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
Author(s): Irina Klimanskaya, Robert Lanza
Series: Methods in Enzymology 418
Edition: 1
Publisher: Academic press
Year: 2006
Language: English
Pages: 408
Series editors......Page 1
Dedication......Page 2
Table of Contents......Page 3
Contributors to Volume 418......Page 5
Preface......Page 8
Foreword......Page 10
Previous Volume List......Page 12
Historical Overview......Page 36
Factors Affecting the Efficiency of Mouse ES Cell Establishment......Page 38
Factors Affecting the Contribution of Mouse ES Cell to Chimeric Embryos......Page 39
Source Embryos......Page 40
Attachment......Page 41
Expansion of First ES Cell-Like Colonies......Page 43
Characterization......Page 45
Materials and Equipment......Page 46
Method......Page 47
Material and Equipment......Page 48
Disaggregation of Outgrowth......Page 49
Beyond the Basic Derivation Protocol......Page 50
Passaging Mouse ES Cell Cultures......Page 51
References......Page 52
[2] Bovine Embryonic Stem Cells......Page 54
Introduction......Page 55
Media Recipes and Solutions......Page 57
MEFs Treatment......Page 60
In Vitro Fertilization......Page 61
Somatic Cell Nuclear Transfer or Cloning......Page 62
Mechanical Isolation of the Inner Cell Mass......Page 63
Maintenance of Bovine Embryonic Stem Cells......Page 64
Detection of Alkaline Phosphatase......Page 66
Immunostaining of Surface Markers......Page 67
Immunocytochemical Analysis of Differentiated Cultures......Page 68
References......Page 69
Introduction......Page 71
Chicken ES Cell Culture Medium......Page 72
Media Formulations......Page 73
Selection of Breeds......Page 74
Isolation of Blastodermal Cells......Page 75
Maintenance of Chicken Embryonic Stem Cells......Page 78
Morphology of Chicken Embryonic Stem Cells......Page 79
Genetic Modification of cES Cells......Page 82
Maximizing the Level of Chimerism......Page 84
Preparation and Use of Surrogate Shells......Page 86
Preparation and Injection of cES Cells......Page 87
Incubation of Recipient Embryos......Page 90
Acquisition, Storage, and Quality Control of Eggs......Page 92
Day 15......Page 93
Efficiency of Chimera Production Using Embryonic Stem Cells......Page 94
References......Page 95
Introduction......Page 97
Reagents......Page 99
Drug-Resistant Feeder Cells......Page 100
Overview......Page 101
Protocol for Initiation of ES Cell Culture from Zebrafish Blastulas......Page 103
Protocol for Initiation of ES Cell Cultures from Zebrafish Gastrula Stage Embryos......Page 105
Overview......Page 106
Protocol for ES Cell Electroporation and Selection of Homologous Recombinants......Page 107
Acknowledgments......Page 108
References......Page 109
Introduction......Page 111
Planning and Considerations......Page 112
Dissection and Primary Culture of MEFs......Page 115
Isolation of Inner Cell Mass......Page 116
Dispersion of Inner Cell Mass......Page 117
Enzymatic Dissociation with Trypsin......Page 118
Freezing hES Cells......Page 119
Thawing hES Cells......Page 120
Culture with Human Feeder Cells......Page 121
References......Page 122
Introduction......Page 126
Materials and Methods......Page 127
Procedure......Page 128
Commentary to Protocol......Page 129
Characteristics of Morula-Derived hESC Lines......Page 130
Cell Cycle Analysis of Morula-Derived hESC Cell Lines......Page 133
Karyotyping of Morula-Derived hESC Lines......Page 135
Equipment......Page 136
Procedure......Page 137
Required Equipment......Page 138
Procedure for Freezing Human Embryonic Stem Cells......Page 139
References......Page 140
[7] Embryonic Stem Cells from Single Blastomeres......Page 141
Introduction......Page 142
Protocol......Page 143
Analysis of Cell Lines......Page 146
References......Page 149
Introduction......Page 150
Mouse Parthenogenetic ESCs......Page 155
Nonhuman Primate Parthenogenetic ESCs......Page 157
The Importance of Choosing the Right Activation Protocol......Page 159
Imprinting and Parthenogenesis......Page 161
Mouse......Page 162
Nonhuman Primate......Page 164
References......Page 165
Introduction......Page 168
Chemicals, Media, and Equipment......Page 169
Media Preparation......Page 171
Oocyte Isolation......Page 172
Removing the Spindle-Chromosome Complex (SCC)......Page 173
Introduction of Nuclei......Page 174
Oocyte Activation......Page 176
Derivation of ES Cells from Cloned Blastocysts......Page 177
References......Page 179
Introduction......Page 181
The Monolayer Protocol Is Not Based on Cell Selection......Page 182
Protocol: Conversion of Mouse ES Cells to Neural Cells in Adherent Monolayer......Page 183
Monitoring Neural and Neuronal Differentiation......Page 185
Nonneural Differentiation......Page 186
Protocol: Conversion of Human ES Cells to Neural Cells in Adherent Monolayer......Page 187
Cell Death......Page 188
Overview......Page 189
Protocol: NS Cell Derivation from ES Cells......Page 190
Derivation of NS Cells Using Sox1-Lineage Selection......Page 191
Derivation of NS Cells from Any ES Cell Line......Page 192
Protocol: Expansion, Freezing, and Thawing of NS Cells......Page 193
Troubleshooting: Mouse NS Cells......Page 194
ES Cell Medium......Page 195
N-2 Supplement......Page 196
Summary......Page 197
References......Page 198
Introduction......Page 199
Differentiation of hES Cells to RPE......Page 201
Materials and Equipment......Page 203
Equipment and Cell Culture Disposables......Page 204
Media Components and Other Reagents......Page 206
hES Cell Culture and Differentiation Culture Setup......Page 207
Method 1. Handpicking Pigmented Cells......Page 209
Method 2. Outgrowth of RPE from EBs......Page 210
Media and Reagents......Page 211
Characterization of hES-RPE Cells......Page 212
Immunofluorescence and Western Blot......Page 213
Primers Used in RT-PCR and Quantitative PCR (QPCR)......Page 214
Freezing ES-RPE......Page 215
Preparation of 4% Paraformaldehyde Solution......Page 216
RT-PCR and Quantitative Real-Time PCR......Page 217
RT-PCR Using Qiagen One-Step RT-PCR Kit......Page 218
PCR Using Amplitaq Gold......Page 219
Real-Time PCR Program......Page 220
References......Page 221
Introduction......Page 224
Developmental Considerations......Page 225
Adult Mesenchymal Stem Cells (MSCs)......Page 226
Mouse ESC-Derived Mesenchymal Tissues......Page 227
Mesenchymal Cells Derived from Human ESCs......Page 228
Derivation, Isolation, and Differentiation of hESC-Derived Mesenchymal Precursors......Page 229
Induction of Mesodermal and Mesenchymal Fates......Page 230
Characterization of hESMPCs......Page 231
Differentiation of hESMPCs into Specialized Mesenchymal Derivatives......Page 232
Skeletal Muscle Differentiation......Page 233
Summary and Perspectives for the Future......Page 234
References......Page 235
[13] Hematopoietic Cells......Page 238
Early ESC Development......Page 239
mESC Stroma Coculture Systems......Page 241
T-Cell Development......Page 242
In Vitro Engraftment of mESC-Derived Hematopoietic Cells......Page 243
Primate ESC-Derived Hematopoiesis......Page 244
Hematopoietic Differentiation of Human ESC Lines......Page 245
Immune Cell Generation......Page 246
Hematopoiesis and Homeobox Gene Expression in ESC......Page 247
Maintenance of Human Embryonic Stem Cells......Page 249
Maintenance of Stromal Cell Lines......Page 250
OP9......Page 251
Stromal-Supported hESC Differentiation......Page 252
Stromal Plus Cytokine-Supplemented Cultures......Page 255
Hematopoietic Differentiation of hESC Using Stromal-Conditioned Medium......Page 257
Aggregation Methods for EB Formation......Page 258
Erythroid Differentiation......Page 259
Megakaryocyte Differentiation......Page 260
Macrophage Differentiation......Page 261
In Vivo Transplantation of EB-Derived CD34+ Cell into Immunodeficient Mice......Page 262
Analysis of NOD/SCID Mouse Hematopoietic Engraftment......Page 263
Production of a GFP/Luciferase-Expressing Lentivirus......Page 264
In Vivo Luciferase Imaging......Page 265
References......Page 266
In Vitro Hematopoiesis from Nonhuman Primate ES Cells......Page 273
Embryonic Mesoderm Regulators and Hematopoietic Onset of Rhesus Monkey ES Cells......Page 274
Reagents for Propagation and Storage of Undifferentiated ES Cells......Page 275
Propagation of Undifferentiated Rhesus Monkey ES Cells......Page 276
Overview......Page 277
Methods......Page 278
References......Page 280
Derivation and Characterization of Embryonic Stem (ES) Cells......Page 282
Differentiation of Embryonic Stem Cells......Page 283
Development of the Blood Vascular System......Page 284
Vascular Developmental Potential of Embryonic Stem Cells......Page 285
Mesodermal Specification in Two-Dimensional Culture on Type IV Collagen-Coated Dishes......Page 286
Embryonic Stem Cell-Derived Endothelial Cells Cultured on Type IV Collagen......Page 288
Vascular Cell Generation from Human Embryonic Stem Cells......Page 289
Dissociation of Human ES Cells......Page 290
Cell Seeding Concentration......Page 291
Lineage-Specific Differentiation......Page 292
References......Page 294
Introduction......Page 297
Embryoid Body (EB) Generation......Page 298
Mouse ES Cell Dissociation......Page 299
Suspension Culture......Page 301
Scalable Production of EBs in a Bioreactor......Page 302
Autoclave Method......Page 305
Cardiomyocyte Differentiation......Page 308
Enrichment of Cardiomyocytes......Page 309
Conclusion......Page 311
References......Page 312
Introduction......Page 314
General Technical Comments......Page 318
Medium and Reagents......Page 319
ESC Differentiation and the Emergence of Germ Cells......Page 320
Detection of Early Stage Germ Cells......Page 321
Formation of Follicle-Like Structures......Page 322
Medium and Reagents......Page 324
Oocyte Growth......Page 325
Cleavage Stage Embryos......Page 326
Analytical Methods......Page 327
Hormone Measurements......Page 328
Immunocytochemistry......Page 329
Reagents and Solutions......Page 331
Conclusions......Page 333
References......Page 334
Introduction......Page 337
ES Cell Derivation and Culture......Page 338
Embryoid Body Differentiation......Page 339
Gelatin Embedding and EB Sectioning for Immunohistologic Examination......Page 340
EB Dissociation for Isolation of Embryonic Germ Cells......Page 341
Discussion......Page 342
References......Page 343
Introduction......Page 345
Generation of mES Cell-Derived Multilineage Progenitor Cells......Page 348
Analysis of Differentiated Phenotypes......Page 350
Semiquantitative RT-PCR Analysis......Page 351
Immunofluorescence Analysis......Page 356
Semiquantitative Determination of Immunofluorescence Signals......Page 357
Insulin ELISA......Page 358
Results......Page 359
Acknowledgments......Page 360
References......Page 361
Introduction......Page 363
Development and Organization of Pulmonary Epithelium......Page 364
Lung Stem Cells......Page 365
Circulating Stem Cells......Page 366
In Vitro Generation of Pulmonary Epithelium from Stem Cells......Page 367
Derivation of Distal Lung Epithelial Cells from Murine Embryonic Stem Cells (mESC) Using Medium Supplementation......Page 368
Selection of Lung Epithelial Progenitors with Lung-Specific Medium......Page 369
Transfection......Page 370
ES Cell Culture......Page 371
Direct Contact EB/Lung Mesenchyme Cocultures......Page 372
Basic Human Embryonic Stem Cell Culture......Page 373
References......Page 374
Author Index......Page 380
Subject Index......Page 411
