This is the second 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 focusing on adult stem cells, and written 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. Read more... Abstract: This is the second 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 focusing on adult stem cells, and written 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
Author(s): Irina Klimanskaya, Robert Lanza
Series: Methods in Enzymology 419
Edition: 1
Publisher: Academic press
Year: 2006
Language: English
Pages: 557
Series Editors......Page 1
Dedication......Page 2
Table of Contents......Page 3
Contributors to Volume 419......Page 5
Preface......Page 8
Foreword......Page 10
List of Previous Volumes......Page 12
Introduction......Page 36
Tissue Culture Equipment......Page 38
Commercial Medium Components......Page 39
Medium Preparation Components......Page 40
Stock Solutions......Page 41
Dissection of Embryonic Tissue......Page 42
Harvesting of Embryonic Brain Tissue......Page 43
Establishing Primary Embryonic Cultures......Page 44
Setup......Page 45
Dissection of Adult Periventricular Region......Page 47
Dissociation Protocol......Page 48
Comments......Page 49
Passaging Neurosphere Cultures......Page 50
Differentiation of Whole Neurospheres......Page 51
Differentiation of Dissociated Cells......Page 53
Flow Cytometric Enrichment of Adult Neural Stem Cells......Page 54
Acknowledgments......Page 55
References......Page 0
[2] Neural Stem Cells and Their Manipulation......Page 56
Introduction......Page 57
Adult Niches for Stem Cells In Vivo......Page 58
Proliferation......Page 60
Survival/Apoptosis......Page 64
Symmetric and Asymmetric Cell Division......Page 65
Differentiation......Page 68
Conclusions and Projections......Page 74
Culture of Human NSCs......Page 76
References......Page 78
[3] Retinal Stem Cells......Page 85
Retinal Stem/Progenitor Cells During Development......Page 86
Retinal Stem Cells and the Ciliary Marginal Zone......Page 88
Ciliary Epithelium......Page 91
Muumlller Glia......Page 92
Harvest and Culture of Retinal Stem/Progenitor Cells......Page 93
Substrate for Adherent Culture of Retinal Stem/Progenitor Cells......Page 95
Analysis of Culture of Retinal Stem/Progenitor Cells......Page 96
Dissection and Fixation of Tissues......Page 100
References......Page 102
Introduction to Skin Epithelial Stem Cells......Page 106
Epithelial Skin Organization and the Hair Cycle......Page 109
Characteristics of Skin Epithelial Stem Cells......Page 110
Epithelial SCs and the Frequency of Their Division......Page 111
Protocol for Labeling and Detection of BrdU LRCs in Whole Mount Epidermis......Page 113
Putative Stem Cell Markers: Genes Preferentially Expressed by Bulge Cells......Page 115
Gene Circuitry in the Stem Cell Niche......Page 116
Protocol for Detecting Gene Expression of Putative Bulge-Expressed Genes......Page 117
Contribution of Epithelial SCs to Tissue Morphogenesis and Wound Repair In Vivo......Page 118
Protocol for Immunofluorescence Staining of Skin Sections......Page 120
Epithelial Stem Cells in Culture......Page 121
Self-Renewal and Differentiation of Epithelial SCs......Page 123
References......Page 127
Introduction......Page 132
Identification of Dental Pulp Stem Cells......Page 134
Isolation of DPSCs......Page 136
Protocol for DPSC Isolation......Page 137
Differentiation of DPSCs......Page 138
Protocol for DPSC Transplantation......Page 141
References......Page 142
Introduction......Page 147
Skeletal Stem Cells and Mesenchymal Stem Cells......Page 148
Stromal Nature......Page 149
Multipotency......Page 150
Self-Renewal......Page 151
Phenotype......Page 153
Origin and Nature of Skeletal Stem Cells......Page 155
Plasticity of BMSCs......Page 156
Role of Skeletal Stem Cells in Disease......Page 158
Molecular Engineering......Page 159
Potential Use of Skeletal Stem Cells in Tissue Engineering and Regenerative Medicine......Page 160
Overview......Page 162
Nonclonal BMSC Culture Protocol......Page 163
Overview......Page 164
Clonal BMSC Culture Protocol......Page 165
Overview......Page 166
Colony-Forming Efficiency Protocol......Page 167
Osteogenic Differentiation Protocol......Page 168
Adipogenic Differentiation Protocol......Page 169
Overview......Page 170
Subcutaneous Ossicles Formed by HA/TCP Protocol......Page 173
Subcutaneous Ossicle Formed with Denatured Collagen Sponges Protocol......Page 174
References......Page 175
Introduction......Page 179
Cell Surface Markers......Page 180
Side Population Assay......Page 182
Fluorescent Substrates for Cytosolic Aldehyde Dehydrogenase Activity......Page 183
Protocol for Isolation of Candidate Human HSCs......Page 184
Surrogate In Vivo Assays......Page 187
Human Hematopoietic Repopulating Cell Assay Protocol......Page 190
Overview......Page 194
Protocol for Derivation of Human Hematopoietic Progenitor Cell Lines......Page 196
References......Page 198
Introduction......Page 209
Hematopoietic and Endothelial Differentiation of Single Cell-Derived EGFP+ Clones......Page 210
Phenotypical and Functional Studies of CD133-Derived Cells during Ex Vivo Expansion and Differentiation......Page 211
Isolation of CD133+ Cells from Frozen Leukapheresis Products......Page 212
Retroviral Transduction of Expanding Cells......Page 213
Immunocytochemical Staining of Freshly Isolated CD133+ Cells and Cultured CD133-Derived Cells......Page 214
Assessment of Functionality of CD133-Derived Endothelial Cells In Vivo......Page 215
Isolation of CD133+ Cells from Frozen Leukapheresis Products......Page 216
Differentiation Cultures......Page 217
Retroviral Transduction of Expanded CD133+ Cells......Page 218
Clonogenic Assay for Hematopoietic Progenitor Cells......Page 219
Immunocytochemical Staining of Freshly Isolated CD133+ Cells and Cultured CD133-Derived Cells......Page 220
Flow Cytometric Analysis of Freshly Isolated and Expanded Cells......Page 221
Functionality of CD133-Derived Endothelial Cells In Vivo......Page 222
References......Page 223
Introduction......Page 224
Analysis of Steady State Tubular Epithelium Maintenance......Page 225
Regeneration of Experimentally Injured Kidney Tubules......Page 226
Extratubular Progenitor Cells......Page 231
Culture of Human Tubular Progenitor Cells......Page 233
References......Page 235
Introduction......Page 238
Origin of Primordial Germ Cells......Page 239
Formation of Human Fetal Oocytes and Primordial and Primary Follicles......Page 240
Immune System-Related Cells and Oogenesis in Fetal Ovaries......Page 241
Fetal Programming of Follicular Renewal during Adulthood......Page 244
Role of Ovarian Tunica Albuginea-OSE Transitions......Page 248
Follicular Renewal......Page 250
Follicular Atresia......Page 254
"Ovary within the Ovary" Pattern and Thy-1 Differentiation Protein......Page 256
Cessation of Adult Oogenesis......Page 257
Oogenesis and Follicular Renewal in Adult Ovaries......Page 258
Working Hypothesis on the Role of the Gonadal Environment......Page 261
Animal Models......Page 263
Potential Treatment of Ovarian Infertility by Producing New Primary Follicles In Vivo......Page 266
Mystery of the Origin of Oocytes in Adult Mammalian Ovaries......Page 267
Oogenesis In Vitro......Page 271
Pluripotency of Ovarian Stem Cells......Page 273
Collection and Culture of OSE Stem Cells......Page 274
Fixation of Cells and Peroxidase Immunohistochemistry......Page 276
Lessons from Ovarian Tissue Cultures......Page 277
Suitability of Patients for Clinical Trial......Page 278
Potential Pitfalls......Page 279
Conclusion......Page 280
References......Page 282
Introduction......Page 289
Spermatogonial Stem Cells......Page 290
SSC Transplantation......Page 293
Choice of Donor Animals......Page 294
Busulfan-Treated Adult Recipients......Page 296
Microinjection of Donor Cell Suspension......Page 297
Microinjection......Page 298
Analysis of Recipient Testes for Donor-Derived Spermatogenesis......Page 299
SSC Culture......Page 300
Testis Digestion......Page 303
MACS Thy1 Selection......Page 304
STO Feeder Cell Monolayer Preparation......Page 305
Subculturing Practices That Optimize Expansion of SSC Numbers......Page 306
Implications......Page 308
References......Page 309
Introduction......Page 313
Anatomical and Cellular Diversity of Adult Lung......Page 315
Stem Cell Phenotypes and Niches in Adult Lung......Page 318
Overview......Page 321
Mouse Naphthalene Injury and BrdU-Labeling Protocol......Page 326
Overview......Page 328
Human Bronchial Xenograft Protocol......Page 332
Medium A......Page 336
Trypsin Inhibitor Buffer......Page 337
Overview......Page 338
In Vitro Mouse Tracheal Epithelial Cell Colony-Forming Efficiency Assay Protocol......Page 340
Overview......Page 342
In Vitro Invasion and Tubulogenesis Assay Using Mouse Tracheal Epithelial Cells......Page 343
References......Page 345
Introduction......Page 350
Pancreas Development......Page 351
Preexisting Beta Cells......Page 354
Acini......Page 356
Adult Stem Cells......Page 357
Summary and Perspective......Page 358
Methods: Design of a Lineage-Tracing Experiment in Mice......Page 359
Tissue Specificity of Cre Recombinase......Page 360
References......Page 361
[14] Intestinal Epithelial Stem Cells and Progenitors......Page 365
General Histology......Page 366
Where Are the Stem Cells?......Page 368
Assaying Intestinal Stem Cells, Progenitors, and Their Lineages In Vivo......Page 369
Regeneration Assays......Page 370
Labeled Phagosomes......Page 372
Protocol......Page 373
beta-Galactosidase (lacZ) Mutation......Page 375
Methylation......Page 376
Mitochondrial Genes......Page 377
Stem, Progenitor, and Lineage Markers......Page 378
Label Retention......Page 379
beta1-Integrin......Page 380
Intestinal Epithelium In Vitro......Page 381
Are They Stem Cells?......Page 383
Primary Cultures......Page 384
Epithelial Isolation Using Mechanical Approaches......Page 385
Microexplants......Page 386
Epithelial Isolation by Chelation......Page 387
Epithelial Isolation with MatriSperse......Page 389
Epithelial Isolation by Enzymatic Digestion......Page 390
Culturing organoids......Page 391
Culture Systems for Studying Mesenchymal-Endodermal Interaction During Development......Page 392
Implants: From Tissues to Tissue Engineering......Page 393
Tissue Engineering......Page 394
Soft Agarose Colony-Forming Assay......Page 395
Side Population......Page 396
Epithelial Isolation Protocols......Page 397
Perfusion Protocol to Obtain Large Amounts of Pure Epithelium for Biochemical or Physiological Studies......Page 398
Immersion Protocol Using Chelation to Obtain Crypts and Cell Clumps for Culture or Other Procedures......Page 399
References......Page 400
Introduction......Page 412
Reagents......Page 414
Culture Media......Page 415
Preparation of MMC-Treated Feeder Cell Stocks......Page 416
Overview......Page 417
TS Cell Line Establishment from Blastocyst-Stage Embryo......Page 419
Overview......Page 421
Passage of TS Cells......Page 422
Overview......Page 423
References......Page 424
[16] Pluripotent Stem Cells from Germ Cells......Page 425
Introduction......Page 426
Germ Cell Development......Page 427
Overview......Page 428
Growth Medium Components......Page 430
Initial Disaggregation and Plating......Page 431
Subsequent Passage of EGC Cultures......Page 432
Troubleshooting......Page 433
Overview......Page 434
Plating Feeder Cells Before Inactivation......Page 436
Overview......Page 437
Sex Determination of Human Gonads by Fluorescence In Situ Hybridization......Page 441
Overview......Page 442
Overview......Page 443
mRNA Expression Profiles......Page 445
References......Page 446
Introduction......Page 451
Amniotic Fluid and Placenta in Developmental Biology......Page 452
Amniotic Fluid and Placenta for Cell Therapy......Page 453
Isolation and Characterization of Progenitor Cells......Page 454
Adipocytes......Page 455
Hepatocytes......Page 457
Neuronal Cells......Page 458
Hepatic Lineage Differentiation Protocol......Page 459
Conclusion......Page 460
References......Page 461
Introduction: Cord Blood Transplantation......Page 464
Hematopoietic Stem and Progenitor Cells......Page 466
Endothelial Progenitor Cells......Page 468
Methods for Enumerating Human Myeloid Progenitor Cells in Umbilical Cord Blood by In Vitro Colony Formation......Page 472
Procedure......Page 473
Procedure......Page 475
Materials/Reagents......Page 476
Plating Procedure for Harvested Colony Culture......Page 477
SAG (Serine/Asparagine/Glutamine)......Page 478
Procedure......Page 479
In Vitro Hematopoietic Stem Cell Assays for Cord Blood......Page 480
Methods for CAFCs and LTC-ICs......Page 481
ML-IC Methods......Page 482
In Vivo Animal Models for Assay of Cord Blood-Derived Hematopoietic Stem Cells......Page 483
Materials and Major Equipment for Transplantation......Page 484
Materials and Major Equipment for Assessment of Engraftment......Page 485
How to Use Collected Data for Human Cell Engraftment to Evaluate the Preceding Assay Assessments......Page 486
Notes......Page 487
Collection of Umbilical Cord Blood......Page 488
Collection of Umbilical Cord Blood......Page 489
Preparation and Culture of Mononuclear Cells......Page 490
Notes......Page 491
References......Page 492
Author Index......Page 499
Subject Index......Page 548
