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Methods in
Molecular Biology 1307
Kursad Turksen Editor
Human
Embryonic Stem
Cell Protocols
Third Edition
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes: http://www.springer.com/series/7651

.
Human Embryonic Stem
Cell Protocols
Third Edition
Edited by
Kursad Turksen
Ottawa Hospital Research Institute
Ottawa, ON, Canada
Editor
Kursad Turksen
Ottawa Hospital Research Institute
Ottawa, ON, Canada
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-2667-1 ISBN 978-1-4939-2668-8 (eBook)
DOI 10.1007/978-1-4939-2668-8
Library of Congress Control Number: 2015947127
Springer New York Heidelberg Dordrecht London

# Springer Science+Business Media New York 2016
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Preface
In spite of ethical and political concerns regarding the use of human embryonic stem cells,
their potential to advance not only regenerative medicine applications but also our funda-
mental understanding of stem cell biology continues to drive interest in research with these
cells. In both cases, delineating the steps in and identifying the molecules responsible for
the commitment and differentiation of these pluripotent cells to different cell fates remains
crucial. With this in mind, I felt that it would be timely to collect some of the most
interesting and useful protocols that have emerged during the last several years. I am
therefore extremely pleased and thankful to those groups who agreed to share their
valuable protocols with others interested in exploring stem cell biology questions in a
research setting.
Once again, I would like to acknowledge John Walker, Editor in Chief of the series, for
continuously fostering my efforts in this series. I am also grateful to Patrick Marton,
Executive Editor of the series, for encouraging me throughout the editorial process. In
addition, I thank David Casey for keeping me on track and pointing out all those details
that make contributions to the series consistent and dependable.
Ottawa, ON, Canada Kursad Turksen
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Derivation of Human Embryonic Stem Cell Lines from Vitrified

Human Blastocysts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Cara K. Bradley, Julia Schaft, Tammie K. Roy, Biljana Dumevska,
and Teija T. Peura
Characterizing Pluripotent Stem Cells Using the TaqMan® hPSC
ScorecardTM Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Jeffrey Fergus, Rene Quintanilla, and Uma Lakshmipathy
Growth of Human Pluripotent Stem Cells Using Functional Human
Extracellular Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Andres Sanz-Garcia, Miodrag Stojkovic, and Carmen Escobedo-Lucea
Efficient Expansion of Dissociated Human Pluripotent Stem Cells
Using a Synthetic Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Eihachiro Kawase
Microarray Approach to Identify the Signaling Network Responsible
for Self-Renewal of Human Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Noboru Sato and Ali Brivanlou
Monitoring Stemness in Long-Term hESC Cultures by Real-Time PCR. . . . . . . . . . 89
Amparo Galán and Carlos Simón
Study of Gap Junctions in Human Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . 105
Alice Pébay and Raymond C.B. Wong
Immunofluorescence Microscopy and mRNA Analysis of Human
Embryonic Stem Cells (hESCs) Including Primary Cilia Associated
Signaling Pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Maj Linea Vestergaard, Aashir Awan, Caroline Becker Warzecha,
Søren Tvorup Christensen, and Claus Yding Andersen
Analysis of Intracellular Calcium Signaling in Human Embryonic
Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Adrienn Péntek, Katalin Pászty, and Ágota Apáti
Genetic Manipulation of Human Embryonic Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . 149
Rachel Eiges
Genetic Modification in Human Pluripotent Stem Cells
by Homologous Recombination and CRISPR/Cas9 System . . . . . . . . . . . . . . . . . . . . 173
Haipeng Xue, Jianbo Wu, Shenglan Li, Mahendra S. Rao,
and Ying Liu
Use of Multicolor Flow Cytometry for Isolation of Specific Cell
Populations Deriving from Differentiated Human Embryonic Stem Cells . . . . . . . . 191
Isabella Mengarelli, Andrew Fryga, and Tiziano Barberi
vii
viii Contents
Definitive Endoderm Differentiation of Human Embryonic Stem

Cells Combined with Selective Elimination of Undifferentiated Cells
by Methionine Deprivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Tomonori Tsuyama, Nobuaki Shiraki, and Shoen Kume
Derivation of Endothelial Cells and Pericytes from Human
Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Sravanti Kusuma and Sharon Gerecht
Differentiation of Human Embryonic Stem Cells on Periodontal
Ligament Fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Y. Murat Elçin, Bülend İnanç, and A. Eser Elçin
Derivation of Epithelial Cells from Human Embryonic Stem Cells
as an In Vitro Model of Vocal Mucosa. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Vlasta Lungova, Ciara Leydon, and Susan Thibeault
Human Embryonic and Hepatic Stem Cell Differentiation Visualized
in Two and Three Dimensions Based on Serial Sections . . . . . . . . . . . . . . . . . . . . . . . . 245
Peter S. Vestentoft, Christian B. Brøchner, Niels Lynnerup,
Claus Yding Andersen, and Kjeld Møllgård
Derivation of Chondrogenic Cells from Human Embryonic
Stem Cells for Cartilage Tissue Engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Wei Seong Toh and Tong Cao
Microgrooved Surface Modulates Neuron Differentiation
in Human Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
David Lu, Chi-Shuo Chen, Chao-Sung Lai, Sushant Soni,
Taranze Lam, Clarence Le, Eric Y.-T. Chen, Thien Nguyen,
and Wei-Chun Chin
Directed Differentiation of Human Embryonic Stem Cells
into Neural Progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Erin Banda and Laura Grabel
Direct Conversion of Pluripotent Human Embryonic Stem
Cells Under Defined Culture Conditions into Human Neuronal
or Cardiomyocyte Cell Therapy Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Xuejun H. Parsons
Generation of Epithelial Cell Populations from Human
Pluripotent Stem Cells Using a Small-Molecule Inhibitor
of Src Family Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Joshua A. Selekman, Xiaojun Lian, and Sean P. Palecek
Dual-SMAD Inhibition/WNT Activation-Based Methods to Induce
Neural Crest and Derivatives from Human Pluripotent Stem Cells. . . . . . . . . . . . . . . 329
Stuart M. Chambers, Yvonne Mica, Gabsang Lee, Lorenz Studer,
and Mark J. Tomishima
Accelerated Three-Dimensional Neuroepithelium Formation
from Human Embryonic Stem Cells and Its Use for Quantitative
Differentiation to Human Retinal Pigment Epithelium . . . . . . . . . . . . . . . . . . . . . . . . . 345
Yu Zhu, Sven Schreiter, and Elly M. Tanaka
Contents ix
Efficient Production of Photoreceptor Precursor Cells from Human

Embryonic Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Anat Yanai, Christopher Laver, Aaron W. Joe, and Kevin Gregory-Evans
Generation of Megakaryocytes and Platelets from Human
Marjorie Pick
A Simple Protocol for the Generation of Cardiomyocytes from Human
Glen Lester Sequiera, Ashish Mehta, and Winston Shim
Erratum to: Microgrooved Surface Modulates Neuron Differentiation
in Human Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
David Lu, Chi-Shuo Chen, Chao-Sung Lai, Sushant Soni,
Taranze Lam, Clarence Le, Eric Y.-T. Chen, Thien Nguyen,
and Wei-Chun Chin
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Contributors
CLAUS YDING ANDERSEN Laboratory of Reproductive Biology, Rigshospitalet, University

Hospital of Copenhagen, Copenhagen, Denmark
ÁGOTA APÁTI Molecular Biophysics Research Group, The Hungarian Academy of Sciences,
Semmelweis University, Budapest, Hungary
AASHIR AWAN Laboratory of Reproductive Biology, Rigshospitalet, University Hospital of
Copenhagen, Copenhagen, Denmark
ERIN BANDA Department of Biology, Hall-Atwater Laboratories, Wesleyan University,
Middletown, CT, USA
TIZIANO BARBERI Australian Regenerative Medicine Institute, Monash University,
Clayton, VIC, Australia
CARA K. BRADLEY Genea Biomedx, Sydney, NSW, Australia
ALI BRIVANLOU Laboratory of Stem Cell Biology and Molecular Embryology, The
Rockefeller University, New York, NY, USA
CHRISTIAN B. BRØCHNER Department of Cellular and Molecular Medicine, Faculty of
Health and Medical Sciences, University of Copenhagen, Copenhagen N, Denmark;
Laboratory of Reproductive Biology, Rigshospitalet, University Hospital of Copenhagen,
Copenhagen, Denmark
TONG CAO Graduate School for Integrative Sciences and Engineering, National
University of Singapore, Singapore, Singapore; Tissue Engineering Program, Life Sciences
Institute, National University of Singapore, Singapore, Singapore
STUART M. CHAMBERS Developmental Biology Program, Center for Stem Cell Biology,
Sloan-Kettering Institute, New York, NY, USA; Department of Neurology and
Neuroscience, Institute for Cell Engineering, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
CHI-SHUO CHEN Bioengineering, University of California, Merced, CA, USA
ERIC Y-T CHEN Bioengineering, University of California, Merced, CA, USA; Center for
Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan
WEI-CHUN CHIN Bioengineering, University of California, Merced, CA, USA
SØREN TVORUP CHRISTENSEN Department of Biology, University of Copenhagen,
Copenhagen, Denmark
BILJANA DUMEVSKA Genea Biocells, Sydney, NSW, Australia
RACHEL EIGES Stem Cell Research Laboratory, Medical Genetics Institute, Shaare Zedek
Medical Center, Jerusalem, Israel
A. ESER ELÇIN Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory,
Ankara University Stem Cell Institute, Ankara, Turkey; Faculty of Science, Ankara
University, Ankara, Turkey
Y. MURAT ELÇIN Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory,
Faculty of Science, Ankara University, Ankara, Turkey; Stem Cell Institute, Ankara
University, Ankara, Turkey
CARMEN ESCOBEDO-LUCEA Tissue Engineering Group, Division of Pharmaceutical
Biosciences, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland; Institute
of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University,
Tokyo, Japan
xi
xii Contributors
JEFFREY FERGUS Life Sciences Solutions, Thermo Fisher Scientific, Carlsbad, CA, USA
ANDREW FRYGA Nursing and Health Sciences, FlowCore, Faculty of Medicine, Monash
University, Clayton, VIC, Australia
AMPARO GALÁN Gene Expression and RNA Metabolism, Prince Felipe Research Center
(CIPF), Valencia, Spain
SHARON GERECHT Department of Materials Science and Engineering, Johns Hopkins
University, Baltimore, MD, USA; Department of Chemical and Biomolecular
Engineering, Johns Hopkins University, Baltimore, MD, USA; Johns Hopkins Physical
Sciences-Oncology Center, Institute for NanoBioTechnology, Johns Hopkins University,
Baltimore, MD, USA
LAURA GRABEL Department of Biology, Hall-Atwater Laboratories, Wesleyan University,
Middletown, CT, USA
KEVIN GREGORY-EVANS Department of Ophthalmology and Visual Sciences, Faculty of
Medicine, University of British Columbia, Vancouver, BC, Canada
BÜLEND İNANÇ Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory,
Faculty of Science, Ankara University, Ankara, Turkey; Department of Periodontology,
Faculty of Dentistry, Yüzüncü Yil University, Van, Turkey
AARON W. JOE Department of Ophthalmology and Visual Sciences, Faculty of Medicine,
University of British Columbia, Vancouver, BC, Canada
EIHACHIRO KAWASE Facility of Cell Processing, Department of Embryonic Stem Cell
Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
SHOEN KUME Department of Stem Cell Biology, Institute of Molecular Embryology and
Genetics (IMEG), Kumamoto University, Kumamoto, Japan; HIGO (Health life science;
Interdisciplinary and Glocal Oriented) Program, Program for Leading Graduate Schools,
Kumamoto, Japan; Graduate School of Bioscience and Biotechnology, Tokyo Institute of
Technology, Yokohama, Kanagawa, Japan
SRAVANTI KUSUMA Department of Biomedical Engineering, Johns Hopkins University,
Baltimore, MD, USA; Department of Chemical and Biomolecular Engineering, Johns
Hopkins Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, MD,
USA; Institute for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA
CHAO-SUNG LAI Department of Electronic Engineering, Chang Gung University,
Taoyuan, Taiwan
UMA LAKSHMIPATHY Life Sciences Solutions, Thermo Fisher Scientific, Carlsbad, CA, USA
TERANZE LAM School of Nature Sciences, University of California, Merced, CA, USA
CHRISTOPHER LAVER Department of Ophthalmology and Visual Sciences, Faculty of
Medicine, University of British Columbia, Vancouver, BC, Canada
CLARENCE LE Bioengineering, University of California, Merced, CA, USA
GABSANG LEE Department of Neurology and Neuroscience, Institute for Cell Engineering,
Johns Hopkins University School of Medicine, Baltimore, MD, USA
CIARA LEYDON Department of Speech Language Pathology, Sacred Heart University,
Fairfield, CT, USA
SHENGLAN LI Department of Neurosurgery, Center for Stem Cell and Regenerative
Medicine, Medical School, University of Texas Health Science Center at Houston, Houston,
TX, USA; The Senator Lloyd & B.A. Bentsen Center for Stroke Research, The Brown
Foundation Institute of Molecular Medicine for the Prevention of Human Diseases,
University of Texas Health Science Center at Houston, Houston, TX, USA
XIAOJUN LIAN Department of Chemical and Biological Engineering, University of
Wisconsin, Madison, WI, USA
Contributors xiii
YING LIU Department of Neurosurgery, Center for Stem Cell and Regenerative Medicine,
Medical School, University of Texas Health Science Center at Houston, Houston, TX,
USA; The Senator Lloyd & B.A. Bentsen Center for Stroke Research, The Brown
DAVID LU Bioengineering, University of California, Merced, CA, USA
VLASTA LUNGOVA Division of Otolaryngology-Head and Neck Surgery, Department of
Surgery, University of Wisconsin, Madison, WI, USA
NIELS LYNNERUP Department of Forensic Medicine, Faculty of Health and Medical
Sciences, University of Copenhagen, Copenhagen, Denmark
ASHISH MEHTA Research and Development Unit, National Heart Centre, Singapore,
Singapore
ISABELLA MENGARELLI Department of Experimental Cardiology, Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands
YVONNE MICA Developmental Biology Program, Center for Stem Cell Biology, Sloan-
Kettering Institute, New York, NY, USA; Life Technologies, Carlsbad, CA, USA
KJELD MØLLGÅRD Department of Cellular and Molecular Medicine, Faculty of Health
and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
THIEN NGUYEN Department of Neurology, The Johns Hopkins University School of
Medicine, Baltimore, MD, USA
SEAN P. PALECEK Department of Chemical and Biological Engineering, University of
XUEJUN H. PARSONS San Diego Regenerative Medicine Institute, San Diego, CA, USA;
Xcelthera, Inc., San Diego, CA, USA
KATALIN PÁSZTY Molecular Biophysics Research Group, Hungarian Academy of Sciences,
Budapest, Hungary; Department of Biophysics, Semmelweis University, Budapest,
Hungary
ALICE PÉBAY Department of Ophthalmology, Centre for Eye Research Australia, Royal
Victorian Eye and Ear Hospital, University of Melbourne, Melbourne East, VIC,
Australia
ADRIENN PÉNTEK Institute of Molecular Pharmacology, Research Centre for Natural
Sciences, Hungarian Academy of Sciences, Budapest, Hungary
TEIJA T. PEURA Genea Biomedx, Sydney, NSW, Australia
MARJORIE PICK Department of Hematology, Hadassah-Hebrew University Medical
Center, Ein Kerem, Jerusalem, Israel
RENE QUINTANILLA Life Sciences Solutions, Thermo Fisher Scientific, Carlsbad, CA, USA
MAHENDRA S. RAO Center for Regenerative Medicine, NIH, Bethesda, MD, USA
TAMMIE K. ROY Genea Biomedx, Sydney, NSW, Australia
ANDRES SANZ-GARCIA Tissue Engineering Group, Division of Pharmaceutical Biosciences,
Faculty of Pharmacy, University of Helsinki, Helsinki, Finland; Institute of Advanced
Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan
NOBORU SATO Department of Biochemistry, University of California, Riverside, CA, USA
JULIA SCHAFT Genea Biocells, Sydney, NSW, Australia
SVEN SCHREITER DFG Research Center for Regenerative Therapies, Technische Universit€ at
Dresden, Dresden, Germany
JOSHUA A. SELEKMAN Department of Chemical and Biological Engineering, University of
xiv Contributors
GLEN LESTER SEQUIERA Research and Development Unit, National Heart Centre,
Singapore, Singapore; DUKE-NUS Graduate Medical School, Singapore, Singapore
WINSTON SHIM Research and Development Unit, National Heart Centre, Singapore,
Singapore; DUKE-NUS Graduate Medical School, Singapore, Singapore
NOBUAKI SHIRAKI Department of Stem Cell Biology, Institute of Molecular Embryology and
Genetics (IMEG), Kumamoto University, Kumamoto, Japan
CARLOS SIMÓN IVI Foundation, Instituto Universitario IVI, University of Valencia,
Valencia, Spain
SUSHANT SONI Bioengineering, University of California, Merced, CA, USA
MIODRAG STOJKOVIC SPEBO Medical, Leskovac, Serbia; Department of Human Genetics,
Faculty of Medicine, University of Kragujevac, Kragujevac, Serbia
LORENZ STUDER Developmental Biology Program, Department of Neurosurgery, Center
for Stem Cell Biology, Sloan-Kettering Institute, New York, NY, USA
ELLY M. TANAKA DFG Research Center for Regenerative Therapies, Technische
Universit€ at Dresden, Dresden, Germany
SUSAN THIBEAULT Division of Otolaryngology-Head and Neck Surgery, Department of
Surgery, University of Wisconsin, Madison, WI, USA
WEI SEONG TOH Discipline of Oral Sciences, Faculty of Dentistry, National University of
Singapore, Singapore, Singapore
MARK J. TOMISHIMA Developmental Biology Program, SKI Stem Cell Research Facility,
Center for Stem Cell Biology, Sloan-Kettering Institute, New York, NY, USA
TOMONORI TSUYAMA Department of Stem Cell Biology, Institute of Molecular Embryology
and Genetics (IMEG), Kumamoto University, Kumamoto, Japan; HIGO (Health life
science; Interdisciplinary and Glocal Oriented) Program, Program for Leading Graduate
Schools, Kumamoto, Japan
PETER S. VESTENTOFT Dako Denmark A/S, Glostrup, Copenhagen N, Denmark
MAJ LINEA VESTERGAARD Laboratory of Reproductive Biology, Rigshospitalet, University
Hospital of Copenhagen, Copenhagen, Denmark
CAROLINE BECKER WARZECHA Department of Biology, University of Copenhagen,
Copenhagen, Denmark
RAYMOND C. B. WONG Department of Ophthalmology, Centre for Eye Research Australia,
Royal Victorian Eye and Ear Hospital, University of Melbourne, Melbourne East, VIC,
Australia
JIANBO WU Department of Neurosurgery, Center for Stem Cell and Regenerative
HAIPENG XUE Department of Neurosurgery, Center for Stem Cell and Regenerative
ANAT YANAI Department of Ophthalmology and Visual Sciences, Faculty of Medicine,
University of British Columbia, Vancouver, BC, Canada
YU ZHU Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Methods in Molecular Biology (2015) 1307: 1–23
DOI 10.1007/7651_2014_85
© Springer Science+Business Media New York 2014
Published online: 25 June 2014
Derivation of Human Embryonic Stem Cell Lines

from Vitrified Human Blastocysts
Cara K. Bradley, Julia Schaft, Tammie K. Roy, Biljana Dumevska,
and Teija T. Peura
Abstract
Human embryonic stem cells are pluripotent cells typically derived from blastulating embryos that have
become excess to clinical needs in assisted reproduction programs. They provide cellular models for
embryonic development and disease, and are thought to be useful for future cell replacement therapies
and regenerative medicine. Here we describe methods to derive human embryonic stem cell lines.
This includes blastocyst cryopreservation using a highly efficient vitrification protocol, the production
and use of fibroblast feeder cells, embryo plating and passaging of resulting cellular outgrowths, and
cryopreservation of putative stem cells lines.
Keywords: Blastocyst, Cryopreservation, Derivation, Embryonic stem cell, Fibroblast feeder cells,
Human, Vitrification
1 Introduction
Human embryonic stem cells (hESCs) were first described by

Thomson and colleagues in 1988 (1) and are most commonly
derived from the pluripotent inner cell mass (ICM) of blastulating
embryos. The source of donated embryos is typically excess blas-
tocysts from assisted reproduction programs, whether because a
couple has completed their family or because the embryo was
found to be clinically unsuitable for transfer due to severe genetic
condition. Their unique characteristics include the ability to grow
in vitro indefinitely and the capacity to differentiate into specialized
somatic cell types from the three germ layers. This makes them and
their differentiated products valuable in vitro models of embryonic
development and disease, as well as tools for drug discovery and
toxicity screening (2). hESCs are also widely considered to be
clinically useful for still to be perfected cell replacement therapies
and regenerative medicine (3).
This chapter covers the steps from the moment a human
embryo is assigned for cryostorage, to be later possibly used for
1
2 Cara K. Bradley et al.
stem cell derivation, up until a new hESC line has been generated
and secured by cryopreservation. This includes embryo cryopreser-
vation using a highly efficient modified Cryotop vitrification
method (4), the production and mitotic inactivation of primary
fibroblast cells used as a feeder layer for hESCs, and derivation of
putative hESC lines including embryo plating, initial embryonic
outgrowth passages, and cryopreservation. The methods for
human IVF are not described, nor are the methods to expand and
characterize the putative hESC line once obtained, as they fall
outside the aims of this chapter.
2 Materials
2.1 Embryo 1. Biological safety cabinet.

Vitrification 2. Stereomicroscope with heating stage.
and Warming
3. Embryo incubator (K-MINC-1000 mini-incubator, Cook IVF,
Cat No. K-MINC-1000).
4. CVM Vitrification Block with handle and lid (Cryologic, Cat
No. CVB, Victoria, Australia). The block should be sterilized
using dry heat 140 C for 6 h or 160 C for 2 h.
5. CVM Cryobath with lid (Cryologic, Cat No. LNB).
6. Cryotop vitrification device (Kitazato, Cat No. 81111 to
81115, Japan).
7. 4-well plates (Nunc, Cat No. 144444).
8. Indirect mouth hose (preventing open airway contact with
solution) and embryo handling pipettes. Alternatively, hand-
held micromanipulation pipettes can be used (Flexipet, Cook
IVF, Cat No. K-MPH-1000).
9. P2, P20, P200, and P1000 pipettes and tips.
10. Liquid nitrogen storage dewar, canes, and goblets.
11. Sydney IVF Blastocyst Vitrification Kit (Cook IVF, Cat No.
K-SIBV-5000) (see Note 1) consisting of a Solution 1 (Cryo-
base buffer), Solution 2 (used during vitrification after addition
of DMSO to make a final solution of 8 % ethylene glycol and
8 % DMSO in Cryobase), Solution 3 (used during vitrification
after addition of DMSO to make a final solution of 0.68 M
trehalose, 16 % ethylene glycol, and 16 % DMSO in Cryobase)
and Solution 4 (DMSO).
12. Blastocyst warming solutions: 1) Cryobase, 2) 1 M trehalose in
Cryobase and 3) 0.5 M trehalose in Cryobase. Cryobase is
HEPES buffered medium containing 20 mg/mL human
serum albumin and 0.01 mg/mL gentamicin (Cook IVF).
Human Embryonic Stem Cell Derivation 3
13. Sydney IVF Blastocyst Medium (Cook IVF, Cat No. K-SIBM-20)
(see Note 1).
14. Embryo culture grade oil (COOK IVF, Cat No. K-SICO-200).
15. Liquid nitrogen.
2.2 Fibroblast Feeder 1. Class II biological safety cabinet.

Cell Preparation 2. Cell culture incubator.
3. Microscope.
4. Gamma irradiation device.
5. Liquid nitrogen dewar with box storage.
6. Liquid waste container or aspirator system.
7. P2, P20, P200, and P1000 pipettes and tips.
8. Cryo label printer and cryo labels or a permanent marker.
9. Forceps and dissecting scissors.
10. Scalpel blades and scalpel blade handle.
11. Cell counter.
12. 0.22 μm sterile filters.
13. 1, 5, 10, 25, and 50 mL pipettes.
14. 15 and 50 mL centrifuge tubes.
15. 1.2 mL cryovials.
16. Culture vessels, e.g., 1-well organ culture dishes (BD Falcon,
Cat No. 353037), 4- or 6-well plates (Nunc, Cat No. 176740
and 140675, respectively), and T25, T75, or T175 flasks (BD
Falcon, Cat No. 353014, 353024, and 353028, respectively).
17. Human tissue sample, aseptically and freshly collected, prefera-
bly at least a few grams (or approximately 5 5 mm in size).
Alternatively, an established commercially sourced feeder cell
line can be used (e.g., CCL-110 human fibroblast line, ATCC).
18. Feeder medium consisting of Dulbecco’s Modified Eagle’s
Medium (DMEM high glucose, no L-glutamine, no sodium
pyruvate, Life Technologies, Gibco, Cat No. 11960-044)
supplemented with 10 % fetal calf serum (Cat No. 16141-079),
1 MEM nonessential amino acids (Cat No. 11140-050), 2 mM
Glutamine (Cat No. 25030-149), 1 mM Sodium Pyruvate
(Cat No. 11360-070), and 50 U/mL penicillin and 50 μg/mL
streptomycin (Cat No. 15140-122). The medium is sterile
filtered and stored at +4 C for a maximum of 1 month.
19. Magnesium and calcium-free PBS (PBS ( )).
20. 0.25 % trypsin.
21. 0.1 % gelatin (Millipore, Cat No. ES-006-B).
22. DMSO.
23. 70 % ethanol.
2.3 hESC Derivation 1. Class II biological safety cabinet.

2. Stereomicroscope with heating stage and eyepiece extension
making it suitable for working inside a class II biological safety
cabinet.
3. Cell culture incubator (or embryo incubator).
4. Indirect mouth hose (preventing open airway contact with
solution) and embryo handling pipettes. Alternatively, hand-
held micromanipulation pipettes can be used (Flexipet, Cook
IVF, Cat No. K-MPH-1000).
5. P2, P20, P200, and P1000 pipette and tips.
6. Ultra sharp splitting blades (e.g., Bioniche Animal Health, Cat
No. ESE020), washed and sterilized. These can be used several
times until becoming blunt.
7. Mitotically inactivated feeder cell plate (see Section 3.2, plated
1–7 days prior to use) with hESC medium.
8. Blastocyst culture medium (Cook IVF, Cat No. K-SIBM-20).
9. Embryo handling medium (Cook IVF, Cat No. K-SIFB-100).
10. Embryo culture grade oil (Cook IVF, Cat No. K-SICO-200).
11. hESC culture medium consisting of Knock Out-DMEM (Life
Technologies, Gibco, Cat No. 10829-018) supplemented with
2 mM glutamine (Cat No. 25030-081), 50 U/mL penicillin
and 50 μg/mL streptomycin (Cat No. 15140-122), 1 MEM
nonessential amino acids (Cat No. 11140-050), 0.1 mM β-
mercaptoethanol (Cat No. 21985-023), and 20 % Knock-out
Serum Replacement (Cat No. 10828-028). The medium is
sterile filtered and stored at +4 C for a maximum of 1 month.
Just prior to use add bFGF (Chemicon, Cat No. GF-003) to a
final concentration of 20 ng/mL.
12. 70 % ethanol.
2.4 Vitrification 1. Class II biological safety cabinet.

and Warming 2. Stereomicroscope with heating stage and eyepiece extension
of Putative Early hESCs making it suitable for working inside a class II biological safety
cabinet.
3. Cell culture incubator.
4. Liquid nitrogen storage dewar, canes, and goblets.
5. P20, P200, and P1000 pipette and tips.
6. Cryo label printer, cryo labels, and permanent marker.
7. Open pulled straws (OPS), 0.5 mL (outer) straws, and straw
putty.
8. 4-well plates (Nunc, Cat No. 144444).
9. 0.22 μm filters.
10. Sterile forceps.

11. Ultra sharp splitting blades (e.g., Bioniche Animal Health,
Cat No. ESE020), washed and sterilized. These can be used
several times until becoming blunt.
12. Mitotically inactivated feeder cell plates (see Section 3.2, plated
1–7 days prior to use) with hESC medium.
13. 2 M sucrose solution in DMEM-HEPES.
14. DMEM-HEPES (without phenol red).
15. Fetal calf serum (FCS).
16. DMSO.
17. Ethylene glycol.
18. Liquid nitrogen.
3 Methods
3.1 Embryo Vitrification is a method of cryopreservation which uses a high

Vitrification concentration of cryoprotectants and ultra-rapid cooling to freeze
and Warming cells in a glass-like state. Unlike conventional slow freezing, vitrifi-
cation avoids detrimental ice crystal formation resulting in excellent
(>90 %) survival rates after embryo thawing. However, prolonged
exposure to high concentrations of cryoprotectants increases
embryotoxicity, making vitrification as demanding as it is successful
when properly executed. The method described here utilizes solu-
tions from the Sydney IVF Blastocyst Vitrification Kit (see Note 1)
with a modified Cryotop protocol whereby vitrification occurs on
the Cryotop device by contact with a metal block precooled with
liquid nitrogen. This procedure minimizes embryo contact with
liquid nitrogen to prevent potential cross-contamination and has
been found to result in pregnancy rates upon warming equivalent
to that of fresh embryo transfers (4).
All protocols involving embryos should be performed in a
biological safety cabinet under sterile conditions using aseptic tech-
niques and preferably disposable consumables. Embryo incubation
should be performed in a controlled humidified atmosphere of 6 %
CO2, 5 % O2, and 89 % N2 at 37 ºC, preferably in a mini-incubator
with separately controlled culture chambers, and embryos should
be removed from these conditions only for a minimum length of
time required for a given procedure.
3.1.1 Embryo Vitrification 1. Appropriately label top and bottom of 4-well dish(es). One dish
can be used to vitrify two embryos from a single patient.
Prepare each vitrification dish as follows using solutions from
the Sydney IVF Blastocyst Vitrification Kit (see Note 1):
(a) To well #1 and #2 add 460 μL Solution 2 and 40 μL

Solution 4 and mix thoroughly.
(b) To well #3 and #4 add 420 μL Solution 3 and 80 μL
Solution 4 and mix thoroughly.
(c) Allow the dish to come to room temperature for 10 min
and use within 60 min.
2. Appropriately label Cryotop vitrification device(s) (one for each
embryo to be vitrified), as well as cane(s) for liquid nitrogen
storage.
3. Fill the cryobath with liquid nitrogen to approximately half the
height of the metal vitrification block. Slowly lower the metal
block with lid into the bath and allow cooling for at least 5 min
before adding further liquid nitrogen to the level just below the
top of the block.
4. Fill a liquid nitrogen container with liquid nitrogen and place
the labeled cane(s) into the bucket to precool.
5. Place Cryotop sheath(s) into the metal block for cooling prior
to use.
6. Move a single embryo to the top of the medium in well #1.
Observe the embryo for collapse of the blastocoel cavity
(see Note 2) and subsequent re-expansion. The embryo is
ready to be moved to the next solution after 12 mins if the
embryo has expanded to >80 % of its initial volume and has not
exhibited any further expansion within a 1 min timeframe, or if
15 min has expired.
7. If freezing an additional embryo this may be transferred to well
#2 of the vitrification dish 3–5 min after the first embryo.
Additional embryos from the same patient can be transferred
to additional vitrification dishes at 3–5 min intervals.
8. The following steps from 9 to 12 should be completed in
60–90 s.
9. When equilibration is complete, transfer the embryo in a mini-
mal volume to the surface of solution in well #3. A swirling
motion whilst dispelling the embryo can aid in the removal of
the previous solution. Wash the embryo thoroughly by moving
it around the well.
10. Transfer embryo in a minimal volume to the top of the solution
in well #4. Wash the embryo as per step 9.
11. Transfer the embryo in a minimal volume from well #4 onto
the Cryotop device (logo side up) near the black marking.
Aspirate excess solution with pipette (see Note 3).
12. Transfer the Cryotop device to the vitrification block and place
the end of the Cryotop onto an allocated spot on the block
surface for 5–7 s.
13. Quickly insert the Cryotop device into a chilled sheath

and gently press down to seal. During this process keep the
Cryotop as close as possible to the metal block to avoid
warming.
14. Move Cryotop to the labeled cane in liquid nitrogen.
15. Repeat steps 6 onwards for remaining embryos. When all
embryos from the patient have been vitrified they can be trans-
ferred to long-term liquid nitrogen storage. This should be
done as quickly as possible to avoid temperature fluctuations
(see Note 4).
3.1.2 Embryo Warming 1. At least 4 h or overnight prior to warming, prepare blastocyst

culture dish(es) containing drops of Sydney IVF Blastocyst
Medium (see Note 1) covered with equilibrated mineral oil,
and incubate at 37 C with 6 % CO2. Also include a “wash” well
containing 500 μL of Blastocyst Medium.
2. Prepare one 35 mm dish per patient containing 2.5 mL of 1 M
trehalose in Cryobase and warm to 37 C for at least 10 min.
3. Prepare one 4-well dish per patient as follows and allow to
come to room temperature:
(a) To well #1 add 500 μL 0.5 M trehalose in Cryobase.
(b) To well #3 and #4 add 500 μL Cryobase.
4. Transfer Cryotop device containing embryo to be thawed to a
liquid nitrogen container and place next to microscope.
5. Focus the microscope (warming stage off) view on the bottom
of the 35 mm dish containing 1 M trehalose in Cryobase.
6. Using tweezers with the tips cooled in liquid nitrogen, hold the
Cryotop protective cover under liquid nitrogen and twist Cryo-
top to loosen. Extract Cryotop, using care not to contact the
liquid nitrogen, and immediately place end into the 35 mm
dish, stirring it in a small circle until the embryo falls off the
Cryotop (see Note 5).
7. After 1 min, quickly move the embryo to the bottom of well #1
of the 4-well dish. Leave the embryo undisturbed (even if it
floats to the surface) for 3 min.
8. Transfer the embryo to the bottom of well #3 of the 4-well
dish. Leave embryo undisturbed for 5 min.
9. Transfer the embryo to well #4 of the 4-well dish. Leave
embryo undisturbed for 1 min.
10. Transfer the embryo to the wash well of the prepared blastocyst
culture dish and move around gently to remove any remaining
warming solution.
11. Transfer the warmed embryo into culture drop in the prepared
blastocyst culture plate, place in embryo incubator, and allow
to recover for at least a few hours prior to hESC derivation.

If desired, the embryo can be left overnight before plating.
However, this has to be determined on a case by case basis,
depending on the appearance and developmental stage of the
embryo.
3.2 Fibroblast Feeder hESCs are commonly cultured on a layer of primary fibroblast cells,
Cell Preparation the so-called “feeder cells,” to promote hESCs attachment and
provide factors important for proliferation and pluripotency.
Although it is possible to culture hESCs on semi-defined extracel-
lular matrices, the high demands of deriving a new hESC line
(supporting the proliferation of a small number of pluripotent
cells within the embryo) reduce the temptation of using these
matrices during derivation attempts. Feeder cells should ideally be
from human origin and can be initiated from primary tissue samples
such as neonatal foreskin or from commercially sourced established
primary cell lines. Whether using commercial or in-house prepared
feeders, it is advisable to prepare and freeze “master stocks” of early
passage cells to serve as a starting point for subsequent expansion of
“working stocks” for mitotic inactivation. Mitotic inactivation of
feeder cells is essential prior to derivation and culture of hESCs and
can be achieved efficiently and conveniently by gamma irradiation
as reported here, or by Mitomycin C treatment as described
previously (5).
All protocols involving feeder cell culture should be performed
in a biological safety cabinet under sterile conditions using aseptic
techniques and preferably disposable consumables. Incubation of
feeder cell cultures should be performed in a controlled humidified
atmosphere of 5 % CO2 in air at 37 ºC.
3.2.1 Establishing 1. Obtain the sample of human tissue (see Note 6) using sterile
a Primary Feeder Cell Line techniques and proceed with processing as soon as possible.
2. Transfer the tissue using sterile forceps to a 35 mm Petri dish
containing 3–4 mL of PBS( ) (see Note 7).
3. Agitate the dish gently to wash the tissue, removing adhering
blood or mucus if possible.
4. Repeat steps 2 and 3 until satisfied the tissue is as clean as
possible (usually 2–4 washes), then transfer the tissue to a
new empty dish (the tissue should still be moist with PBS).
5. Finely cut the tissue into pieces of at least 1 mm2 or as small as
possible using a scalpel blade. Ideally the pieces should be of
similar size so that attachment will occur at the same rate.
6. Transfer a minimum of 6–8 small pieces of tissue (see Note 8) to
the bottom of an empty T25 cell culture flask lying horizontally.
Replace the cap on the flask and stand upright for 15–20 min
(see Note 9). Prepare multiple flasks this way to ensure enough
material for the cultures and to safeguard against accidental

contamination or other loss of a single flask.
7. Add 5 mL of warm feeder medium to the bottom of each flask
in an upright position taking care to avoid the attached tissue
pieces.
8. Gently tilt the flask until the first tissue pieces touch the
medium and if they remain attached continue tilting until the
flask lies in a horizontal position. Carefully transfer the flask
into the incubator avoiding movement that may dislodge the
pieces. Alternatively, the upright flasks can be transferred to the
incubator immediately after adding the medium and then tilted
in the incubator to minimize movement.
9. If the first tissue pieces detach upon contact with the medium,
return the flask to the upright position and allow the remaining
pieces to dry for another 5 min before attempting to tilt the
flask. Repeat as necessary. Having just a few pieces detaching
and floating is not detrimental to the subsequent culture, but if
the source material is scarce these pieces can be removed from
the flask, transferred to an empty flask, and the attachment
procedure repeated.
10. Flasks should not be disturbed for a minimum of 48 h, after
which they can be assessed under a microscope for the out-
growth of fibroblast cells from the attached tissue piece.
Depending on the origin of the tissue, other cell types may
also be observed such as epithelial cells.
11. If the tissue pieces remain attached and outgrowth of cells is
visible, replace the culture medium with fresh medium to
remove any free floating pieces, red blood cells, or debris. If
the tissue pieces have not attached and the source tissue is
scarce, it may be worthwhile to attempt reattachment using
the procedure described above.
12. The medium should be changed on the flasks approximately
every 6 days until they are ready for passaging for further
expansion or cryopreservation. This occurs when the cells ori-
ginating from the attached tissue pieces are 70–80 % confluent
and still in the exponential growth phase, which may take
approximately 5–10 days (from plating) depending on the
amount of tissue plated.
3.2.2 Passaging Feeder 1. Examine the cultures under a microscope to determine the split
Cells ratio. For expanding fibroblast feeder cells this is usually
between 1:2 and 1:5 (i.e., one flask of cells split to two to five
new flasks).
2. Warm the required volume of PBS( ), 0.25 % trypsin, and
feeder medium for passaging and ongoing culture (see Table 1;
Table 1
Passaging of human fibroblast feeder cells
Feeder medium Feeder medium

Vessel PBS( ) 0.25 % trypsin (passaging) (culturing)
T25 3 1 4 7
T75 10 2 8 20
T175 20 4 16 50
Volumes (mL) of PBS, 0.25 % trypsin and feeder medium required for cell passaging for
commonly used culture vessels
note that feeder medium is also required during passaging for

trypsin inactivation).
3. Transfer required reagents and consumables to a biological
safety cabinet followed by the feeder flask(s) to be passaged.
Ideally no more than 4 flasks should be passaged simulta-
neously (see Note 10).
4. Aspirate medium from flask(s) to a waste container and add
PBS( ) to wash the cells (see Table 1).
5. Aspirate PBS( ), add 0.25 % trypsin (see Table 1), and gently
agitate to evenly distribute.
6. Incubate the cells in trypsin at 37 C for 2–3 min then tap the
side of flask(s) several times to dislodge the cells. Ensure the
cells have completely detached by visualizing under a
microscope.
7. Once cells have detached, add approximately 4 volume of
feeder medium (see Table 1) to the flask(s) and mix. Pipette
the cells several times and transfer to a 50 mL tube.
8. Collect any remaining cells in the flask by addition of new
feeder medium (this same volume can be transferred into each
flask sequentially) and add to the 50 mL tube. The cells from
several flasks can be combined to one or more tubes depending
on the number of flask processed.
9. Centrifuge the tube(s) for 4 min at 300 g.
10. Remove supernatant being careful not to disrupt the cell pellet.
11. Resuspend cells in an appropriate volume of feeder medium
depending on the number of flasks to be seeded. This can be
calculated on the basis of 1–2 mL of cell suspension for each
new flask, or alternatively, if splitting cells to only a few flasks,
the cells can be resuspended in the final volume of feeder
medium (see Table 1).
12. Pipette the required volume of cell suspension to each new flask
and if required add feeder medium to achieve the final culture
volume.
13. Transfer newly plated flasks into incubator until ready for fur-
ther passaging or freezing. Generally media change is not
required during this time (see Note 11) unless culturing for
longer than 7–8 days.
3.2.3 Freezing 1. Fibroblast feeder cells are ready to be frozen when they have
Feeder Cells reached 70–80 % confluence and are still in an exponential
growing phase. Typically cells are frozen at a concentration of
1 106/mL in 0.5 mL or 1 mL aliquots. This may have to be
performed in several batches depending on the number of flasks
(and cells) to be frozen (see Note 12).
2. In preparation for freezing, label the estimated number of
cryovials with cell line details and passage number, the freezing
date, and the number of cells for cryopreservation (see Note
13). Place cryovials in a tube rack and cool by storing at 20 C
until required.
3. Trypsinize the cells as per passaging protocol (steps 2–10 in
Section 3.2.2).
4. After centrifugation, resuspend cells in a suitable volume of
room temperature feeder medium to enable accurate counting.
For example, 1–3 mL per T75 flask containing 70–80 %
confluent cells if using a hemocytometer.
5. Determine the total number of viable cells in the cell
suspension.
6. Calculate the volume required to dilute cells to the desired
concentration taking into account the volume of feeder
medium in which the cells are currently suspended and that
10 % of the final volume is to consist of DMSO (see Note 14).
7. Add the remaining volume of feeder medium to the cell sus-
pension, mix gently, and place tube on ice.
8. Retrieve the tube rack containing labeled cryovials from the
freezer and loosen the lids in preparation for use.
9. Add required volume of DMSO to the tube containing the cell
suspension so that the final freezing medium composition is
90 % feeder medium and 10 % DMSO. Mix thoroughly keeping
the tube on ice.
10. Pipette the appropriate volume of freeze medium containing
resuspended cells into each cryovial, mixing the remaining cells
in the 50 mL tube frequently to ensure that the aliquoted cell
numbers remain consistent throughout the process. Work
quickly keeping the cell mixture on ice and the cryovials in
the precooled rack and handling them as little as possible to

minimize warming.
11. Quickly transfer the cryovials into a 80 C freezer overnight
or maximum for a few days. Long-term storage at 80 C is
not recommended as viability can be compromised.
12. Quickly transfer the cryovials from the 80 C freezer into
liquid nitrogen storage. This should be done in a way that
ensures that harmful warming of the vials does not occur.
3.2.4 Thawing 1. Determine the number of vials to be thawed. Depending on the

Feeder Cells fibroblast feeder cell line, the plating density for further expan-
sion can be as low as 5,000 cells per cm2 surface area.
2. In preparation for thawing, fill a suitable container with
37–40 C water or set up a water bath to this temperature
range.
3. Remove the cryovials(s) from liquid nitrogen storage and trans-
fer to the laboratory in a suitable container filled with liquid
nitrogen, dry ice or nothing, depending on time until proces-
sing; cryovial(s) can be transported at room temperature for a
minute if processed immediately. Depending on the number of
cyrovials required, thawing may have to be performed in several
batches (see Note 15).
4. Pipette a minimum of 7 mL of warm feeder medium per every
0.5 mL of frozen cells to be thawed into a 50 mL tube.
5. Place the cryovials(s) into warm water and thaw until only a
sliver of ice can be seen in the vial. This usually takes a few
minutes and is faster if the vial is moved through the water.
6. Quickly clean the outside of the cryovial(s) with 70 % ethanol
and transfer to the biological safety cabinet.
7. Pipette the contents of the vial(s) slowly and drop-wise into the
50 mL tube containing feeder medium.
8. Pipette 1 mL of feeder medium containing thawed cells back to
the cryovial(s) to recover any remaining cells then transfer to
the 50 mL tube. Depending on the number of vials thawed,
the cells from several vials can be collected to one or more
50 mL tubes.
9. Centrifuge the tube(s) for 4 min at 300 g.
10. Remove supernatant being careful not to disrupt the cell pellet.
11. Resuspend cells in an appropriate volume of feeder medium
depending on the number of flasks to be seeded. This can be
calculated on the basis of 1–2 mL of cell suspension for each
new flask, or alternatively, if plating cells to only a few flasks, the
cells can be resuspended in the final volume of feeder medium
(see Table 1).
12. Pipette the required volume of cell suspension to each new flask
and if required add feeder medium to achieve the final culture
volume.
13. Transfer newly plated flasks into incubator until ready for fur-
ther passaging or mitotic inactivation. Generally media change
is not required during this time (see Note 11) unless culturing
for longer than 7–8 days.
3.2.5 Mitotic Inactivation 1. Fibroblast feeder cells are ready for gamma irradiation when
of Feeder Cells by Gamma approaching confluence (approximately 90 %). We typically
Irradiation prepare a batch of 100–150 million feeder cells for gamma
irradiation (16 T175 flasks) and cryopreservation.
2. Harvest the cells as described for feeder cell passaging (steps
2–10 in Section 3.2.2).
3. After centrifugation, resuspend the pelleted cells in feeder
medium and transfer tubes to ice.
4. Irradiate cell suspension with 4,000 rad gamma irradiation.
5. Determine concentration of viable cells and freeze working
stocks of mitotically inactivated feeder cells at 1 106/mL as
described in Section 3.2.3 (steps 6–11). Mitotically inactivated
feeder cells can also be plated immediately after irradiation for
hESC derivation and culture the following day.
6. After preparation of a batch of gamma irradiated feeder cells, it
is sensible to perform quality control measures to determine
success of mitotic inactivation (see Note 16).
3.2.6 Plating of 1. Coat the surface of the required culture vessels with 0.1 %
Mitotically Inactive gelatin (see Table 2) and incubate at room temperature for a
Feeder Cells minimum of 10 min (and up to several hours).
2. Aspirate 0.1 % gelatin from vessels and allow to dry in a
biological safety cabinet.
3. If using organ culture dishes or 4-well plates, add PBS( ) to the
moat of the dishes or to the centre of the plates (see Note 17).
4. Determine required number of mitotically inactivated feeder
cells (see Table 2 and Note 18).
5. Thaw vials of mitotically inactivated feeder cells as per Sec-
tion 3.2.4 (steps 2–10).
6. Resuspend the cell pellet in a suitable volume of feeder medium
and determine number of viable cells.
7. Pipette the required amount of cell suspension to each vessel
and if required, make up final volume with feeder medium (see
Table 2).
Table 2
Preparation of mitotically inactivated fibroblast feeder cell vessels
for hESC culture
Feeder cell
Growth
area Gelatin Medium
Vessel (cm2) (mL) Density # (106) (mL)
4-well plate 1.9 0.2 High 0.13 0.7
Low 0.045
Organ culture 2.9 0.3 High 0.2 1
dish
Low 0.07
6-well plate 9.5 1 Low 0.23 3
35 mm dish 10 1 Low 0.24 3
T25 flask 25 2 Low 0.6 7
T75 flask 75 5 Low 1.8 20
Volumes of 0.1 % gelatin and medium, and number of mitotically inactivated fibroblast
cells for commonly used culture vessels. A higher density of feeder cells is used when
deriving hESC lines and a lower density when culturing established hESC lines. Values
given for 4-well and 6-well plates are for a single well
8. Transfer culture vessels to incubator. The mitotically inacti-

vated feeder cell cultures are ready for use the following day,
and no later than 3 days after plating for hESC derivation and 7
days for established hESC lines.
3.3 hESC Derivation hESC lines are typically derived from excess cryopreserved blasto-
cysts created for assisted conception. The scarcity of donated
human embryos has meant that the methods for hESC derivation
have not changed significantly since the first reports of primate
embryonic stem cell generation (6) and still rely on cultivation on
top of a fibroblast feeder cell layer and mechanical passaging. This
section describes the techniques of hESC line derivation including
embryo zona pellucida removal, bisection, and plating, which are
followed by first critical manual passages of early embryonic out-
growths. These methods have been used for successful derivation of
over 100 hESC lines (see www.geneabiocells.com), including
GMP-level clinical grade lines (7), PGD-derived lines (8, 9), and
lines derived from poor quality clinically unsuitable embryos (10).
All protocols involving embryo and cell culture work should
be performed in a biological safety cabinet under sterile conditions
using aseptic techniques and preferably disposable consumables.
Cell culture incubation should be performed in a controlled
humidified atmosphere of 5 % CO2 in air at 37 ºC or, preferably,
in 6 % CO2, 5 % O2, and 89 % N2 at 37 ºC.
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degrees F.—and are immediately succeeded by marked increase in
the severity of the symptoms, both mental and physical, especially if
the attacks follow each other in rapid succession or last for a number
of days. They may be due to hemorrhage, embolism, or effusion,
and be marked by any or all of the usual symptoms and sequences
of those conditions, permanent or transient. General and aural
vertigo are not uncommon.
The muscular tremor before the last stages varies in different

muscles—excessive perhaps in the tongue, moderate in the fingers,
and so on. It may also seem slight as compared with the other
symptoms, or, on the other hand, be enormously exaggerated in
certain groups of muscles out of all proportion to all other indications.
At the end extreme and constant tremulousness accompanies every
voluntary movement.
Spastic paralysis, muscular tension, contractures, rigidity of the most

persistent character seem at times to be under the influence of the
will, although of cortical origin and in a certain sense automatic, like
convulsions.
The knee-jerk is changed in somewhat more than half the cases, a

little oftener exaggerated than abolished; but sometimes the reflexes
are enormously increased all over the body, so that a strong puff of
air in the face even will set the arms and legs going like a jumping-
jack. I have twice seen the patellar reflex abolished in one leg, and
so marked in the other as to seem to me exaggerated.24 I have also
known it to disappear absolutely in both legs two weeks after it had
been found to be excessively exaggerated. It also varies under
conditions of rest, fatigue, excitement, etc. Intense pain in the joints
occurs, and I have found it where the knee-jerk was exaggerated, in
one case giving rise in a physician to the delusion that his arms and
elbows had been resected. This may disappear in time. Charcot's
joint disease has been observed.
24 There was no evidence, and there had been no history, of a hemiplegic attack in
either case.
In the final stages the bones are fragile and easily break;
hemorrhages under the periosteum or perichondrium arise from
trifling force or injury, giving rise to hæmatomata, the most common
of which are on parts exposed to pressure, etc., as the ear. The
patient is confined to his bed, fed like a small child, demented, hardly
able to articulate the extravagant delusions which form such a
grotesque contrast to his actual state, until the mind is as incapable
of forming or receiving ideas as of expressing thoughts; and the
body is simply a filthy, helpless mass of humanity, dying of
exhaustion or decay, unless lung gangrene, bed-sores superficial
and deep, necrobiosis, exhausting diarrhœa, pneumonia, pulmonary
consumption, perhaps asphyxia from an epileptic fit or choking, have
followed incontinence of urine and feces to the fatal end, or heart
failure or apoplexy have closed the scene.
PATHOLOGY AND MORBID ANATOMY.25—General paralysis of the insane

is, according to Mendel, following Rokitansky's idea, a connective-
tissue disease, affecting the nerve-cells and tissues secondarily,
while Tuczek and Wernicke think that the primary disease is of the
nerve-elements (primäre Atrophie der Nervenelemente)—a diffuse
interstitial cortical encephalitis on the one hand, or a diffuse
parenchymatous cortical encephalitis on the other. There is also, in
well-marked cases, atrophy of the white substance, due, according
to general opinion of pathologists, to primary interstitial encephalitis
ending in sclerosis.
25 For a detailed statement of the post-mortem appearances in general paralysis
compare Spitzka's Insanity, pp. 218-243; Beiträge zur pathologischen Anatomie und
zur Pathologie der Dementia Paralytica, von Dr. Franz Tuczek; Die Progressive
Paralyse der Irren, von Dr. E. Mendel; Lehrbuch der Gehirnkrankheiten, von Dr. C.
Wernicke, iii. pp. 536-541. Westphal's classical work is not referred to, as his latest
views and others of interest are given in a report of a discussion by the German
Association of Alienists in the Allgemeine Zeitschrift für Psychiatrie, iv. 1883, pp. 634-
638 and 648-654. In the third number of the Neurol. Centralblatt, Mendel reports an
autopsy of a patient diagnosticated to have melancholia, who died a violent death,
where he thought that he found evidence of the early stage of general paralysis in
moderate opacity of the pia mater, with nodules as large as a pin's head in both
parietal regions, and in slight indications of diffuse interstitial inflammation of the
cortex, the blood-vessels in the frontal convolutions being extensively filled with white
blood-corpuscles.
In the majority of cases there is pachymeningitis, often extensive and

excessive, with hemorrhages, but which may be no more than is
quite commonly found in persons dying of phthisis or chronic
nephritis. There is also, usually, leptomeningitis, with adhesions to
the cortex, especially of the anterior and antero-lateral portions, so
firm that the arachnoid cannot be removed without tearing off
portions of the brain; but it is sometimes scarcely observed, and may
be no more than is found in persons dying simply of old age. The pia
may be in places thickened, opaque, and without adhesions.
Ependymitis is usual.
In the terminal stage of general paralysis there is well-marked

atrophy (with compensatory serous effusion), which is, as a rule,
most marked in the cortex of the brain, but which is of varying
degrees in its different portions. Rarely there is scarcely any atrophy
of the cortex. The central portion of the brain may be of leathery
consistence, but usually shows marked sclerosis, which also may
affect its different portions and the different ganglia very differently.
The changes resulting from inflammatory, degenerative, and atrophic
processes are general and profound.
An opinion is beginning to obtain that general paralysis is primarily a

disease of the small cerebral blood-vessels, functional or vaso-
motor; and Meynert holds that the transition line between that stage,
which he considers curable, and organic disease may be recognized
clinically.
In general paralysis, as in other mental diseases, the nervous

discharge is accompanied by a greater disturbance in the structure
of the gray substance of the brain, a more extensive decompounding
of it, and consequently by a more complete exhaustion of nervous
force than in healthy mental processes. Longer periods of rest and
improved nutrition are therefore necessary to restore healthy
function. In general paralysis, as in all other mental diseases
dependent upon destructive disease of the brain, there is not only
decompounding, but decomposing and disintegrating, of the
structure of the brain.
Posterior spinal sclerosis is frequently found. If alone or

predominating over sclerosis of the lateral columns of the cord, the
knee-jerk is abolished if the morbid process has gone far enough. If
descending degeneration of the lateral columns is chiefly found, and
is sufficiently advanced, the knee-jerk is increased. At least one of
these forms of sclerosis exists in the vast majority of cases.
There is also a distinctly syphilitic disease of the smaller cerebral

arteries, together with a diffuse parenchymatous and interstitial
encephalitis of syphilitic origin. At present we have no means of
differentiating it at the autopsy from general paralysis following a
subacute or chronic course, except inferentially from the presence of
other evidences of syphilis. It is not always possible, therefore, to
distinguish between syphilis and a syphilitic diathesis as the chief
factor in diffuse encephalitis.
DIAGNOSIS.—Although a well-marked case of general paralysis is

unmistakable, the diagnosis in the early stages or in obscure cases
may be extremely difficult. The varying degrees in which the various
portions of the cortex, medullary portion, and different ganglia of the
brain may be involved in the morbid process naturally give rise to a
great variety in the symptoms, mental and physical, sensory and
motor, emotional and intellectual, and in the relative preponderance
of one or another in individual cases. The usual symptoms of any
form of mental disease may for a time obscure the dementia which
sooner or later must appear in general paralysis, and which, as has
already been said, is the only mental symptom universally present in
all cases. This mental impairment must also be associated with
progressive muscular loss of power, although the relation of the two
symptoms to each other, the degree to which a given amount of the
one leads to a fair inference of a certain amount of the other, is liable
to the greatest variation, the range of which can only be learned by
observation and experience. There is a certain quality to the
dementia, as already described, which is often sufficient of itself to
establish the diagnosis with a practised physician.
The early mental symptoms may simulate those of cerebral

neurasthenia, in which the patient thinks that there is decided mental
impairment, although there is no progressive dementia. The tremor
in neurasthenia is greater and more universal than in the stage of
general paralysis with which it might be confounded, and the
subjective symptoms are much more prominent.
Muscular malaise and pains throughout the body give rise to the
diagnosis of malaria or rheumatism, in which there may be loss of
power, but no ataxia or dementia.
The sclerosis may be predominating or pronounced in the basal

ganglia and bulbar nuclei, giving occasion for a hasty diagnosis of
labio-glosso-pharyngeal paralysis, until it is found that the clinical
history of that disease is not followed. In the same way, any motor or
sensory ganglia or nerve-roots may be so early implicated in the
degenerative process as to mislead the physician into giving
attention to only the local symptoms.
Once I have known the early convulsions of general paralysis in a

very self-conscious woman mistaken for hysteria, the mental
impairment and physical weakness having been overlooked on
account of the prominence of the convulsive attacks and the
hysterical symptoms, which may be a complication of any form of
insanity in young and middle-aged people, particularly women.
It is not uncommon for the attacks to so thoroughly resemble

epilepsy as to be mistaken for it, the dementia not being observed or
being supposed to be the ordinary mental deterioration generally
following epilepsy. In such cases the progressive dementia, ataxia,
and muscular weakness may advance so slowly as to entirely
escape observation for a long time, and give rise to the confident
diagnosis of epilepsy for five or six years. Epilepsy, however, arising
in a vigorous, middle-aged person without evident cause, should
always suggest the suspicion of syphilis, cerebral tumor, or general
paralysis, when careful scrutiny of all the symptoms will show where
it belongs.
Embolisms, hemorrhages, cerebral effusions, more or less diffuse

encephalitis from an injury to the head, sometimes give rise to the
suspicion of general paralysis, until it is found that its characteristic
progressive symptoms do not appear, but chiefly when the history of
the case has not been definitely ascertained, or when the usual
symptoms of those conditions are not well marked.
Chronic endarteritis, arterio-sclerosis, atheroma of the cerebral

arteries may be so diffused as to simulate general paralysis,
especially in drunkards and syphilitics, but the symptoms do not
advance in the manner characteristic of that disease.
Multiple cerebro-spinal sclerosis of the descending form may be

confounded with general paralysis while the symptoms are obscure
and consist in change of character, when, indeed, organic disease
can only be suspected to be present.
Lead has been known to attack the central nervous system in such a
way as to produce an intellectual apathy and muscular weakness
somewhat resembling the early stage of the demented form of
general paralysis, but without its ataxic symptoms and its regular
progress. The presence of lead in the urine, and the marked
improvement from the use of iodide of potassium, tonics, and
electricity, are sufficient to establish the diagnosis.
Chronic and persistent alcoholism is always attended with some

mental impairment, which may so resemble the dementia of general
paralysis, with marked moral perversion, mental exaltation, grand
delusions, muscular tremor, ataxic symptoms, and impaired
muscular power, as to make the diagnosis doubtful for several
months, until removal of the cause (alcohol) in the course of time
causes the symptoms to so abate as to make the real character of
the disease evident.
I have once seen chronic interstitial nephritis without its usual
prominent symptoms and with mild uræmic convulsions mistaken for
general paralysis.
A tumor of the brain, if not attended with the common symptom of

vomiting, may be the cause of convulsions and headache
resembling those often seen in general paralysis. Optic neuritis or
atrophy is usual in cerebral tumor, but rare in a stage of general
paralysis so early that the diagnosis might be doubtful.
Hemorrhagic pachymeningitis also now and then simulates an

obscure case of general paralysis in the early stage, but a few weeks
at most settle any doubts in the matter.
Although diffuse cerebral syphilis is more apt to be associated with

distinctly localized symptoms than the demented form of general
paralysis, and although it is characterized by a mental apathy and
physical torpor which follow a more regular course with more definite
symptoms, resulting in a slow decay, yet there may be doubtful
cases in which the differential diagnosis is impossible, and in which
antisyphilitic treatment does not throw any light on the subject.
Syphilitic new growths, endarteritis, and meningitis may so far
improve from the use of mercury or the iodide of potassium as to end
in an apparent cure, but in those cases the symptoms are not so
marked as to make an exact diagnosis always possible. A distinct
syphilitic cachexia is presumptive evidence of syphilitic encephalitis
when there is doubt whether the syphilis is the cause or the
diathesis.
Profound melancholia is not so often as varying gloom or moderate

despondency a symptom of general paralysis. When it is such, there
are developed in time the other marks of that disease, and it will only
be necessary to hold the diagnosis in reserve for their appearance.
The melancholia masks the dementia unless it is very carefully
sought for, and the tremor may be as marked in melancholia as in
the early stage of general paralysis, but more universal.
Acute mania is not uncommonly mistaken for general paralysis,
when, as often happens, the delusions are as expansive and the
tremor as great in the mania as in general paralysis; and it may be
several months before the differential diagnosis can be made with
certainty. In the presence of a high degree of maniacal excitement,
with great emotional agitation and muscular tremor, it is difficult to
establish the fact of the existence or not of dementia in doubtful
cases until it is well developed. Acute mania has been known to
constitute the prodromal period of general paralysis for a number of
years.
Primary mental deterioration cannot be always differentiated from

general paralysis of the demented type in its early stage. After the
age of sixty the probabilities are in favor of primary mental
deterioration in doubtful cases, but general paralysis occurs—
seldom, to be sure—up to the age of sixty-five.
Early senile dementia may simulate general paralysis of the

subacute form, but has not its clinical history. General paralysis of
the quiet, insidious type and primary mental deterioration have been
called premature senility. The three diseased conditions have certain
points of similarity, and the pathological processes involved in them
do not differ sufficiently to authorize the assumption that they are not
closely related, if not simply variations, due to age and other causes,
in one morbid process.
Finally, the mental impairment caused by the prolonged use of

bromide of potassium and hydrate of chloral has been mistaken for
general paralysis, until a critical examination unmistakably showed
the presence of the well-known symptoms of those drugs.
In examining the patient it is especially important to avoid leading

questions, as in general paralysis and in those conditions which
simulate its early stage the mind is in a condition to readily fall into
the train of thought suggested to it. The fact should be kept in mind,
too, that the symptoms in early general paralysis are so variable as
to be sometimes quite evident, and at other times not to be got at
with certainty at all or only after long and patient examination; that
they sometimes quite disappear under the influence of complete
mental and bodily rest; and that in all stages, until near the end, such
complete remissions may occur as to make the diagnosis,
independent of the history of the case, difficult if not impossible.
A gentleman once committed an offence characteristic of general

paralysis in marrying a pretty servant-girl while temporarily away
from his home. His wife, daughters, and friends saw that the act was
so contrary to his natural character that he was placed in an insane
asylum and kept there several weeks under observation for an
opinion as to his responsibility. He appeared so well in the absolute
quiet and rest that he was declared sane, tried, and sentenced to the
State prison, where he showed his marked mental impairment as
soon as he was set to work. He could not concentrate his mind
sufficiently for the simplest labor, and a couple of years later he was
sent to the insane asylum to die, a complete mental and physical
wreck, in the late stage of general paralysis.
PROGNOSIS.—The very few reported cures in so common a disease

as general paralysis, and the circumstances under which they have
been reported, lead to the suspicion that there was an error in
diagnosis or that the mistake was made of supposing a remission to
be a cure, as has often happened. The course of the disease is more
rapid in men than women, and in young persons than in the older.
From the galloping cases of a couple of months to those slowly
advancing, with long remissions, over twenty years, the average,
including the prodromal period, is probably not far from five (perhaps
six) years. Collected from asylum statistics, it is given as from two to
three years. When I am sure of the diagnosis, I generally say that the
patient may die within twenty-four hours (of paralysis of the heart,
from suffocation by an accident in an epileptic attack, from choking,
from cerebral hemorrhage or effusion, or suddenly with cerebral
symptoms of which the autopsy gives no satisfactory explanation),
within a short time of intercurrent disease, especially diarrhœa or
pneumonia, or that he may live several years, as he probably will,
and possibly have a remission, during which he may lead for a while
somewhat the same kind of life as other people.
Persons presenting symptoms which can in no way be positively
distinguished from those at the beginning of the prodromal period of
general paralysis recover, but not many come under the physician's
care so early. We are not yet in a position to say whether they were
suffering from a mild, transient illness or from what would otherwise
have become serious organic disease.
TREATMENT.—Life may be prolonged in general paralysis, and usually

is prolonged, by the use of such measures as contribute to the
patient's comfort, and which in a general way have already been
considered under the head of treatment of mental disease on a
previous page.
In my experience, stimulating tonics, wine, and even coffee, increase

the morbid cerebral energy of the early stage of the disease, but are
sometimes of use later. Cod-liver oil and the hypophosphites do
better, and many of the disagreeable symptoms of the period of loss
of control over the involuntary muscles are relieved by strychnia.
Ergot and the judicious use of the bromides abate the cerebral
congestion. Gastro-intestinal disorders, when not controlled by
attention to diet, require the usual treatment.
Iodide of potassium in the large or small dose, and mercury, I have

never found to benefit those cases of general paralysis with a
previous history of syphilis. On the contrary, they have proved
debilitating and harmful.
When furious excitement is not relieved by prolonged warm baths,

with cool applications to the head if possible, and quiet, chloral is of
use, and sometimes opium and its preparations.
Frequently-repeated violent convulsions, the epileptic state, are

usually at once mitigated by chloral given by the rectum; the
inhalation of nitrite of amyl is reported also to have been of use.
There are few cases in which I find that morphine does not quiet
restlessness, calm delusions, abate distressing hallucinations, and
make the patient generally more comfortable; and I give it freely,
seldom more than twice a day, often almost daily, for two or three
years. In this way it can be used in quite moderate doses. Coca also
relieves symptoms.
Rest and quiet are most important in all stages of the disease. This
can be best accomplished in a quiet private house in the country,
which can be made a virtual hospital, and next in a private asylum.
But such care is beyond the reach of the vast majority of the insane,
to whom the public asylum becomes a necessity. Wherever they are,
an orderly life is best for them, with as little irritating interference with
their ways or control of them as is possible.
If the results of treatment are in the highest degree unsatisfactory,

and consist chiefly in meeting symptoms as they come up, without
hope of permanent recovery, it is not impossible that when we can
put the patient under treatment at the very beginning of his disease,
as we can now do in pulmonary consumption, the prognosis in the
former disease may change as much for the better as it has changed
in the latter.
A general paralytic is at any time liable to congestive or maniacal

attacks of short duration, and so is always, potentially, a dangerous
person. In the prodromal period the risk is small; in all stages there
will, in the majority of cases, be some warning; but in the developed
disease the only safe way is to have some responsible person near
at hand, both to prevent the patient from doing harm to others and to
save him from injuring himself, whether by intent or through not
knowing better than to wander off or fall into all sorts of accidents. In
many conditions several should be readily available, or else the
security of an asylum must be sought.
In the treatment of general paralysis by society the same rule should

obtain as in all forms of insanity—that distinct mental disease is
presumptive proof of irresponsibility, or at least of limited
responsibility; that a diseased mind means lessened intellectual
power throughout and diminished ability to choose the right and
avoid the wrong; that there are changes in circulation or nutrition, or
some unknown condition in the brain, especially in general paralysis,
by virtue of which the mental state and power of self-control vary
from time to time, and as a result of which a person seeming
responsible one day may have been quite irresponsible some
previous day.
INSANITY FROM GROSS LESIONS OF THE BRAIN (tumors, new growths of

all kinds, exostoses, spicules or portions of depressed bone,
embolisms, hemorrhages, wounds, injuries, cysticerci, etc.) is
attended with the usual indications of those conditions which may
determine diffuse disorders of the brain, giving rise to any of the
symptoms of the various psycho-neuroses and cerebro-psychoses.
The lowered mental and moral tone after cerebral hemorrhages is a
matter of common observation, and after one an individual is rarely
observed to be fully himself again.
The PROGNOSIS is very unfavorable. Although there are rare cases of

improvement, the tendency is toward profound dementia.
CEREBRAL SYPHILITIC INSANITY comes either under the head of the

insanity last described or belongs to the slowly-advancing dementia
with final paralysis already referred to under the head of Diagnosis in
General Paralysis, and called by some authorities on mental disease
pseudo-paralytic dementia from syphilis.
Antisyphilitic treatment is of value in the first class of cases, and

although most of the recoveries end in relapses and incurability, the
prolonged use of iodide of potassium seems sometimes to effect a
permanent cure. It is claimed that similar treatment is followed by the
same result in the cases of dementia with paresis, but the weight of
authority, and certainly my own experience, are against that
statement.
CHRONIC ALCOHOLIC INSANITY depends upon the vascular and other

changes due to abuse of alcohol so long continued that the
pathological condition has become organic and incurable. It is
commonly associated with delusions of suspicion or persecution. It
may be a purely moral insanity, with gross beliefs rather than
distinctly insane delusions, and it rarely fails to be at least that when
the persistent excessive drinking is kept up until the age of beginning
dissolution of the brain. It then gives rise to all sorts of embarrassing
complications in regard to property, family relations, and wills.
Chronic alcoholic insanity may take the form of mild dementia, by
virtue of which the patient cannot control himself, but can be easily
kept within bounds of reasonable conduct by various degrees of
restraint, from the constant presence of a responsible person to the
seclusion of an asylum. In well-marked cases this dementia is
associated with muscular weakness, tremor, and exhilaration to such
an extent as to simulate general paralysis. It is then called by some
—especially French—writers pseudo-paralytic dementia from
alcohol.
The condition is susceptible of improvement by removal of the

cause, alcohol, and by a carefully-regulated life, hydropathic
treatment, etc., but complete recoveries cannot be expected.
SECONDARY DELUSIONAL INSANITY is slowly developed from various

mental diseases, incurable or uncured, where the progress to
marked dementia is slow, by the persistence of delusions in those
forms of insanity characterized by delusions. It is chronic and
incurable. In melancholia and mania the mental depression and the
exaltation and motor excitement disappear to a great extent, and
there are left a slowly-advancing dementia, confusion, and
expanding delusions, with apathy or with agitation, for which the
asylum is the only safe place unless physical weakness makes the
patient harmless. It is either a terminal state in which many forms of
insanity end, or a stage through which they pass to terminal
dementia. It depends upon incurable, and therefore organic,
changes in the brain, like all incurable insanity, although those
changes are not yet determined exactly. It might be a question
whether chronic delusional insanity properly belongs under the head
of Organic Mental Diseases, and a similar criticism may be made
regarding terminal dementia. But in this paper no definite
classification of insanity is attempted, because our knowledge of the
subject is still so indefinite, although the several mental diseases are
grouped in a certain order for convenience to the reader and the
writer; and this order of course approximately follows natural lines.
TERMINAL DEMENTIA is the end to which most of the insanity not

resulting in recovery finally comes. The features marking the disease
in its early stages for the most part disappear, leaving all the
functions of the mind impaired in all degrees up to total extinction—
the whole character on a lower plane. It is the disease which to so
great an extent crowds the wards of insane asylums and
almshouses with the (1) agitated or (2) apathetic chronic insane, the
worst of whom are mental and physical wrecks, squatting on floors,
uttering an unintelligible jargon, noisy, filthy, without intelligence for
the simplest natural wants. Their chief function, under the prevalent
methods of construction and management of lunatic hospitals in
most places, is to blight with a certain feeling of hopelessness many
of the curable insane who are obliged to go for rest and quiet to
institutions where the overwhelming majority of the inmates are
manifestly and painfully incurable.
French writers include a great part of chronic delusional insanity

(secondary confusional insanity, Wahnsinn, secundäre Verrücktheit)
and terminal dementia (Blödsinn) under one head, démence; and
with much reason, as it is not always possible to differentiate
between the two.
The proper TREATMENT of the incurable, demented insane should

provide not only that they be not at large, where they annoy the
strong and the well, but also that they shall not disturb the insane
who are acutely ill and in need of treatment suited to sick people,
and whose chances of recovery at best are none too favorable.
Experiments, now quite numerous, have shown that the lives and
occupations of many of them may be made not entirely unlike those
to which they were reared, and that nearly all may be suitably
provided for without the expensive hospitals and appliances
necessary for the proper treatment of acute mental disease.
A comparison of countries in which there is and is not a

comprehensive system of State supervision of the insane by a
competent board seems to me to reveal so unquestionably the fact
that such a system alone provides the proper protection for the
insane, and the needed variety and uniformly high standard of
excellence in the provisions for their treatment, that I hope to see the
medical profession using its vast influence upon public opinion to
secure it.
If we meet in the wards of our insane asylums hopeless mental and

physical wrecks, if we find there the extremity of human
wretchedness, the supreme control of all that is evil or vile in our
nature, the worst antitypes of all the virtues, so, on the other hand,
nowhere else do we see such struggles for the mastery of the better
impulses, such efforts against such odds to hold back the mind in an
unequal fight. Nowhere else, too, are developed finer sympathy,
more beautiful unselfishness, more generous charity, or more heroic
resignation where no hope in life remains but for death.
The State has taken charge of these most unfortunate people,

shutting up behind the same locked doors and barred windows
people of all social grades, often mingling together in one presence
the so-called criminal insane, insane criminals, idiots, imbeciles,
epileptics, paralytics, the chronic insane, and the demented, with
patients suffering from acute mental disease. Some of them are
unconscious of their condition, many are better off than ever before,
but others are painfully alive to their situation and surroundings, fully
aware of the gravity of their illness, keenly sensitive to the
distressing sights and associations, disturbed by the noises, and
discouraged by the many chances of becoming like the worst
incurables around them. The State cannot evade the responsibility of
seeing that their confinement is made the least rigorous, wretched,
and injurious possible.
HYSTERIA.
BY CHARLES K. MILLS, M.D.
DEFINITION.—Hysteria is a functional disease of the cerebro-spinal

axis, characterized either by special mental symptoms or by motor,
sensory, vaso-motor, or visceral disorders related in varying degree
to abnormal psychical conditions.
This, like all other definitions of hysteria, is imperfect. No absolutely

satisfactory definition can well be given. It is not abnormal ideation,
although this is so often prominent; it is not emotional exaltation,
although this may be a striking element; it is not perversion of
reflexes and of sensation, although these may be present. Some
would make it a disease of the womb, others an affection of the
ovaries; some regard it as of spinal, others as of cerebral origin;
some hold it to be a disease of the nerves, others claim that it is a
true psychosis; but none of these views can be sustained.
Sir James Paget1 says of hysterical patients that they are as those
who are color-blind. They say, “I cannot;” it looks like “I will not,” but it
is “I cannot will.” Although, however, much of the nature of hysteria is
made clear in this explanation, hysteria is not simply paralysis of the
will. A true aboulomania or paralysis of the will occurs in non-
hysterical patients, male and female, and of late years has been
studied by alienists.
1 “Clinical Lecture on the Nervous Mimicry of Organic Diseases,” Lancet for October,
November, and December, 1873.
In many definitions the presence of a spasmodic seizure or

paroxysm is made the central and essential feature; but, although
convulsions so frequently occur, typical hysterical cases pass
through the whole course of the disorder without suffering from
spasm of any kind.
In a general neurosis a definition, well considered, should serve the
purpose of controlling and guiding, to a large extent at least, the
discussion of the subject.
The definition given asserts that hysteria is a functional disease. In

the present state of knowledge this is the only ground that can be
taken. It is claimed that in a strict sense no disease can be regarded
as functional; but it is practically necessary to use such terms as
functional in reference to affections in which disordered action
without recognizable permanent alteration of structure is present.
Temporary anatomical changes must sometimes be present in
hysteria; organic disease may be a complication in special cases;
post-mortem appearances may occasionally be found as accidents
or coincidences; it is possible that structural alterations may result
from hysteria; but no pathologist has as yet shown the existence of a
special morbid anatomy underlying as a permanent basis the
hysterical condition.
The mental, motor, sensory, and other phenomena of hysteria

cannot be explained except by regarding the cerebro-spinal nervous
system as the starting-point or active agency in their production.
The term vaso-motor is used in a broad sense to include not only

peripheral vascular disturbances, but also cardiac, respiratory,
secretory, and excretory affections of varying type. Some of these
disorders are also visceral, but under visceral affections are also
included such hysterical phenomena as abdominal phantom tumors,
hysterical tympanites, and the like.
That all hysterical phenomena are related in varying degree to

abnormal psychical conditions may perhaps, at first sight, be
regarded as open to dispute and grave doubt. It is questionable
whether in every case of hysteria the relation of the symptoms to
psychical states could be easily demonstrated. I certainly do not look
upon every hysterical patient as a case of insanity in the technical
sense, but hold that a psychical element is or has been present,
even when the manifestations of the disorder are pre-eminently
physical. James Hendrie Lloyd,2 in a valuable paper, has ably
sustained this position, one which has been held by others, although
seldom, if ever, so clearly defined as by this writer.
2 “Hysteria: A Study in Psychology,” Journal of Nervous and Mental Disease, vol. x.,
No. 4, October, 1883.
The alleged uterine origin of hysteria has been entirely disregarded

in the definition. This has been done intentionally. It is high time for
the medical profession to throw off the thraldom of this ancient view.
The truth is, as asserted by Chambers,3 that hysteria “has no more
to do with the organs of reproduction than with any other of the
female body; and it is no truer to say that women are hysterical
because they have wombs, than that men are gouty because they
have beards.”
3 Brit. Med. Journ., December 21, 1861, 651.
SYNONYMS.—Hysterics, Vapors. Many Latin and other synonyms

have been used for hysteria: most of these have reference to the
supposed uterine origin of the disease, as, for instance, Uteri
adscensus, Asthma uteri, Vapores uterini, Passio hysterica,
Strangulatio uterina seu Vulvæ. Some French synonyms are Maladie
imaginaire, Entranglement, and Maux ou attaques de nerfs. Other
French synonyms besides these have been used; most of them are
translations from the Latin, having reference also to the uterine
hypothesis. In our language it is rare to have any other single word
used as a synonym for hysteria. Sir James Paget4 introduced the
term neuromimesis, or nervous mimicry, and suggested that it be
substituted for hysteria, and neuromimetic for hysterical.
Neuromimesis is, however, not a true synonym. Many cases of
hysteria are cases of neuromimesis, but they are not all of this
character. Among the desperate attempts which have been made to
originate a new name for hysteria one perhaps worthy of passing
notice is that of Metcalfe Johnson,5 who proposes to substitute the
term ganglionism, as giving a clue to the pathology of hysteria. His
main idea is that hysteria exhibits a train of symptoms which are
almost always referable to the sympathetic or ganglionic nervous
system. This is another of those half truths which have misled so
many. The term hysteria, from the Greek ὑστερα, the uterus,
although attacked and belabored, has come to stay; it is folly to
attempt to banish it.
4 Op. cit.
5 Med. Times and Gaz., 1872, ii. 612.
METHOD OF DISCUSSING THE SUBJECT.—It is hard to decide upon the

best method of discussing the subject of hysteria. One difficulty is
that connected with the question whether certain affections should
be considered as independent disorders or under some subdivisions
of the general topic of hysteria. Certain great phases of hysteria are
represented by hystero-epilepsy, catalepsy, ecstasy, etc.; but it will
best serve practical ends to treat of these in separate articles. They
have distinctive clinical features, and are capable of special definition
and discussion.
HISTORY AND LITERATURE.—To give a complete history of hysteria it

would be necessary to traverse the story of medicine from the time
of Hippocrates to the present. A complete bibliography would require
an immense volume. Volume vi. of the Index Catalogue of the
Library of the Surgeon-General's Office, United States Army, which
has appeared during the present year (1885), contains a
bibliography of nearly seventeen double-column pages, most of it in
the finest type. The references are to 318 books and 914 journals.
The number of books and articles cited as having appeared in
different languages is as follows: Latin, 99; Greek, 2; German, 180;
British, 177; American, 159; French, 449; Italian, 75; Spanish, 45;
Swedish, 12; miscellaneous, 34. Even this wonderful list probably
only represents a tithe of the works written on this subject. Those
desirous of studying it from a bibliographical point of view can do so
by consulting this great work.
Many as are the names and voluminous as is the literature, certain

names and certain works are pre-eminent—Sydenham, Laycock,
and Skey in England; Tissot, Briquet, Charcot, and Landouzy in
France; Stahl, Frank, Eulenburg, and Jolly in Germany; and in

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Derivation of Human Embryonic Stem Cell Lines from Vitrified

Definitive Endoderm Differentiation of Human Embryonic Stem

Efficient Production of Photoreceptor Precursor Cells from Human

CLAUS YDING ANDERSEN  Laboratory of Reproductive Biology, Rigshospitalet, University

Derivation of Human Embryonic Stem Cell Lines

Human embryonic stem cells (hESCs) were first described by

2.1 Embryo 1. Biological safety cabinet.

2.2 Fibroblast Feeder 1. Class II biological safety cabinet.

2.3 hESC Derivation 1. Class II biological safety cabinet.

2.4 Vitrification 1. Class II biological safety cabinet.

10. Sterile forceps.

3.1 Embryo Vitrification is a method of cryopreservation which uses a high

(a) To well #1 and #2 add 460 μL Solution 2 and 40 μL

13. Quickly insert the Cryotop device into a chilled sheath

3.1.2 Embryo Warming 1. At least 4 h or overnight prior to warming, prepare blastocyst

to recover for at least a few hours prior to hESC derivation.

material for the cultures and to safeguard against accidental

Feeder medium Feeder medium

note that feeder medium is also required during passaging for

the precooled rack and handling them as little as possible to

3.2.4 Thawing 1. Determine the number of vials to be thawed. Depending on the

8. Transfer culture vessels to incubator. The mitotically inacti-

The muscular tremor before the last stages varies in different

Spastic paralysis, muscular tension, contractures, rigidity of the most

The knee-jerk is changed in somewhat more than half the cases, a

PATHOLOGY AND MORBID ANATOMY.25—General paralysis of the insane

In the majority of cases there is pachymeningitis, often extensive and

In the terminal stage of general paralysis there is well-marked

An opinion is beginning to obtain that general paralysis is primarily a

In general paralysis, as in other mental diseases, the nervous

Posterior spinal sclerosis is frequently found. If alone or

There is also a distinctly syphilitic disease of the smaller cerebral

DIAGNOSIS.—Although a well-marked case of general paralysis is

The early mental symptoms may simulate those of cerebral

The sclerosis may be predominating or pronounced in the basal

Once I have known the early convulsions of general paralysis in a

It is not uncommon for the attacks to so thoroughly resemble

Embolisms, hemorrhages, cerebral effusions, more or less diffuse

Chronic endarteritis, arterio-sclerosis, atheroma of the cerebral

Multiple cerebro-spinal sclerosis of the descending form may be

Chronic and persistent alcoholism is always attended with some

CLAUS YDING ANDERSEN Laboratory of Reproductive Biology, Rigshospitalet, University