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 xvPreface xix1 Neural Differentiation of Pluripotent Stem Cells 1Mohan C. Vemuri1.1 Introduction 11.2 NSC Derivation from Rosette Formation of Embryoid Bodies 11.3 Rosette Free NSC in a Monolayer Culture 21.4 Pathways Involved in Neural Tube and Neural Crest Lineages 21.5 Differentiation and Gene Expression in Human Brain Development 31.6 Conclusions 6References 62 The History and Design of Assays for the Identification and Characterization of Neural Stem Cells 9Sharon A. Louis and Brent A. Reynolds2.1 Introduction 92.2 Materials 122.3 Methods 142.4 Notes 21Acknowledgments 23References 233 Culture of Human Neural Stem Cells 25Soojung Shin, Yiping Yan and Mohan C Vemuri3.1 Introduction 253.2 Materials 253.3 Methods 303.4 Notes 333.5 Expected Results 33References 344 GMP4-Compatible Production and Expansion of Human Neural Stem Cells 35David J. Brick, Alexander E. Stover, Hubert E. Nethercott, Maria G. Banuelos and Philip H. Schwartz4.1 Introduction 354.2 Materials 364.3 Methods 404.4 Notes 50Acknowledgments 52References 525 Primary Rat Neural Cultures 55Navjot Kaur5.1 Introduction 555.2 Materials 555.3 Methods 575.4 Notes 59References 596 Cryopreservation of Human Neural Stem and Progenitor Cells 61Profico Daniela Celeste, Sgaravizzi Giada, Projetti Pensi Massimo, Muzi Gianmarco, Ricciolini Claudia, Gelati Maurizio and Vescovi Angelo Luigi6.1 Introduction 616.2 Materials 626.3 Methods 626.4 Notes 65References 657 Cryopreservation and Recovery of Primary Rat Neural Cells 67Rhonda Newman and Navjot Kaur7.1 Introduction 677.2 Materials 677.3 Methods 687.4 Notes 70References 708 Efficient Induction and Scale up Generation of Neural Stem Cells from Human Pluripotent Stem Cells 71Yiping Yan, Soojung Shin and Mohan C. Vemuri8.1 Introduction 718.2 Materials 718.3 Methods 738.4 Notes 78References 799 Directed Differentiation of Human NSC/NPC into Dopaminergic Neurons 81Andrzej Swistowski and Xianmin Zeng9.1 Introduction 819.2 Materials 829.3 Methods 839.4 Notes 88References 8810 In vitro Differentiation of Pluripotent Stem Cells towards either Forebrain GABAergic or Midbrain Dopaminergic Neurons 91Brigham J. Hartley, Bradley Watmuff, Cameron P.J. Hunt, John M. Haynes and Colin W. Pouton10.1 Introduction 9110.2 Materials 9110.3 Methods 9410.4 Notes 9710.5 Typical Results 97References 9811 Generation of Astrocytes from Human Pluripotent Stem Cells using a Defined System 101Atossa Shaltouki and Xianmin Zeng11.1 Introduction 10111.2 Materials and Methods 10111.3 Notes 108References 10812 Directed Differentiation of Human PSC into Oligodendrocytes 111Anu Hyysalo and Susanna Narkilahti12.1 Introduction 11112.2 Materials 11112.3 Methods 11312.4 Notes 116Acknowledgments 117References 11713 Directed Differentiation towards Human Neural Retinal Cells 119Deepak A. Lamba13.1 Introduction 11913.2 Materials 12013.3 Methods 121References 12614 Directed Differentiation of Photoreceptors and Retinal Pigment Epithelium from Adult Mouse Retinal Stem Cells 129Brian G. Ballios, Valeria Marigo and Derek van der Kooy14.1 Introduction 12914.2 Materials 13014.3 Methods 13114.4 Notes 134Acknowledgments 134References 13415 Induction of Schwann Cells from Rat Bone Marrow Mesenchymal Stem Cells 137Shohei Wakao, Masaaki Kitada and Mari Dezawa15.1 Introduction 13715.2 Materials 13815.3 Method 14115.4 Notes 149References 15116 Neural Cell Viability Assays 155Bonnie J. Hanson and Michael K. Hancock16.1 Introduction 15516.2 Materials 15716.3 Methods 15816.4 Notes 165References 16517 Genetic Modification of Pluripotent Stem Cell Derived Human Neural Progenitor Cells 167Jamie M. Chilton and Steven L. Stice17.1 Introduction 16717.2 Materials 16817.3 Methods 17017.4 Notes 176Acknowledgments 178References 17818 Electrophysiology of Neurons 179Christine M. Emnett and Steven Mennerick18.1 Introduction 17918.2 Materials 18018.3 Methods 18118.4 Notes 185References 18719 Induction and Transplantation of Neural Stem Cells from Human Pluripotent Stem Cells in Experimental Models (Rat) of Parkinson’s disease 189Makoto Motono, Asuka Morizane and Jun Takahashi19.1 Introduction 18919.2 Materials 18919.3 Methods 19119.4 Notes 194Acknowledgments 194References 19420 Transplantation of Human Neural Stem Cells and Progenitors in Animal Models of Disease 197Joseph F. Bonner and Itzhak Fischer20.1 Introduction 19720.2 General Principles of Effective Transplantation 19820.3 Different Methods of Transplantation 20420.4 Materials and Method 20520.5 Conclusions 21120.6 Notes 212Acknowledgments 212References 21221 Morphometric Assays for Neural Differentiation and Synaptic Development of Human iPSC-Derived NSC 215Barbara Calabrese, Leif Dehmelt and Shelley Halpain21.1 Introduction 21521.2 Materials 21621.3 Method 21821.4 Notes 222Acknowledgments 223References 22322 BacMam Transduction of Neural Stem Cells 225Rene Quintanilla and Uma Lakshmipathy22.1 Introduction 22522.2 Materials 22522.3 Methods 22722.4 Notes 228Acknowledgments 229References 22923 The Proximity Ligation Assay: A High Throughput Technique for Protein Analysis in Neuroscience 231Michela Zaltieri, PierFranco Spano, Cristina Missale and Arianna Bellucci23.1 Introduction 23123.2 Materials 23223.3 Methods 23223.4 Notes 23823.5 Troubleshooting 238Acknowledgments 240References 24024 Quantification of Micro-RNA Expression in Human Neural Stem Cells using TaqMan Assays 241Xavier Nissan and Alexandra Benchoua24.1 Introduction 24124.2 Materials 24224.3 Methods 24424.4 Notes 248References 24925 Genetic Reporter Cell Lines: Tools for Stem Cell Biology and Drug Discovery 251Cameron P.J. Hunt, Bradley Watmuff, Brigham J. Hartley, Colin W. Pouton and John M. Haynes25.1 Pluripotent Stem Cell Reporter Lines 25125.2 Choice of PSC Genetic Reporter Systems 25125.3 Reporter Systems 25225.4 Multiple Reporter Lines 25425.5 Use of Genetic Reporter Escs to Study Neural Differentiation 255References 260Index 261

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 xvPreface xix1 Neural Differentiation of Pluripotent Stem Cells 1Mohan C. Vemuri1.1 Introduction 11.2 NSC Derivation from Rosette Formation of Embryoid Bodies 11.3 Rosette Free NSC in a Monolayer Culture 21.4 Pathways Involved in Neural Tube and Neural Crest Lineages 21.5 Differentiation and Gene Expression in Human Brain Development 31.6 Conclusions 6References 62 The History and Design of Assays for the Identification and Characterization of Neural Stem Cells 9Sharon A. Louis and Brent A. Reynolds2.1 Introduction 92.2 Materials 122.3 Methods 142.4 Notes 21Acknowledgments 23References 233 Culture of Human Neural Stem Cells 25Soojung Shin, Yiping Yan and Mohan C Vemuri3.1 Introduction 253.2 Materials 253.3 Methods 303.4 Notes 333.5 Expected Results 33References 344 GMP4-Compatible Production and Expansion of Human Neural Stem Cells 35David J. Brick, Alexander E. Stover, Hubert E. Nethercott, Maria G. Banuelos and Philip H. Schwartz4.1 Introduction 354.2 Materials 364.3 Methods 404.4 Notes 50Acknowledgments 52References 525 Primary Rat Neural Cultures 55Navjot Kaur5.1 Introduction 555.2 Materials 555.3 Methods 575.4 Notes 59References 596 Cryopreservation of Human Neural Stem and Progenitor Cells 61Profico Daniela Celeste, Sgaravizzi Giada, Projetti Pensi Massimo, Muzi Gianmarco, Ricciolini Claudia, Gelati Maurizio and Vescovi Angelo Luigi6.1 Introduction 616.2 Materials 626.3 Methods 626.4 Notes 65References 657 Cryopreservation and Recovery of Primary Rat Neural Cells 67Rhonda Newman and Navjot Kaur7.1 Introduction 677.2 Materials 677.3 Methods 687.4 Notes 70References 708 Efficient Induction and Scale up Generation of Neural Stem Cells from Human Pluripotent Stem Cells 71Yiping Yan, Soojung Shin and Mohan C. Vemuri8.1 Introduction 718.2 Materials 718.3 Methods 738.4 Notes 78References 799 Directed Differentiation of Human NSC/NPC into Dopaminergic Neurons 81Andrzej Swistowski and Xianmin Zeng9.1 Introduction 819.2 Materials 829.3 Methods 839.4 Notes 88References 8810 In vitro Differentiation of Pluripotent Stem Cells towards either Forebrain GABAergic or Midbrain Dopaminergic Neurons 91Brigham J. Hartley, Bradley Watmuff, Cameron P.J. Hunt, John M. Haynes and Colin W. Pouton10.1 Introduction 9110.2 Materials 9110.3 Methods 9410.4 Notes 9710.5 Typical Results 97References 9811 Generation of Astrocytes from Human Pluripotent Stem Cells using a Defined System 101Atossa Shaltouki and Xianmin Zeng11.1 Introduction 10111.2 Materials and Methods 10111.3 Notes 108References 10812 Directed Differentiation of Human PSC into Oligodendrocytes 111Anu Hyysalo and Susanna Narkilahti12.1 Introduction 11112.2 Materials 11112.3 Methods 11312.4 Notes 116Acknowledgments 117References 11713 Directed Differentiation towards Human Neural Retinal Cells 119Deepak A. Lamba13.1 Introduction 11913.2 Materials 12013.3 Methods 121References 12614 Directed Differentiation of Photoreceptors and Retinal Pigment Epithelium from Adult Mouse Retinal Stem Cells 129Brian G. Ballios, Valeria Marigo and Derek van der Kooy14.1 Introduction 12914.2 Materials 13014.3 Methods 13114.4 Notes 134Acknowledgments 134References 13415 Induction of Schwann Cells from Rat Bone Marrow Mesenchymal Stem Cells 137Shohei Wakao, Masaaki Kitada and Mari Dezawa15.1 Introduction 13715.2 Materials 13815.3 Method 14115.4 Notes 149References 15116 Neural Cell Viability Assays 155Bonnie J. Hanson and Michael K. Hancock16.1 Introduction 15516.2 Materials 15716.3 Methods 15816.4 Notes 165References 16517 Genetic Modification of Pluripotent Stem Cell Derived Human Neural Progenitor Cells 167Jamie M. Chilton and Steven L. Stice17.1 Introduction 16717.2 Materials 16817.3 Methods 17017.4 Notes 176Acknowledgments 178References 17818 Electrophysiology of Neurons 179Christine M. Emnett and Steven Mennerick18.1 Introduction 17918.2 Materials 18018.3 Methods 18118.4 Notes 185References 18719 Induction and Transplantation of Neural Stem Cells from Human Pluripotent Stem Cells in Experimental Models (Rat) of Parkinson’s disease 189Makoto Motono, Asuka Morizane and Jun Takahashi19.1 Introduction 18919.2 Materials 18919.3 Methods 19119.4 Notes 194Acknowledgments 194References 19420 Transplantation of Human Neural Stem Cells and Progenitors in Animal Models of Disease 197Joseph F. Bonner and Itzhak Fischer20.1 Introduction 19720.2 General Principles of Effective Transplantation 19820.3 Different Methods of Transplantation 20420.4 Materials and Method 20520.5 Conclusions 21120.6 Notes 212Acknowledgments 212References 21221 Morphometric Assays for Neural Differentiation and Synaptic Development of Human iPSC-Derived NSC 215Barbara Calabrese, Leif Dehmelt and Shelley Halpain21.1 Introduction 21521.2 Materials 21621.3 Method 21821.4 Notes 222Acknowledgments 223References 22322 BacMam Transduction of Neural Stem Cells 225Rene Quintanilla and Uma Lakshmipathy22.1 Introduction 22522.2 Materials 22522.3 Methods 22722.4 Notes 228Acknowledgments 229References 22923 The Proximity Ligation Assay: A High Throughput Technique for Protein Analysis in Neuroscience 231Michela Zaltieri, PierFranco Spano, Cristina Missale and Arianna Bellucci23.1 Introduction 23123.2 Materials 23223.3 Methods 23223.4 Notes 23823.5 Troubleshooting 238Acknowledgments 240References 24024 Quantification of Micro-RNA Expression in Human Neural Stem Cells using TaqMan Assays 241Xavier Nissan and Alexandra Benchoua24.1 Introduction 24124.2 Materials 24224.3 Methods 24424.4 Notes 248References 24925 Genetic Reporter Cell Lines: Tools for Stem Cell Biology and Drug Discovery 251Cameron P.J. Hunt, Bradley Watmuff, Brigham J. Hartley, Colin W. Pouton and John M. Haynes25.1 Pluripotent Stem Cell Reporter Lines 25125.2 Choice of PSC Genetic Reporter Systems 25125.3 Reporter Systems 25225.4 Multiple Reporter Lines 25425.5 Use of Genetic Reporter Escs to Study Neural Differentiation 255References 260Index 261

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 xvPreface xix1 Neural Differentiation of Pluripotent Stem Cells 1Mohan C. Vemuri1.1 Introduction 11.2 NSC Derivation from Rosette Formation of Embryoid Bodies 11.3 Rosette Free NSC in a Monolayer Culture 21.4 Pathways Involved in Neural Tube and Neural Crest Lineages 21.5 Differentiation and Gene Expression in Human Brain Development 31.6 Conclusions 6References 62 The History and Design of Assays for the Identification and Characterization of Neural Stem Cells 9Sharon A. Louis and Brent A. Reynolds2.1 Introduction 92.2 Materials 122.3 Methods 142.4 Notes 21Acknowledgments 23References 233 Culture of Human Neural Stem Cells 25Soojung Shin, Yiping Yan and Mohan C Vemuri3.1 Introduction 253.2 Materials 253.3 Methods 303.4 Notes 333.5 Expected Results 33References 344 GMP4-Compatible Production and Expansion of Human Neural Stem Cells 35David J. Brick, Alexander E. Stover, Hubert E. Nethercott, Maria G. Banuelos and Philip H. Schwartz4.1 Introduction 354.2 Materials 364.3 Methods 404.4 Notes 50Acknowledgments 52References 525 Primary Rat Neural Cultures 55Navjot Kaur5.1 Introduction 555.2 Materials 555.3 Methods 575.4 Notes 59References 596 Cryopreservation of Human Neural Stem and Progenitor Cells 61Profico Daniela Celeste, Sgaravizzi Giada, Projetti Pensi Massimo, Muzi Gianmarco, Ricciolini Claudia, Gelati Maurizio and Vescovi Angelo Luigi6.1 Introduction 616.2 Materials 626.3 Methods 626.4 Notes 65References 657 Cryopreservation and Recovery of Primary Rat Neural Cells 67Rhonda Newman and Navjot Kaur7.1 Introduction 677.2 Materials 677.3 Methods 687.4 Notes 70References 708 Efficient Induction and Scale up Generation of Neural Stem Cells from Human Pluripotent Stem Cells 71Yiping Yan, Soojung Shin and Mohan C. Vemuri8.1 Introduction 718.2 Materials 718.3 Methods 738.4 Notes 78References 799 Directed Differentiation of Human NSC/NPC into Dopaminergic Neurons 81Andrzej Swistowski and Xianmin Zeng9.1 Introduction 819.2 Materials 829.3 Methods 839.4 Notes 88References 8810 In vitro Differentiation of Pluripotent Stem Cells towards either Forebrain GABAergic or Midbrain Dopaminergic Neurons 91Brigham J. Hartley, Bradley Watmuff, Cameron P.J. Hunt, John M. Haynes and Colin W. Pouton10.1 Introduction 9110.2 Materials 9110.3 Methods 9410.4 Notes 9710.5 Typical Results 97References 9811 Generation of Astrocytes from Human Pluripotent Stem Cells using a Defined System 101Atossa Shaltouki and Xianmin Zeng11.1 Introduction 10111.2 Materials and Methods 10111.3 Notes 108References 10812 Directed Differentiation of Human PSC into Oligodendrocytes 111Anu Hyysalo and Susanna Narkilahti12.1 Introduction 11112.2 Materials 11112.3 Methods 11312.4 Notes 116Acknowledgments 117References 11713 Directed Differentiation towards Human Neural Retinal Cells 119Deepak A. Lamba13.1 Introduction 11913.2 Materials 12013.3 Methods 121References 12614 Directed Differentiation of Photoreceptors and Retinal Pigment Epithelium from Adult Mouse Retinal Stem Cells 129Brian G. Ballios, Valeria Marigo and Derek van der Kooy14.1 Introduction 12914.2 Materials 13014.3 Methods 13114.4 Notes 134Acknowledgments 134References 13415 Induction of Schwann Cells from Rat Bone Marrow Mesenchymal Stem Cells 137Shohei Wakao, Masaaki Kitada and Mari Dezawa15.1 Introduction 13715.2 Materials 13815.3 Method 14115.4 Notes 149References 15116 Neural Cell Viability Assays 155Bonnie J. Hanson and Michael K. Hancock16.1 Introduction 15516.2 Materials 15716.3 Methods 15816.4 Notes 165References 16517 Genetic Modification of Pluripotent Stem Cell Derived Human Neural Progenitor Cells 167Jamie M. Chilton and Steven L. Stice17.1 Introduction 16717.2 Materials 16817.3 Methods 17017.4 Notes 176Acknowledgments 178References 17818 Electrophysiology of Neurons 179Christine M. Emnett and Steven Mennerick18.1 Introduction 17918.2 Materials 18018.3 Methods 18118.4 Notes 185References 18719 Induction and Transplantation of Neural Stem Cells from Human Pluripotent Stem Cells in Experimental Models (Rat) of Parkinson’s disease 189Makoto Motono, Asuka Morizane and Jun Takahashi19.1 Introduction 18919.2 Materials 18919.3 Methods 19119.4 Notes 194Acknowledgments 194References 19420 Transplantation of Human Neural Stem Cells and Progenitors in Animal Models of Disease 197Joseph F. Bonner and Itzhak Fischer20.1 Introduction 19720.2 General Principles of Effective Transplantation 19820.3 Different Methods of Transplantation 20420.4 Materials and Method 20520.5 Conclusions 21120.6 Notes 212Acknowledgments 212References 21221 Morphometric Assays for Neural Differentiation and Synaptic Development of Human iPSC-Derived NSC 215Barbara Calabrese, Leif Dehmelt and Shelley Halpain21.1 Introduction 21521.2 Materials 21621.3 Method 21821.4 Notes 222Acknowledgments 223References 22322 BacMam Transduction of Neural Stem Cells 225Rene Quintanilla and Uma Lakshmipathy22.1 Introduction 22522.2 Materials 22522.3 Methods 22722.4 Notes 228Acknowledgments 229References 22923 The Proximity Ligation Assay: A High Throughput Technique for Protein Analysis in Neuroscience 231Michela Zaltieri, PierFranco Spano, Cristina Missale and Arianna Bellucci23.1 Introduction 23123.2 Materials 23223.3 Methods 23223.4 Notes 23823.5 Troubleshooting 238Acknowledgments 240References 24024 Quantification of Micro-RNA Expression in Human Neural Stem Cells using TaqMan Assays 241Xavier Nissan and Alexandra Benchoua24.1 Introduction 24124.2 Materials 24224.3 Methods 24424.4 Notes 248References 24925 Genetic Reporter Cell Lines: Tools for Stem Cell Biology and Drug Discovery 251Cameron P.J. Hunt, Bradley Watmuff, Brigham J. Hartley, Colin W. Pouton and John M. Haynes25.1 Pluripotent Stem Cell Reporter Lines 25125.2 Choice of PSC Genetic Reporter Systems 25125.3 Reporter Systems 25225.4 Multiple Reporter Lines 25425.5 Use of Genetic Reporter Escs to Study Neural Differentiation 255References 260Index 261