vaporized isoflurane and will fall asleep within 1–2 min.
(c) Once mice are asleep, they can be easily transferred to a nose cone delivering vaporized isoflurane to
remain unconscious. (d, d′) Once mice are anesthetized and placed into the nose cone, insert a cold, sterile
needle containing tumor cells suspended in 30 % Matrigel into the anorectal transition zone. Carefully move
the tip of the needle from side to side to create a space for the cells to reside (arrow). (e) After the cells are
dispensed, a swelling will appear in the injection site. A small bubble (white arrow) may escape from the
puncture wound, but this typically does not affect tumor formation (color figure online)
4. Before dispensing the cells, ensure that there is a space for the
cells to reside by carefully moving the tip of the needle from
side to side (Fig. 1d, d′).
5. Dispense 150 μl of cells in 30 % Matrigel. If the injection is
correctly done, no cells should come out from the injection
site (see Note 5, Fig. 1e).
6. Remove anesthetized mice from the nose cone and place in a
clean, sterile cage. Mice generally recover from isoflurane anes-
thesia within 1–2 min.
Fig. 2 Verification of location of transplanted EYFP + cells by immunofluorescent antibody staining. (a) Three to
5 days post-transplantation of tumor cells, the injected anorectal region of the mouse appears normal.
Dissection, fixation and staining of this tissue are required to verify the location of transplanted fluorescently
labeled cells. (b) EYFP + anorectal SCC cells injected into the anorectal region of a recipient mouse localize
precisely to the anorectal transition zone, as seen by immunofluorescent antibody staining (image taken using
a 10× objective lens). α6-integrin (red; BD Bioscience) stains the transplanted EYFP + cells. It is present in the
basal layer of the stratified epithelia of the anal canal. DAPI (blue) labels the nuclear chromatin
3.3.2 Harvesting, Depending on the aggressiveness of the tumor cell line, number of
Dissociation of Tumor, and cells injected and genetic background of recipient mice, tumor
Preparation of Tumor Cell latency can vary. 100,000 cells originating from an aggressive
Suspensions cancer cell line generally form a palpable and visible tumor in an
immunocompromised nude mouse by 1 week post-transplantation
(see Note 7, Fig. 3a).
1. Sacrifice tumor-bearing mice by CO2 inhalation according to
standard protocol, after which death is determined by cessa-
tion of breathing and pinching the limbs. These procedures are
consistent with the recommendations of the Panel of Euthanasia
of the American Veterinary Medical Association.
2. Carefully dissect the tumor, ensuring that it is separated from
skin, fat, muscle, and other contaminating tissue (Fig. 3b).
Remove any necrotic tissue.
Serial Orthotopic Transplantation of Epithelial Tumors in Single-Cell Suspension 241
Fig. 3 Harvesting, dissociation of tumor and preparation of tumor cell suspensions. (a) Between 1 and 6 weeks
post-transplantation, anorectal tumors will begin to grow. (b) Dissect the tumor, separating it from skin, fat and
other contaminating tissue and place it in a 10 cm plate containing 1× PBS. (c, c′) Large tumors (greater than
1 × 1 cm) can be separated into pieces that can be fixed and embedded for antibody staining as well as dis-
sociated for transplantation assays. (d) The piece of tumor must be minced into small pieces, approximately
1 mm in diameter. (e) After incubation with collagenase, the tumor mixture will appear very viscous. (f) After
treatment with DNAse I, the viscosity of the tumor mixture will be greatly reduced
4 Notes
1. The strain and the age of mice used for transplantation assays
can affect the results of tumorigenic assays. Using immuno-
compromised mice may result in faster and more aggressive
tumor formation, but can produce more artificial results than
transplantation into immunocompetent (syngeneic) mice.
The age of the nude mice may influence the reproducibility of
the graft. To keep this parameter consistent, we maintain our
own nude colony by mating heterozygous Nu+/Nu females,
Serial Orthotopic Transplantation of Epithelial Tumors in Single-Cell Suspension 243
Acknowledgments
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Chapter 21
Abstract
The design of tissue culture conditions that faithfully reproduce the characteristics of cells in their native
environment remains one of the main challenges of cancer stem cell (CSC) biology. Here we describe a
detailed methodology for the isolation and expansion of both human colon CSCs and mouse intestinal
adenoma together with a brief differentiation and coculture method that proved to be valuable to study
the concept of CSCs plasticity.
Key words Colorectal cancer, Adenoma, Cancer stem cells, CD133, Wnt signaling, Ultralow adherent,
EGF, Isolation
1 Introduction
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_21, © Springer Science+Business Media, LLC 2013
247
248 Pramudita R. Prasetyanti et al.
2 Materials
2.1 General 1. Laminar flow cabinet certified for handling biosafety level 2
Equipment and (BSL-2) specimens.
Material List 2. Humidified tissue culture incubator capable for maintaining
37 °C and 5 % CO2 atmosphere.
3. Low-speed centrifuge (e.g., Hettich Rotanta 460).
4. Inverted and phase contrast light microscopes equipped with
10×, 20×, and 40× magnification.
5. Hemocytometer counting chamber.
6. 37 °C waterbath.
7. Trypan blue solution 0.4 % (Sigma).
8. Pipet-aid.
9. Vortex.
10. Sterile disposables plastics: 100 μl filter tips, 1,000 μl long
reach pipette tips (VWR), pipettes 1-, 2-, 10-, and 25-ml
volume, 15 and 50 ml polypropylene sterile tubes (Falcon),
10 cm petri dish.
11. Cell culture vessels: 24-, 6-wells plate ultralow adherent
plate (Corning), 25 cm2 ultralow adherent flask (Corning),
24-, 12-, 6-wells adherent plate (Greiner), 25-, 75-cm2 adherent
plate (Greiner).
Isolation and Propagation of Colon Cancer Stem Cells 249
Table 1
Overview of reagents for CSC medium and isolation enzymes
2.2 Tissue Digestion 1. Equipment: closed container certified to transport BSL-2 speci-
of Human men, medical waste container, sharps waste disposal container.
Tumor Tissue 2. Autoclaved dissection equipment (thumb dressing forceps,
dissecting scissors straight 10 cm, scalpel handle, and disposable
blade number 22).
3. Sterile disposable plastics: 1.8 ml cryovial tube (Nunc), 70 μM
nylon mesh filter (Nunc).
4. Hank’s Balanced Salt Solution 1× (HBSS) without Ca2+ and
Mg2+ (Invitrogen).
5. Antibiotic and antimycotic solution: 100 U/ml penicillin,
100 μg/ml streptomycin, 10 mg/ml gentamycin, and 2.5 μg/ml
amphotericin-B (see Note 1).
6. Culture medium, digestion enzymes and growth factor (see
Table 1 and Note 2).
7. Digestion buffer: digestion enzymes (see Table 1) in culture
medium (see Note 3).
8. Ficoll gradient reagents (e.g., Lympholyte—Miltenyi)
2.3 Culture and 1. Phosphate-Buffered Saline (PBS) without Ca2+ and Mg2+.
Differentiation of 2. Trypsin 10× solution (Lonza). To prepare working solution,
Isolated Human CSCs dilute the trypsin 1: 40 in PBS. Store at 4 °C.
250 Pramudita R. Prasetyanti et al.
Table 2
Overview of reagents for Adenoma culture medium
Working
Component Supplier Stock solution dilution Storage
Adenoma culture medium
Advanced DEMEN/F12 (ADF) medium Invitrogen 4 °C
B27 supplement Invitrogen 50× 1 in 50 −20 °C
N2 supplement Invitrogen 100× 1 in 100 −20 °C
BSA 20 % 1 in 50 4 °C
GlutaMAX-I Invitrogen 100× (200 mM) 1 in 100 RTa
Hepes Invitrogen 1M 1 in 100 4 °C
N-acetyl cysteine Sigma-Aldrich 500 mM 1 in 500 4 °C
Antimycotic/Antibiotic Invitrogen 100× 1 in 100 −20 °C
Mouse EGF Prepro-Tech 1 μg/ml 1 in 20 −20 °C
Mouse noggin Prospec 10 μg/ml 1 in 100 −20 °C
a
RT room temperature
2.4 Reagents for 1. Sterile surgical tools for dissection (scissors, tweezers, round-
Mouse Adenoma tipped curved oral gavage for rats).
Isolation and 2. Fresh Intestinal adenomas derived from mice.
Passaging
3. Sterile disposable plastics: 10 ml syringe, 50 and 15 ml poly-
propylene sterile tubes (Falcon), 15 cm tissue culture dishes,
24-well tissue culture plate, 70 μm nylon mesh filter.
4. Phosphate-Buffered Saline (PBS) without Ca2+ and Mg2+.
5. 2.5 % 10× trypsin, no phenol red (Invitrogen catalog #
15090-046).
6. Advanced DMEM/F12 (ADF) medium (Invitrogen catalog #
12634-010) (see Note 4).
7. Growth Factor Reduced Matrigel (BD Bioscience, catalog #
354230) (see Note 5).
8. See Table 2 for adenoma culture medium.
Isolation and Propagation of Colon Cancer Stem Cells 251
3 Methods
3.1 Isolation 1. After surgical resection, immediately transfer the tumor piece
of Colon CSCs into sterile 50 ml tubes containing HBSS. As indicated in
BSL-2 practice, use appropriate container to transport the
specimen to the laminar flow cabinet (see Note 6).
2. Place the tumor piece in a new 50 ml tube containing 20 ml of
HBSS and incubate at room temprature for fifteen minutes.
Shake the tube vigorously to wash the remaining feces and
blood from the tumor pieces. Allow the tumor piece to sedi-
ment and discard the supernatant. Repeat this step twice.
3. Transfer the tumor piece in a 5 cm petri dish containing 5 ml
of HBSS. Using a sterile blade, cut the tumor piece into two
parts and remove as much necrotic tissue as possible.
4. Transfer the tumor fragments into a new 50 ml tube containing
20 ml of HBSS. Wash the tumor fragment (see points
Subheading 3.1, step 2) and repeat this step a minimum of ten
times or until the supernatant is clear.
5. Cut the tumor fragment into small pieces of approximately
0.5 mm3 (see Note 7).
6. Transfer the minced tumor into a 50 ml tube containing diges-
tion buffer. Digest the tumor for 30–60 min at 37 °C with
occasional agitation (see Note 8).
7. Allow the digested fragments to sediment to the bottom of
the tube. Transfer the supernatant, which now contains the
dissociated single cells, into a new 50 ml tube and centrifuge at
600 × g for 5 min. The supernatant can be discarded or used for
a second round of digestion (see Note 9).
8. Resuspend and pass the supernatant through a 70 μm nylon
mesh filter. Pass additional 10 ml of culture medium to wash
the remaining cells away.
9. Centrifuge the cells suspension at 600 × g for 5 min.
10. Discard the supernatant and resuspend the cell pellet with
10 ml of culture medium and centrifuge the cells again at
600 × g for 5 min. Repeat this step to remove any trace of
digestion enzymes.
11. Resuspend the pellet in a minimal volume of pre-warmed culture
medium (usually 1 ml) supplied with growth factor and aliquot
10 μl for cell counting.
252 Pramudita R. Prasetyanti et al.
Fig. 1 Phase contrast micrographs of CSCs derived from stage IIIA colon carcinoma. (a) Enzymatically dissoci-
ated tumor are seeded as suspension culture in ultralow adherent plate (10× magnification) (b) Substantial
increase of dead cells are occasionally observed during the first days of plating (10× magnification) (c) After
30 days, the CSCs will proliferate faster than in the beginning of culture initiation. The solid healthy spheres
appear as balls of cells that can be maintained over passages (40× magnification)
3.2 Culture and 1. Examine the confluency and condition of the cells under in the
Propagation of Colon microscope prior to passaging (see Note 11).
CSCs 2. Transfer the cell suspension into a 15 ml tube. Wash the culture
vessel with 2 ml of PBS to detach the remaining cells and pool
them together in the previous tube.
3. Centrifuge the cells at 450 × g for 3 min and discard the
supernatant.
4. Resuspend the pellet in trypsin and incubate at 37 °C for 5 min
(see Note 12).
5. Stop the trypsin reaction by adding similar volume of trypsin
inhibitor and dissociate the spheres mechanically by pipetting
up and down several times using a long reach pipette tip.
6. Add 5 ml of culture medium on top of the cells suspension and
centrifuge at 500 × g for 3 min. Gently decant the supernatant
without disturbing the pellet.
7. Resuspend the pellet in culture medium supplemented with
growth factor in the desired cell density (see Subheading 3.1,
steps 11–13).
Isolation and Propagation of Colon Cancer Stem Cells 253
Fig. 2 The withdrawal of growth factor and the addition of serum induce CSCs differentiation. After 96 h of
incubation with the differentiation medium, the CSCs sphere structure will completely disappear into a flattened
morphology. This can be observed in a monolayer culture system (a) and in Matrigel (b) (40× magnification)
3.3 Differentiation The conditions described above maintain colon CSCs in an immature
of Colon CSCs undifferentiated state. However, withdrawal of the growth factors
and addition of serum can lead to the differentiation of colon CSCs
into the various differentiated lineages present in the original
malignancy. This differentiation process can be performed at least
in two different ways: in adherent conditions as a monolayer cul-
ture (Fig. 2a) and in a 3D Matrigel culture system (Fig. 2b).
Conversely, the addition of myofibroblasts derived factors prevents
the differentiation potential of colon CSCs (Fig. 3) [7].
3.3.1 CSCs 1. Dissociate the spheres according to the Subheading 3.2 and
Differentiation in Adherent count the cells with the hemocytometer counting chamber.
Cell Culture Vessel 2. Transfer the cells into a 15 ml tube and pellet the cells by cen-
trifugation 600 × g for 3 min.
3. Resuspend the cells in 5 ml of PBS to wash the residual growth
factors. Spin the cells at 500 × g for 3 min and discard the
supernatant. Perform this washing step twice.
4. Resuspend the pellet using p1000 tips in pre-warmed culture
medium containing EGF and seed the cells according to the
recommended density in adherent culture vessels.
254 Pramudita R. Prasetyanti et al.
3.3.3 Generation of 1. Seed 1 × 106 18Co human myofibroblasts cells in a T75 adherent
Myofibroblasts Conditioned Flask in DMEM medium supplemented with 10 % FCS (fetal
Medium (MFCM) calf serum) and 1 % glutamine.
2. The following day, wash the cells two times with PBS to remove
any remaining FCS.
3. Delicately overlay 10 ml of DMEM/F12 on the myofibroblasts
(see Note 14).
4. Collect the myofibroblasts conditioned medium (MFCM)
after 24 h in a 15 ml falcon.
5. Spin the medium collected at 750 × g for 5 min (see Note 15).
6. MFCM can be aliquoted and stored at 4 °C up to 6 months
(see Note 16).
Isolation and Propagation of Colon Cancer Stem Cells 255
3.3.4 Dedifferentiation 1. Follow the steps 1–3 of CSCs differentiation in adherent cell
of Colon-CSCs with MFCM culture vessel as described in (see Subheading 3.3.1).
2. Resuspend the pellet using p1000 tips with MFCM supple-
mented with 2 % heat inactivated FBS (see Note 17).
3. Observe the morphological changes after 48 h (see Fig. 3c).
3.3.5 Dedifferentiation of 1. Seed 7.5 × 105 18Co human myofibroblasts in a T25 flask in
Human Colon-CSCs on a DMEM culture medium supplemented with 10 % FCS and
Feeder Layer of 1 % glutamine.
Myofibroblasts 2. When cells are approximately 90 % confluent, aspirate the
medium and wash the cells twice with PBS.
3. Overlay 4 ml of differentiation medium on the myofibroblasts.
4. Follow steps 1–3 of CSCs differentiation in adherent cell
culture vessel (see Subheading 3.3.1) and resuspend 1 × 105
of CSCs with 1 ml of differentiation medium.
5. Transfer the CSCs suspension onto the T25 flask containing
myofibroblasts and swirl gently.
6. Observe the morphological changes after 48 h.
3.4 Mouse Adenoma Unless otherwise stated all steps are carried out at room temperature
Isolation and do not need to be done in sterile conditions.
1. Carefully remove the small intestine from the mouse (see
Note 18) and place it on a clean flat surface (see Note 19).
2. Flush the intestine (see Note 20) with 4 °C PBS to remove the
remaining feces.
3. Cut the intestine longitudinally open with a sterile scissors
(see Note 21) and open up the intestine so that the villi face
upwards.
4. Cut out adenomas using a scissors (see Note 22) and dice the
tissue into small pieces (approximately 0.3 × 0.3 mm).
5. Place adenoma pieces into a 50 ml tube containing 10 ml 4 °C
PBS. From this step onwards the adenoma pieces should be
kept on ice.
6. Wash the adenoma pieces three to five times with 10 ml 4 °C
PBS (see Note 23) or until the supernatant is clear.
7. Remove supernatant and add 5 ml 2.5 % 10× trypsin. Incubate
for 30 min at 37 °C.
8. Shake vigorously and collect the supernatant in a new 50 ml
tube. Keep the supernatant on ice for the remaining steps.
9. Repeat washing steps of the adenoma pieces two to four times
using 10 ml 4 °C ADF medium (see Note 24). After each wash
collect the supernatant in the same 50 ml Falcon tube.
10. Filter the collected supernatant through a 70 μM mesh filter.
256 Pramudita R. Prasetyanti et al.
Fig. 4 The isolation and expansion of Adenoma cultures. The isolation of adenomas can be done by isolating
lesions which are visible either macroscopically (a) or microscopically (b-5× magnification). The adenomas
are already large by day 10 (c) and can be maintained indefinitely (40× magnification)
4 Notes
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INDEX
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8, © Springer Science+Business Media, LLC 2013
261
STEM CELL NICHE: METHODS AND PROTOCOLS
262 Index