Table Of ContentMethods in
Molecular Biology 1074
Ramiro Alberio Editor
Epiblast
Stem Cells
Methods and Protocols
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
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Epiblast Stem Cells
Methods and Protocols
Edited by
Ramiro Alberio
School of Biosciences, University of Nottingham, Loughborough, UK
Editor
Ramiro Alberio
School of Biosciences
University of Nottingham
Loughborough, UK
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-627-6 ISBN 978-1-62703-628-3 (eBook)
DOI 10.1007/978-1-62703-628-3
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Pref ace
The cells of the epiblast give rise to all the tissues of the embryo proper. How epiblast cells
acquire pluripotent features and how they transition to form the trilaminar embryo are cur-
rent topics of intense investigation. A more detailed understanding of these in vivo events
will lead to improved strategies for in vitro cellular differentiation protocols and genetic
modifi cation. In E piblast Stem Cells : M ethods and Protocols , leading researchers provide a
detailed collection of techniques and protocols useful to the study of the biology of the
pluripotent epiblast. As embryo development varies in model organisms, this volume
includes chapters describing techniques used to study epiblast development in different
amniotes. This collection brings contributions from the fi elds of embryology, stem cell
biology and developmental biology together, such that readers can fi nd in a single volume
detailed procedures for the isolation and culture of epiblasts at different stages of develop-
ment and techniques for the study of differentiation into specifi c lineages. Over the past fi ve
years, advances in the molecular characterization of pluripotent cells have led to a new
understanding of how metastable states of pluripotency develop during the transition
between the early inner cell mass to the late epiblast stage. Exploiting this new knowledge
has enabled researchers to develop protocols that capture these novel pluripotent interme-
diates. These intermediate stages of pluripotency are important for modelling the transi-
tions that these cells undergo in vivo and will contribute to improving the development of
robust in vitro differentiation protocols.
Written in the highly successful M ethods in Molecular Biology ™ series format, chapters
include introductions to their respective topics, a complete list of the necessary materials
and reagents, detailed laboratory protocols, and extensive notes providing suggestions on
troubleshooting and how to overcome common diffi culties.
I hope the chapters presented in this volume will serve as an up-to-date source of novel
protocols developed as a result of the new understanding in pluripotency and will contrib-
ute to the incremental use of stem cell technologies in different species.
Comprehensive and cutting-edge E piblast Stem Cells : M ethods and Protocols serves as a
resource to individuals interested in studying the biology of pluripotent cells.
Loughborough, UK Ramiro Alberio
Contents
Preface ............................................................................................................................. v
Contributors .................................................................................................................... ix
1 Generation and Characterization of Epiblast Stem Cells
from Blastocyst-Stage Mouse Embryos ............................................................. 1
Daniel C. Factor, Fadi J. Najm, and Paul J. Tesar
2 Reversion of Mouse Postimplantation Epiblast Stem Cells
to a Naïve Pluripotent State by Modulation of Signalling Pathways ................... 15
Astrid Gillich, Siqin Bao, and M. Azim Surani
3 Converting Mouse Epiblast Stem Cells into Mouse Embryonic Stem Cells
by Using Small Molecules ................................................................................. 31
Wenlin Li and Sheng Ding
4 Isolation and Culture of Rabbit Embryonic Stem Cells ..................................... 39
Arata Honda
5 Goat Embryonic Stem-Like Cell Derivation and Characterization ..................... 51
Esmail Behboodi, Louise Lam, William G. Gavin, Alla Bondareva,
and Ina Dobrinski
6 Derivation and Culture of Canine Embryonic Stem Cells .................................. 69
Ian C. Tobias, Courtney R. Brooks, Jonathan H. Teichroeb,
and Dean H. Betts
7 Isolation and Culture of Porcine Embryonic Stem Cells .................................... 85
Ivan Vassiliev and Mark B. Nottle
8 Isolation and Culture of Pig Epiblast Stem Cells ............................................... 97
Aida Rodriguez, David A. Contreras, and Ramiro Alberio
9 Isolation and Culture of Bovine Embryonic Stem Cells ..................................... 111
Shanbo Cao, Fang Wang, and Lin Liu
10 Analysis of Molecular Markers for Staging Peri-g astrulating
Bovine Embryos ................................................................................................. 125
Isabelle Hue, Severine A. Degrelle, and Christoph Viebahn
11 Chicken Embryonic Stem Cells: Establishment and Characterization ................. 137
Pauline Aubel and Bertrand Pain
12 Manipulating the Avian Epiblast and Epiblast-Derived Stem Cells ..................... 151
Cantas Alev, Mikiharu Nakano, Yuping Wu, Hiroyuki Horiuchi,
and Guojun Sheng
viii Contents
13 Stepwise Differentiation from Naïve State Pluripotent Stem Cells
to Functional Primordial Germ Cells Through an Epiblast-Like State ................ 175
Katsuhiko Hayashi and Mitinori Saitou
14 Isolation and Culture of Porcine Neural Progenitor Cells
from Embryos and Pluripotent Stem Cells ........................................................ 185
Mikkel A. Rasmussen, Vanessa J. Hall, and Poul Hyttel
15 Derivation of Neural Precursors from Bovine Preimplantation Embryos ............ 199
Giovanna Lazzari, Silvia Colleoni, and Cesare Galli
Index ............................................................................................................................... 209
Contributors
RAMIRO ALBERIO • Division of Animal Sciences, School of Biosciences, University of
Nottingham , Loughborough, UK
CANTAS ALEV • Laboratory for Early Embryogenesis, RIKEN Center for Developmental
Biology , Chuo-Ku, Kobe, Japan
PAULINE AUBEL • Inserm, U846, Stem Cell and Brain Research Institute , Bron , France ;
Université de Lyon , Lyon , France
SIQIN BAO • Wellcome Trust/Cancer Research UK Gurdon Institute, University of
Cambridge , Cambridge, UK
ESMAIL BEHBOODI • Department of Comparative Biology and Experimental Medicine,
Faculty of Veterinary Medicine, University of Calgary , Calgary , AB , Canada
DEAN H. BETTS • Department of Physiology and Pharmacology , Schulich School of Medicine
and Dentistry, University of Western Ontario , London, ON , Canada ; Children’s Health
Research Institute, Lawson Health Research Institute , London, ON , Canada
ALLA BONDAREVA • Department of Comparative Biology and Experimental Medicine,
Faculty of Veterinary Medicine, University of Calgary , Calgary , AB , Canada
COURTNEY R. BROOKS • Department of Physiology and Pharmacology , Schulich School of
Medicine and Dentistry, University of Western Ontario , London, ON , Canada
SHANBO CAO • The Third Affi liated Hospital of Guangzhou Medical University ,
Guangzhou, China
SILVIA COLLEONI • Avantea, Laboratory of Reproductive Technologies , Cremona, Italy
DAVID A. CONTRERAS • Division of Animal Sciences, School of Biosciences, University of
Nottingham , Loughborough, UK
SEVERINE A. DEGRELLE • INRA, UMR1198 Biologie du Développement et Reproduction ,
Jouy-en-Josas, France ; ENVA , Maisons Alfort, France ; Department of Immunology,
INSERM, Institut Cochin , Paris , France
SHENG DING • Department of Pharmaceutical Chemistry , Gladstone Institute of
Cardiovascular Disease, University of California , San Francisco, CA , USA
INA DOBRINSKI • Department of Comparative Biology and Experimental Medicine, Faculty
of Veterinary Medicine, University of Calgary , Calgary , AB , Canada
DANIEL C. FACTOR • Department of Genetics and Genome Sciences, Case Western Reserve
University School of Medicine , Cleveland, OH , USA
CESARE GALLI • Avantea, Laboratory of Reproductive Technologies , Cremona, Italy ;
Department of Veterinary Medical Sciences, University of Bologna , Bologna , Italy ;
Fondazione Avantea , Cremona, Italy
WILLIAM G. GAVIN • rEVO Biologics , Framingham, MA , USA
ASTRID GILLICH • Wellcome Trust/Cancer Research UK Gurdon Institute, University of
Cambridge , Cambridge, UK
VANESSA J. HALL • Department of Veterinary Clinical and Animal Sciences, Faculty of
Health and Medical Sciences, University of Copenhagen , Frederiksberg C, Denmark
