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This extensive volume explores areas of intense activity related to the very early commitment of stem cells to particular lineages and the progression of differentiation to mature cell stages. Research on embryonic stem cells continues to move very quickly, thus the kinds of studies continue to expand and diversify, and methodologies are continuously being refined and improved, which this book reflects. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions on their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Comprehensive and fully updated, Embryonic Stem Cell Protocols, Third Edition serves as an ideal reference for researchers investigating this rich area of study.

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1. Maximizing Clonal Embryonic Stem Cell Derivation by ERK Pathway Inhibition

Jennifer Nichols and Thorsten Boroviak

2. A Simple and Efficient Method of Slow Freezing for Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Keitaro Imaizumi, Momoe Iha, Naoki Nishishita, Shin Kawamata, Shinichi Nishikawa, and Teruo Akuta

3. Resolving Heterogeneity: Fluorescence Activated Cell Sorting of Dynamic Cell Populations from Feeder-Free Mouse Embryonic Stem Cell Culture

Jurriaan J. Hölzenspies, Gelo V. Dela Cruz, and Joshua M. Brickman

4. Acquiring Ground State Pluripotency: Switching Mouse Embryonic Stem Cells from Serum/LIF Medium to 2i/LIF Medium

Matteo Tosolini and Alice Jouneau

5. Generation of Embryonic Stem Cells in Rabbits

Marielle Afanassieff, Pierre Osteil, and Pierre Savatier

6. Gene Transfer into Pluripotent Stem Cells Via Lentiviral Transduction

Ortwin Naujok, Ulf Diekmann, and Matthias Elsner

7. Imaging Pluripotency: Time-Lapse Analysis of Mouse Embryonic Stem Cells

Anna Pezzarossa, Ana M.V. Guedes, Domingos Henrique, and Elsa Abranches

8. Expanding the Utility of FUCCI Reporters Using FACS-Based ´Omics Analysis

James Chappell, Ben Boward, and Stephen Dalton

9. Selection of Antibodies Interfering with Cell-Surface Receptor Signalling Using Embryonic Stem Cell Differentiation

Anna N. Melidoni, Michael R. Dyson, and John McCafferty

10. Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells

Lida Radan, Chris S. Hughes, Jonathan H. Teichroeb, Lynne-Marie Postovit, and Dean H. Betts

11. Analysis of mRNA Translation Rate in Mouse Embryonic Stem Cells

Anisa B. Rahim and Leah A. Vardy

12. Generation and Purification of Definitive Endoderm Cells Generated from Pluripotent Stem Cells

Ulf Diekmann and Ortwin Naujok

13. Definitive Endoderm Differentiation of Human Embryonic Stem Cells Combined with Selective Elimination of Undifferentiated Cells by Methionine Deprivation
Tomonori Tsuyama, Nobuaki Shiraki, and Shoen Kume

14. Selective Differentiation into Hematopoietic and Cardiac Cells from Pluripotent Stem Cells Based on the Expression of Cell Surface Markers

Atsumasa Okada, Katsuhisa Tashiro, Tomoko Yamaguchi, and Kenji Kawabata

15. Methods for Derivation of Multipotent Neural Crest Cells Derived from Human Pluripotent Stem Cells

John Avery and Stephen Dalton

16. From Naive to Primed Pluripotency: In Vitro Conversion of Mouse Embryonic Stem Cells in Epiblast Stem Cells

Matteo Tosolini and Alice Jouneau

17. In Vitro Differentiation of Embryonic Stem Cells into Hematopoietic Lineage: Towards Erythroid Progenitor´s Production

Iliana Fauzi, Nicki Panoskaltsis, and Athanasios Mantalaris

18. Dopaminergic Differentiation of Human Embryonic Stem Cells on PA6-Derived Adipocytes

M. Oktar Guloglu and Anna Larsen

19. Intracellular Calcium Measurements for Functional Characterization of Neuronal Phenotypes

Talita Glaser, Ana Regina G. Castillo, µgatha Oliveira, and Henning Ulrich

20. In Vitro Differentiation of Pluripotent Stem Cells into Functional beta Islets Under 2D and 3D Culture Conditions and In Vivo Preclinical Validation of 3D Islets

Bipasha Bose and Sudheer Shenoy P

21. Generation of Corneal Keratocytes from Human Embryonic Stem Cells

Andrew J. Hertsenberg and James L. Funderburgh

22. Maintenance, Transgene Delivery, and Pluripotency Measurement of Mouse Embryonic Stem Cells

Tetsuya S. Tanaka

23. Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool

Rahel Wettstein, Maxime Bodak, and Constance Ciaudo

24. Derivation of Neural Precursor Cells from Human Embryonic Stem Cells for DNA Methylomic Analysis

Ivan Roubal, Sun Joo Park, and Yong Kim

25. 26S and PA28-20S Proteasome Activity in Cytosolic Extracts from Embryonic Stem Cells

Malin Hernebring

26. A Concise Protocol

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