Differentiation of Embryonic Stem Cells

65.pdf......Page 0
Table of Contents......Page 3
Contributors to Volume 353......Page 7
Preface......Page 1
METHODS IN ENZYMOLOGY......Page 12
Introduction......Page 44
Foetal Calf Serum (FCS)......Page 46
Gelatin......Page 49
ES Cell Morphology......Page 51
Preparation of MEDII......Page 52
Adherent Culture......Page 53
Suspension culture......Page 54
Gene Expression in EPL Cells......Page 55
Differentiation of EPL Cells: Formation of Nascent and .........Page 57
Differentiation of EPL Cells: Formation of Nascent and.........Page 60
Reversion of EPL Cells to ES Cells......Page 63
Quality Control of MEDII......Page 64
Concluding Remarks......Page 65
Introduction......Page 67
Overall Strategy......Page 69
Derivation, culture and in vitro gene-targeting.........Page 71
Identification of 39XO Subclones by PCR......Page 72
Production of Tetraploid Embryos......Page 73
Isolation and in vitro Culture of Preimplantation Embryos......Page 74
Electrofusion of Two-cell Embryos......Page 75
Manual Alignment and DC Fusion of Two-cell Embryos......Page 76
Electrofusion by AC Alignment and DC Pulse......Page 77
Embryo Transfer to Recipient Females......Page 78
C-section and Cross-Fostering of ES Cell-Tetraploid Mice......Page 80
Introduction......Page 82
Materials and Reagents......Page 85
Prepared Reagents......Page 86
Prepared Media......Page 89
Step 1: Maintenance of ES cells......Page 92
Step 2: Generation of embryoid bodies......Page 94
Step 3: Analysis of EBs......Page 95
Conclusions......Page 102
Summary......Page 103
The Lateral Plate Mesoderm and Its Derivatives......Page 104
Differentiation of Mesoderm and Mesodermal .........Page 105
Reagents......Page 107
Generation of Smooth Muscle Cells and Vascular .........Page 108
Assessing Gene Expression During in vitro .........Page 109
Differentiation and Isolation of ES-derived Endothelial.........Page 110
In Vitro Formation of Vascular Tubes......Page 111
Primitive and Definitive Hematopoieitic Cultures .........Page 112
Assessing Gene Expression During in vitro .........Page 113
Analysis of Gene Expression Data......Page 115
Introduction......Page 117
OP9 Stromal Cells......Page 118
Maintenance of OP9 Cells......Page 119
Induction of Differentiation into Multipotential .........Page 120
Induction of Differentiation to Megakaryocytes, Osteoclasts, .........Page 123
Lineage-specific Gene Expression Analysis Using OP9 Cells......Page 124
Conditional Gene Expression by the Tet System During.........Page 125
OP9 System vs the EB Method......Page 126
Conclusions......Page 128
Introduction......Page 129
ES Cell Maintenance......Page 132
In Vitro Differentiation......Page 133
Embryoid Body Generation—Method 1 (Dispase)......Page 134
Attachment and Differentiation of Embryoid Bodies......Page 135
Visualization and Quantitation of Blood Vessels......Page 136
Visualization and Quantitation of Hematopoietic Cells......Page 138
Primitive Erythrocyte Differentiation......Page 139
Time-lapse Imaging to Visualize Vascular Development......Page 140
Concluding Remarks......Page 143
Introduction......Page 145
Materials......Page 147
Gelatin Coating of Culture Dishes......Page 151
Preparation of EF......Page 152
Maintenance of Undifferentiated ES Cells (Note 4)......Page 153
Osteoclastogenesis from Undifferentiated ES Cells .........Page 154
Osteoclastogenesis of Undifferentiated ES Cells.........Page 156
Alkaline Phosphatase Staining......Page 157
Immunohistochemical Staining of CD31......Page 158
Notes......Page 159
Introduction......Page 162
Leukemic Engraftment......Page 164
Bcr/Abl-Induction of Hematopoietic Cultures......Page 165
Non-oncogenic Engraftment......Page 167
Lox-in to Derive Inducible ES Cell Lines from .........Page 170
Hanging Drop EB Culturess......Page 172
HoxB4-Induction of Hematopoietic Cultures......Page 173
Therapeutic Repopulation......Page 176
Introduction......Page 178
Neutrophil Differentiation Mediums......Page 181
Sera......Page 182
Plasticware for Tissue Culture......Page 184
Routine culture of CCE ES cells......Page 185
gp-130 Secondary Differentiation Medium......Page 186
Tertiary Neutrophil Differentiation Mix......Page 187
Neutrophil Production in the ES/OP9 Coculture System......Page 188
The Production of Neutrophils From Embryonic .........Page 189
Applications of the ES/OP9 Coculture System for Studying .........Page 190
Introduction......Page 192
Maintenance of ES Cells......Page 194
Maintenance of OP9 Stromal Cells......Page 197
ES Cell Differentiation......Page 198
Retrovirus-Mediated Transduction of Genes into Megakaryocytes......Page 201
Analysis of ES Cell-Derived Megakaryocytes......Page 203
Perspective......Page 208
Introduction......Page 209
Materials......Page 211
Methods......Page 214
Comments......Page 217
Final Remarks......Page 219
Introduction......Page 221
Maintenance of the Parent ES Cell Line......Page 223
Generation of Embryoid Bodies......Page 225
Differentiation of DC from EBs......Page 226
Maturation of esDC......Page 229
Validation of the Differentiation Pathway by Global Gene Expression Profiling......Page 230
Genetic Modification of esDC......Page 233
Discussion......Page 236
Introduction......Page 239
Reagents and Materials......Page 241
ES Differentiation to EBs......Page 243
Differentiation Media......Page 244
Reagents and Materials......Page 245
FACS Analysis......Page 246
Hematopoietic Replating Analysis......Page 251
Cell Sorting and in vitro Culture of Sorted Cell Populations......Page 253
Introduction......Page 256
Production of Retrovirus......Page 258
Differentiation of ES Cells......Page 260
Concluding Remarks......Page 268
Introduction......Page 269
Differentiation Methods......Page 270
Procedure for Endothelial ES Cell Differentiation in Semi-solid Methylcellulose Medium......Page 272
Growth Factors......Page 273
Endothelial Gene Expression Analysis......Page 274
Vascular Morphogenesis in ES-derived EBs......Page 276
Angiogenesis during EB Maturation in Three Dimensional Collagen Matrix......Page 278
Procedure for EB Secondary Culture into Type I Collagen Gels......Page 280
Visualization and Quantitation of Endothelial Sprouting......Page 281
Further Considerations......Page 283
Introduction......Page 284
General Considerations......Page 286
Solutions and Media......Page 288
Primary Embryonic Fibroblast Feeder Cells......Page 289
Inactivation of Primary Embryonic Fibroblast Feeder Cells......Page 291
Cultivation of Undifferentiated ES Cells......Page 292
Differentiation Protocols......Page 293
Hanging Drop Method......Page 294
Mass Culture Procedure......Page 295
Procedure......Page 296
Introduction......Page 298
Materials and Reagents......Page 299
Preparation of EB derived Cell Populations for Plating in the Osteoprogenitor Culture System......Page 301
Fixation and Staining of 3--4 Week Cultures......Page 302
Concluding Remarks......Page 305
Introduction......Page 309
Material, Media, Additiva and Buffer Solutions......Page 310
Procedures......Page 312
Characterization of ES Cell-derived Chondrogenic and Osteogenic Differentiation......Page 317
Detection of Gene Expression by Semiquantitative RT-PCR Analysis......Page 318
Whole-mount Fluorescence in situ Hybridization for Scleraxis or for Collagen X-mRNA Coupled with Immunostaining for Collagen II......Page 321
Isolation of Chondrogenic Cells from EBs......Page 322
Histochemical Stainings......Page 323
Enhancing the Efficiency of ES Cell-derived Chondrogenic Differentiation......Page 324
Introduction......Page 327
Maintenance of ES Cells on Gelatine-coated Tissue Culture Plastic......Page 330
Differentiation of ES Cell-derived Embryoid Bodies into Adipocytes......Page 331
Assessment of ES Cell Differentiation into Adipocytes......Page 334
Introduction......Page 337
Visceral Endoderm and Hepatic Differentiation in Embryoid Bodies......Page 338
Induction of Hepatic Differentiation Without Forming Classical Embryoid Bodies......Page 344
Conclusion and Future Directions......Page 346
Introduction......Page 348
Differentiation of ES cells into Pancreatic and Hepatic Cells......Page 352
ES Cell Differentiation into Pancreatic or Hepatic Cells......Page 353
Analysis of Differentiated Phenotypes......Page 355
Results......Page 360
Differentiation of Pancreatic Cells......Page 361
Differentiation of Hepatic Cells......Page 362
Summary......Page 363
Derivation of Dopaminergic Neurons from Mouse Embryonic Stem Cells......Page 365
Procedure Outline......Page 366
Materials and Reagents......Page 367
Procedure for Generation of Dopaminergic Neurons from ES cells......Page 370
Preparing Cultures of Rodent Fetal Neuroepithelial Stem Cells......Page 376
Materials and Reagents (in addition to those described in ES cell section)......Page 378
Dissecting Tissue from the Rat Fetus......Page 380
Plating and Culturing Rat Fetal Stem Cells......Page 382
Passaging Cells......Page 383
Appendix for Fetal Stem Cell Culture......Page 384
Dissecting Tissue and Plating Cells from the Rat Adult Subventricular Zone......Page 387
Appendix for Adult Stem Cell Culture......Page 388
Introduction......Page 390
ES Cells......Page 392
Culture Medium......Page 393
Monitoring Monolayer Differentiation Using 46C ES Cells......Page 394
Monitoring Neuronal Differentiation......Page 397
Purification and Propagation of Neural Precursors from Monolayer Differentiation......Page 399
Trouble-Shooting Monolayer Differentiation......Page 401
Role of Components in N2B27 and Extrinsic and Intrinsic Factors......Page 402
Summary......Page 404
Introduction......Page 405
Materials......Page 407
Preparation of Embryonic Fibroblast Cells Treated with Mitomycin C for Feeder Cells......Page 408
Preparation of Undifferentiated ES Cells......Page 409
Induction of Differentiation into Melanocytes......Page 410
Comments......Page 412
Introduction......Page 416
Fixatives......Page 417
PGCs Isolation and Culture......Page 418
Cell Proliferation......Page 421
Derivation of EG Cells......Page 422
Picking and Passaging Single EG Colonies......Page 423
Freezing and Thawing EG Cells......Page 424
Notes......Page 425
Introduction......Page 429
ROSA Series Retroviral Gene Traps......Page 431
ES Cell Culture......Page 434
Retroviral Infection and Electroporation......Page 436
Picking, Synchronizing, Freezing, and Thawing ES Cell Clones......Page 437
Cloning cDNA and Genomic DNA Sequences Flanking Gene Trap Insertion Sites......Page 438
Cloning cDNA Flanks with 5' RACE and 3' RACE......Page 439
Cloning Genomic DNA Flanks with Anchoring PCR......Page 442
Monitoring Expression of Trapped Genes by Reporter Assays......Page 443
Production and Analysis of Gene Trap Mouse Strains......Page 444
Gene Trap Mutagenesis Screens......Page 446
Introduction......Page 449
Bifunctional Retroviral Vectors......Page 452
Propagation and Maintenance of ES Cells......Page 455
Retroviral Entrapment Vector Infection and Selection of ES Cells......Page 458
Design of Differential Expression Screen......Page 461
Culture and Assay Conditions......Page 463
Differentiation of ES Cells into Embryoid Bodies......Page 464
Colocalization of AP Reporter Gene and Developmental Marker Expression......Page 465
Colocalization: Proof of Principle......Page 467
Conclusions......Page 468
Introduction......Page 470
Preparation of ENU......Page 472
Picking and Cryopreservation of Mutagenized Clones......Page 473
Gene-based Mutation Screening by Heteroduplex Analysis......Page 474
Generation of Mice Carrying ENU-induced Mutations......Page 476
Concluding Remarks......Page 478
Introduction......Page 482
Establishment of Cynomolgus ES Cell Lines......Page 483
Improved Methods for Maintenance of Monkey ES Cell Lines......Page 484
Protocol......Page 485
Differentiation of ES Cell In Vitro......Page 487
Protocol......Page 489
Teratoma Formation in SCID Mice......Page 490
Prospects of Primate ES Cells......Page 491
Introduction......Page 493
Human embryos......Page 494
Mouse embryo fibroblast feeder cells......Page 495
Establishment and maintenance of ES cells......Page 496
Immunochemical characterization of human ES cells......Page 498
Indirect immunofluorescence......Page 500
Flow cytometry......Page 501
Immunomagnetic isolation of viable ES cells......Page 502
Gene Expression in ES Cells......Page 504
RNA Isolation from ES Cells Using Dynalbeads mRNA DIRECT kit......Page 505
Polymerase Chain Reaction......Page 506
ES Cell Differentiation......Page 507
In vitro differentiation......Page 508
Introduction......Page 511
Spontaneous Differentiation of Human ES Cells......Page 512
Protocol for Formation of EBs......Page 515
Factors Effecting Differentiation of Human ES Cells......Page 517
Assaying for Cellular Differentiation......Page 520
Molecular Markers......Page 521
Protocol for Genetic Labeling of Human ES Cells......Page 522
Tissue Integration In Vivo......Page 524
Conclusions......Page 525
Introduction......Page 527
Feeder Layer Preparation......Page 528
ES Cell Propagation......Page 529
Immunofluorescent Analysis......Page 531
Assessment of Cardiomyocyte Proliferation and Cell-Cycle Regulation......Page 533
Calcium Imaging......Page 535
Multi-electrode Array Recording......Page 536
Pharmacological Studies......Page 538
Concluding Remarks......Page 539
Author Index......Page 540
Subject Index......Page 565