Human Adult Stem Cells (eBook)

Preface 6
Contents 7
Contributors 9
Chapter 1 Neural Progenitors 12
1.1 Primary Derivation and Selection of hNPC 12
1.1.1 Determination of Fetal Age and Developmental Stage 13
1.1.2 Dissection and Digestion of Human Fetal Periventricular Zone 15
1.1.3 Establishing Primary hNPC Cultures 17
1.2 Long Term Expansion and Maintenance of hNPC 22
1.2.1 Freezing hNPC Aliquots 23
1.2.2 Thawing Cryopreserved hNPC 24
1.2.3 Establishing Secondary hNPC Cultures 25
1.2.4 General Feeding of hNPC 25
1.2.5 Dissociation of hNPC 27
1.2.6 Replating Dissociated hNPC for General Expansion 31
1.3 Preservation of Conditioned Medium 31
1.4 “What About Neurospheres?” 32
1.5 Characterization of Undifferentiated hNPC 32
1.6 Functional Diagnostics for Multipotency 34
1.6.1 In Vitro Differentiation 35
1.6.2 In Vivo Differentiation 36
1.7 Equipment, Supplies, Chemicals, Reagents, and Media 37
1.7.1 Equipment and Consumable Supplies 37
1.7.2 Chemicals and Reagents 37
1.7.3 Media 38
1.7.4 Preparation Notes 38
1.7.5 Normal Plating Volumes 39
1.8 Introduction to the Tissue Culture (TC) Hood 40
1.8.1 Preparation of the Tissue Culture (TC) Hood 40
1.8.2 Notes on Sterility and Methods of Sterilization 40
References 42
Chapter 2 Multipotent Stromal Cells (hMSCs) 56
2.1 Introduction 56
2.2 Bone Marrow Procurement and Processing 58
2.3 Freezing and Recovery of MSCs 63
2.4 Expansion of Harvested MSCs in Flasks or Cell Culture Chambers (CCCs) 65
2.5 Characterization of MSCs – Colony Forming Unit Assay and Differentiation 72
2.6 hMSC Surface Epitope Analysis by Flow Cytometry 77
2.7 Potential Therapeutic Applications 79
2.8 Summary 80
References 81
Chapter 3 Endothelium 84
3.1 Introduction and Methodology for Tissue EPC Culture 85
3.2 Methodology for Isolation of Circulating EPCs 89
3.3 Protocols for Evaluating the Function of EPCs 91
3.4 Prospective of Development of EPC Culture System 95
3.5 Concluding Remarks 97
References 97
Chapter 4 Lung 101
4.1 Foreword 101
4.2 Ontogeny, Morphological and Spatial Layers of the Human Lung 102
4.3 “Stemness” and the Adult Human Lung 106
4.4 Protocols 110
References 121
Chapter 5 Eye 123
5.1 Limbal Epithelial Stem Cells Maria Notara, Julie T Daniels 123
5.2 Culture of Human Corneal Stromal Stem Cells Yiqin Du, James L. Funderburgh 131
5.3 Neural Differentiation 139
5.4 Isolation and Culture of M¨ uller Stem Cells from the Adult Neural Retina of the Human Eye G. Astrid Limb 140
References 147
Chapter 6 Colon 153
6.1 Identification of Stem Cell Markers in Colon Tissues 155
6.2 Isolation of Colon Stem Cells 159
6.3 Propagation of Colon Stem Cells 163
6.4 Differentiation of Cells from Colon Spheres 164
Chapter 7 Spermatogonia 167
7.1 Introduction 167
7.2 Protocols Developed for Mouse SSC Culture 168
7.3 Modifications of Mouse Protocols for the Application of SSC Culture from Other Species 174
7.4 Issues to Be Considered for Development of Human SSC Culture 175
7.5 Feeder-Free Culture and Bioreactor: Two Techniques that Need to Be Explored in Animal Models for Clinical Application of Human SSC Culture 177
7.6 Concluding Remarks 178
References 179
Chapter 8 Hair Follicle Pluripotent Stem (hfPS) Cells 181
8.1 Introduction 182
8.2 Discussion 185
8.3 Conclusions 186
8.4 Materials and Methods 187
References 190
Chapter 9 Pancreas 193
9.1 Pancreas Development and Function 194
9.2 Regeneration of ß Cells Occurs Physiologicallyand Pathophysiologically 196
9.3 Evidence that ß Cells Are Generated from PSC 197
9.4 Evidence that Islet ß Cells Are Capableof Self-Replication 200
9.5 The Identity of PSC is Inconclusive 201
9.6 Future Directions for PSC Research 202
References 203
Chapter 10 Prostate 207
10.1 Properties of Stem Cells 207
10.2 Anatomy of the Prostate Gland 208
10.4 Sources of Human Prostate Tissue 209
10.5 Tissue Handling 210
10.6 Tissue Processing 210
10.7 Identification and Characterization of Putative Prostate Stem Cells 210
10.8 Cell Sorting 211
10.9 In Vitro Cell Culture Techniques for Maintenance and Characterization of Stem Cells 211
10.10 In Vitro Differentiation Potential of Self-Renewing Stem Cells 212
10.11 Molecular Characterization of Putative Prostate Stem Cells 212
10.12 Techniques for In Vivo Characterization of Putative Human Prostate Stem Cells 214
References 215