Embryonic Stem Cells
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Table of contents
- Embryonic Stem Cellsiii
- Copyright Pageiv
- Dedication Pagev
- Table of Contentsvii
- Contributors to Volume 418ix
- Prefacexiii
- Forewordxv
- Volumes in Seriesxvii
- Section I: Derivation and Maintenance of Embryonic Stem Cells1
- Chapter 1: Murine Embryonic Stem Cells3
- Historical Overview3
- Factors Affecting the Efficiency of Mouse ES Cell Establishment5
- Factors Affecting the Contribution of Mouse ES Cell to Chimeric Embryos6
- Critical Events During Mouse ES Cell Establishment7
- Freezing of ES Cell Lines12
- Characterization12
- Protocols13
- Acknowledgments19
- References19
- Chapter 2: Bovine Embryonic Stem Cells21
- Introduction22
- Materials24
- Methods24
- References36
- Chapter 3: Avian Embryonic Stem Cells38
- Introduction38
- Selection of Culture Media and Routine Procedures39
- Derivation and Maintenance of Chicken Embryonic Stem Cell Lines41
- Genetic Modification of cES Cells49
- Production of Chimeric Chickens51
- Acquisition, Storage, and Quality Control of Eggs59
- Timeline of Chimera Production60
- Efficiency of Chimera Production Using Embryonic Stem Cells61
- Acknowledgments62
- References62
- Chapter 4: Zebrafish Embryonic Stem Cells64
- Introduction64
- Materials66
- Derivation of Zebrafish ES Cell Cultures68
- Homologous Recombination in ES cells and Selection of Colonies Carrying a Targeted Plasmid Insertion73
- Acknowledgments75
- References76
- Chapter 5: Human Embryonic Stem Cells78
- Introduction78
- Derivation of hES Cell Lines79
- Conclusion89
- Acknowledgments89
- References89
- Chapter 6: Embryonic Stem Cells from Morula93
- Introduction93
- Protocol for Isolation Human ES Cell Line from Morula94
- Characteristics of Morula-Derived hESC Lines97
- Protocol of Immunofluorescence Staining for TRA-2-39 or TRA-2-54 and Oct-4103
- Freezing Cell Lines105
- Protocol for Freezing Morula-Derived hESC Lines105
- Procedure for Freezing Human Embryonic Stem Cells106
- Thawing hESC Lines107
- Acknowledgments107
- References107
- Chapter 7: Embryonic Stem Cells from Single Blastomeres108
- Introduction109
- Derivation of ES Cells from a Single Blastomere110
- Analysis of Cell Lines113
- References116
- Chapter 8: Embryonic Stem Cells from Parthenotes117
- Introduction117
- Mouse Parthenogenetic ESCs122
- Nonhuman Primate Parthenogenetic ESCs124
- The Importance of Choosing the Right Activation Protocol126
- Imprinting and Parthenogenesis128
- Protocols for Derivation of Parthenogenetic ESCs129
- Summary132
- Acknowledgments132
- References132
- Chapter 9: Embryonic Stem Cells Using Nuclear Transfer135
- Introduction135
- Somatic Cell Nuclear Transfer136
- Media Preparation138
- Culture Dishes and Incubator139
- Protocol139
- Derivation of ES Cells from Cloned Blastocysts144
- References146
- Section II: Differentiation of Embryonic Stem Cells149
- Chapter 10: Neural Stem Cells, Neurons, and Glia151
- Introduction151
- Protocols152
- Media and Reagents165
- Summary167
- Acknowledgments168
- References168
- Chapter 11: Retinal Pigment Epithelium169
- Introduction169
- Differentiation of hES Cells to RPE171
- Materials and Equipment173
- Harvesting RPE Cells179
- Culture and Properties of hES-Derived RPE181
- RPE Culture Protocols181
- Characterization of hES-RPE Cells182
- Concluding Remarks186
- Acknowledgments186
- Appendix 1186
- Appendix 2187
- References191
- Chapter 12: Mesenchymal Cells194
- Introduction194
- Developmental Considerations195
- Adult Mesenchymal Stem Cells (MSCs)196
- Mouse ESC-D erived Mesenchymal Tissues197
- Mesenchymal Cells Derived from Human ESCs198
- Derivation, Isolation, and Differentiation of hESC-Derived Mesenchymal Precursors199
- Summary and Perspectives for the Future204
- References205
- Chapter 13: Hematopoietic Cells208
- Introduction209
- Methods219
- References236
- Chapter 14: Hematopoietic Cells from Primate Embryonic Stem Cells243
- Introduction243
- Protocol for Propagation of Rhesus Monkey ES Cell Lines245
- Induction of Hematopoietic Differentiation of Rhesus Monkey ES Cells247
- Technical Notes250
- References250
- Chapter 15: Vascular Cells252
- Derivation and Characterization of Embryonic Stem (ES) Cells252
- Differentiation of Embryonic Stem Cells253
- Development of the Blood Vascular System254
- Vascular Developmental Potential of Embryonic Stem Cells255
- Vascular Differentiation of ES Cells In Vitro256
- References264
- Chapter 16: Cardiomyocytes267
- Introduction267
- Embryoid Body (EB) Generation268
- Hanging Drop Method269
- Human ES Cell Suspension Culture272
- Scalable Production of EBs in a Bioreactor272
- Cardiomyocyte Differentiation278
- Enrichment of Cardiomyocytes279
- Conclusion281
- References282
- Chapter 17: Oocytes284
- Introduction284
- General Technical Comments288
- Embryonic Stem Cell Culture289
- MEF Removal from ESC Cultures290
- ESC Differentiation and the Emergence of Germ Cells290
- Detection of Early Stage Germ Cells291
- Detection of Late Stage Germ Cells292
- Formation of Follicle-Like Structures292
- Oocyte Growth295
- Oocyte Maturation296
- Cleavage Stage Embryos296
- Reoccurring Events297
- Schematic Overview of the In Vitro Derivation of Oocytes from Murine ESCs297
- Conclusions303
- Acknowledgments304
- References304
- Chapter 18: Male Germ Cells307
- Introduction307
- Methods308
- Discussion312
- Acknowledgment313
- References313
- Chapter 19: Insulin-Producing Cells315
- Introduction315
- Materials and Methods318
- Results329
- Summary330
- Acknowledgments330
- References331
- Chapter 20: Pulmonary Epithelium333
- Introduction333
- Development and Organization of Pulmonary Epithelium334
- Lung Stem Cells335
- Circulating Stem Cells336
- In Vitro Generation of Pulmonary Epithelium from Stem Cells337
- References344
- Author Index351
- Subject Index383
This is the first of three planned volumes in the Methods in Enzymology series on the topic of stem cells. This volume is a unique anthology of stem cell techniques written by experts from the top laboratories in the world. The contributors not only have hands-on experience in the field but often have developed the original approaches that they share with great attention to detail. The chapters provide a brief review of each field followed by a “cookbook and handy illustrations. The collection of protocols includes the isolation and maintenance of stem cells from various species using “conventional and novel methods, such as derivation of ES cells from single blastomeres, differentiation of stem cells into specific tissue types, isolation and maintenance of somatic stem cells, stem cell-specific techniques and approaches to tissue engineering using stem cell derivatives. The reader will find that some of the topics are covered by more than one group of authors and complement each other. Comprehensive step-by-step protocols and informative illustrations can be easily followed by even the least experienced researchers in the field, and allow the setup and troubleshooting of these state-of-the-art technologies in other laboratories.
* Provides complete coverage spanning from derivation/isolation of stem cells, and including differentiation protocols, characterization and maintenance of derivatives and tissue engineering
* Presents the latest most innovative technologies
* Addresses therapeutic relevance including FDA compliance and tissue engineering
* Provides complete coverage spanning from derivation/isolation of stem cells, and including differentiation protocols, characterization and maintenance of derivatives and tissue engineering
* Presents the latest most innovative technologies
* Addresses therapeutic relevance including FDA compliance and tissue engineering