Neural Stem Cell Assays

Neural Stem Cell Assays provides a detailed and comprehensive review of the basic methods for neural stem cell cultures. Including an overview of progress in the field over the past decade, Neural Stem Cell Assays is a one-stop reference for consistent methods and reliable tools that span the entire assay work flow, from isolation or generation of neural stem cells to characterization, manipulation and final application of neural stem cells in disease paradigms such as Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis.

An excellent source of information for academic, pharmaceutical and biotechnology researchers who are new to the neural stem cell field, Neural Stem Cell Assays is an invaluable to experienced users who wish to integrate newly developed tools and technologies into their workflow. The book also covers important course material for students at the undergraduate and graduate level who are learning the basics of neural stem cell cultures, and differentiation to sub-neural lineages.

Navjot Kaur, Ph.D, is Staff Scientist in the Life Technologies division of the Invitrogen Corporation. His research experience covers a wide range of cell biology, protein biochemistry and molecular biology concepts, with special expertise in cell culture and cryopreservation media development, cell-based assays, primary cells, and stable cell lines.

Mohan C. Vemuri, Ph.D.,MBA is director of stem cell research and development in the Life Technologies division of Invitrogen & Applied Biosystems. The author of designs of peer-reviewed articles in the field, Dr. Vemuri is also a co-editor of three books on stem cells and stem cell cultures.

Contributors xv

Preface xix

1 Neural Differentiation of Pluripotent Stem Cells 1
Mohan C. Vemuri

1.1 Introduction 1

1.2 NSC Derivation from Rosette Formation of Embryoid Bodies 1

1.3 Rosette Free NSC in a Monolayer Culture 2

1.4 Pathways Involved in Neural Tube and Neural Crest Lineages 2

1.5 Differentiation and Gene Expression in Human Brain Development 3

1.6 Conclusions 6

References 6

2 The History and Design of Assays for the Identification and Characterization of Neural Stem Cells 9
Sharon A. Louis and Brent A. Reynolds

2.1 Introduction 9

2.2 Materials 12

2.3 Methods 14

2.4 Notes 21

Acknowledgments 23

References 23

3 Culture of Human Neural Stem Cells 25
Soojung Shin, Yiping Yan and Mohan C Vemuri

3.1 Introduction 25

3.2 Materials 25

3.3 Methods 30

3.4 Notes 33

3.5 Expected Results 33

References 34

4 GMP4-Compatible Production and Expansion of Human Neural Stem Cells 35
David J. Brick, Alexander E. Stover, Hubert E. Nethercott, Maria G. Banuelos and Philip H. Schwartz

4.1 Introduction 35

4.2 Materials 36

4.3 Methods 40

4.4 Notes 50

Acknowledgments 52

References 52

5 Primary Rat Neural Cultures 55
Navjot Kaur

5.1 Introduction 55

5.2 Materials 55

5.3 Methods 57

5.4 Notes 59

References 59

6 Cryopreservation of Human Neural Stem and Progenitor Cells 61
Profico Daniela Celeste, Sgaravizzi Giada, Projetti Pensi Massimo, Muzi Gianmarco, Ricciolini Claudia, Gelati Maurizio and Vescovi Angelo Luigi

6.1 Introduction 61

6.2 Materials 62

6.3 Methods 62

6.4 Notes 65

References 65

7 Cryopreservation and Recovery of Primary Rat Neural Cells 67
Rhonda Newman and Navjot Kaur

7.1 Introduction 67

7.2 Materials 67

7.3 Methods 68

7.4 Notes 70

References 70

8 Efficient Induction and Scale up Generation of Neural Stem Cells from Human Pluripotent Stem Cells 71
Yiping Yan, Soojung Shin and Mohan C. Vemuri

8.1 Introduction 71

8.2 Materials 71

8.3 Methods 73

8.4 Notes 78

References 79

9 Directed Differentiation of Human NSC/NPC into Dopaminergic Neurons 81
Andrzej Swistowski and Xianmin Zeng

9.1 Introduction 81

9.2 Materials 82

9.3 Methods 83

9.4 Notes 88

References 88

10 In vitro Differentiation of Pluripotent Stem Cells towards either Forebrain GABAergic or Midbrain Dopaminergic Neurons 91
Brigham J. Hartley, Bradley Watmuff, Cameron P.J. Hunt, John M. Haynes and Colin W. Pouton

10.1 Introduction 91

10.2 Materials 91

10.3 Methods 94

10.4 Notes 97

10.5 Typical Results 97

References 98

11 Generation of Astrocytes from Human Pluripotent Stem Cells using a Defined System 101
Atossa Shaltouki and Xianmin Zeng

11.1 Introduction 101

11.2 Materials and Methods 101

11.3 Notes 108

References 108

12 Directed Differentiation of Human PSC into Oligodendrocytes 111
Anu Hyysalo and Susanna Narkilahti

12.1 Introduction 111

12.2 Materials 111

12.3 Methods 113

12.4 Notes 116

Acknowledgments 117

References 117

13 Directed Differentiation towards Human Neural Retinal Cells 119
Deepak A. Lamba

13.1 Introduction 119

13.2 Materials 120

13.3 Methods 121

References 126

14 Directed Differentiation of Photoreceptors and Retinal Pigment Epithelium from Adult Mouse Retinal Stem Cells 129
Brian G. Ballios, Valeria Marigo and Derek van der Kooy

14.1 Introduction 129

14.2 Materials 130

14.3 Methods 131

14.4 Notes 134

Acknowledgments 134

References 134

15 Induction of Schwann Cells from Rat Bone Marrow Mesenchymal Stem Cells 137
Shohei Wakao, Masaaki Kitada and Mari Dezawa

15.1 Introduction 137

15.2 Materials 138

15.3 Method 141

15.4 Notes 149

References 151

16 Neural Cell Viability Assays 155
Bonnie J. Hanson and Michael K. Hancock

16.1 Introduction 155

16.2 Materials 157

16.3 Methods 158

16.4 Notes 165

References 165

17 Genetic Modification of Pluripotent Stem Cell Derived Human Neural Progenitor Cells 167
Jamie M. Chilton and Steven L. Stice

17.1 Introduction 167

17.2 Materials 168

17.3 Methods 170

17.4 Notes 176

Acknowledgments 178

References 178

18 Electrophysiology of Neurons 179
Christine M. Emnett and Steven Mennerick

18.1 Introduction 179

18.2 Materials 180

18.3 Methods 181

18.4 Notes 185

References 187

19 Induction and Transplantation of Neural Stem Cells from Human Pluripotent Stem Cells in Experimental Models (Rat) of Parkinson’s disease 189
Makoto Motono, Asuka Morizane and Jun Takahashi

19.1 Introduction 189

19.2 Materials 189

19.3 Methods 191

19.4 Notes 194

Acknowledgments 194

References 194

20 Transplantation of Human Neural Stem Cells and Progenitors in Animal Models of Disease 197
Joseph F. Bonner and Itzhak Fischer

20.1 Introduction 197

20.2 General Principles of Effective Transplantation 198

20.3 Different Methods of Transplantation 204

20.4 Materials and Method 205

20.5 Conclusions 211

20.6 Notes 212

Acknowledgments 212

References 212

21 Morphometric Assays for Neural Differentiation and Synaptic Development of Human iPSC-Derived NSC 215
Barbara Calabrese, Leif Dehmelt and Shelley Halpain

21.1 Introduction 215

21.2 Materials 216

21.3 Method 218

21.4 Notes 222

Acknowledgments 223

References 223

22 BacMam Transduction of Neural Stem Cells 225
Rene Quintanilla and Uma Lakshmipathy

22.1 Introduction 225

22.2 Materials 225

22.3 Methods 227

22.4 Notes 228

Acknowledgments 229

References 229

23 The Proximity Ligation Assay: A High Throughput Technique for Protein Analysis in Neuroscience 231
Michela Zaltieri, PierFranco Spano, Cristina Missale and Arianna Bellucci

23.1 Introduction 231

23.2 Materials 232

23.3 Methods 232

23.4 Notes 238

23.5 Troubleshooting 238

Acknowledgments 240

References 240

24 Quantification of Micro-RNA Expression in Human Neural Stem Cells using TaqMan Assays 241
Xavier Nissan and Alexandra Benchoua

24.1 Introduction 241

24.2 Materials 242

24.3 Methods 244

24.4 Notes 248

References 249

25 Genetic Reporter Cell Lines: Tools for Stem Cell Biology and Drug Discovery 251
Cameron P.J. Hunt, Bradley Watmuff, Brigham J. Hartley, Colin W. Pouton and John M. Haynes

25.1 Pluripotent Stem Cell Reporter Lines 251

25.2 Choice of PSC Genetic Reporter Systems 251

25.3 Reporter Systems 252

25.4 Multiple Reporter Lines 254

25.5 Use of Genetic Reporter Escs to Study Neural Differentiation 255

References 260

Index 261

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