Cover -- TItle Page -- Copyright -- Table of Contents -- Contributors -- Preface -- Chapter 1 Neural Differentiation of Pluripotent Stem Cells -- 1.1 Introduction -- 1.2 NSC Derivation from Rosette Formation of Embryoid Bodies -- 1.3 Rosette Free NSC in a Monolayer Culture -- 1.4 Pathways Involved in Neural Tube and Neural Crest Lineages -- 1.5 Differentiation and Gene Expression in Human Brain Development -- 1.5.1 Differentiation to Forebrain Neurons from Pluripotent Stem Cells -- 1.5.2 Differentiation to Midbrain Dopaminergic Neurons from Pluripotent Stem Cells -- 1.5.3 Differentiation to Motor Neurons from Human Pluripotent Stem Cells -- 1.6 Conclusions -- References -- Chapter 2 The History and Design of Assays for the Identification and Characterization of Neural Stem Cells -- 2.1 Introduction -- 2.1.1 Stem Cells -- 2.1.2 Stem Cells in the Central Nervous System -- 2.1.3 The Neurosphere Assay -- 2.2 Materials -- 2.2.1 General Equipment -- 2.2.2 Tissue Culture Equipment (Neurosphere Culture) -- 2.2.3 Media, Supplements and Associated Reagents for Neurosphere Cultures -- 2.2.4 Media, Supplements, and Tissue Culture Equipment for Neural Colony Forming Cell Assay -- 2.3 Methods -- 2.3.1 Neurosphere Cultures from Primary Embryonic or Adult CNS Tissues -- 2.3.2 Neural Colony Forming Cell (NCFC) Assay for Enumerating Neural Stem Cells and Neural Progenitor Cells -- 2.3.3 Categorizing NCFC Colonies and Scoring NCFC Colonies -- 2.3.4 Application of the NCFC Assay for Estimation of NSCs or Neural Progenitors -- 2.4 Notes -- Acknowledgments -- References -- Chapter 3 Culture of Human Neural Stem Cells -- 3.1 Introduction -- 3.2 Materials -- 3.2.1 Reagents List -- 3.2.2 Preparing Stock Solution and Complete Medium -- 3.2.3 Preparing Matrix Coat Culture Vessels -- 3.3 Methods.

3.3.1 Thawing Frozen Neural Stem Cells -- 3.3.2 Passaging Neural Stem Cells (Adherent Culture) -- 3.3.3 Passaging Neural Stem Cells (Suspension Culture) -- 3.3.4 Cryopreserving Neural Stem Cells -- 3.4 Notes -- 3.5 Expected Results -- References -- Chapter 4 GMP4-Compatible Production and Expansion of Human Neural Stem Cells -- 4.1 Introduction -- 4.2 Materials -- 4.2.1 PSC Transition Reagents and Supplies -- 4.2.2 NSC Induction Reagents and Supplies -- 4.2.3 NSC Immunocytochemical Characterization -- 4.2.4 NSC Flow Cytometry Characterization -- 4.2.5 NSC Expansion and Cryopreservation -- 4.3 Methods -- 4.3.1 Transitioning PSCs from Co-Culture with Feeder Cells to StemPro Defined Culture on Matrigel -- 4.3.2 Preparing Matrigel -- 4.3.3 Plating Matrigel -- 4.3.4 Single-Cell Passaging of PSCs onto Matrigel with Accutase -- 4.3.5 Defined Chemical (DMH1 and SB) NSC Induction from Defined Adherent PSC StemPro Cultures -- 4.3.6 NSC Characterization by Immunocytochemistry -- 4.3.7 NSC Characterization by Flow Cytometry -- 4.3.8 NSC Expansion and Cryopreservation -- 4.3.9 Recovering NSCs in modified GM -- 4.4 Notes -- Acknowledgments -- References -- Chapter 5 Primary Rat Neural Cultures -- 5.1 Introduction -- 5.2 Materials -- 5.2.1 Media and Reagents -- 5.2.2 Equipment -- 5.3 Methods -- 5.3.1 Plate Coating -- 5.3.2 Homogenous Cell Preparation -- 5.3.3 Cells Plating -- 5.3.4 Neuronal Characterization -- 5.4 Notes -- References -- Chapter 6 Cryopreservation of Human Neural Stem and Progenitor Cells -- 6.1 Introduction -- 6.2 Materials -- 6.3 Methods -- 6.3.1 Freezing -- 6.3.2 Thawing -- 6.3.3 Differentiation -- 6.4 Notes -- References -- Chapter 7 Cryopreservation and Recovery of Primary Rat Neural Cells -- 7.1 Introduction -- 7.2 Materials -- 7.3 Methods -- 7.3.1 Media Preparation -- 7.3.2 Homogenous Cell Preparation.

7.3.3 Cryopreservation of Neural Cells -- 7.3.4 Recovery of Frozen Neural Cells -- 7.4 Notes -- References -- Chapter 8 Efficient Induction and Scale up Generation of Neural Stem Cells from Human Pluripotent Stem Cells -- 8.1 Introduction -- 8.2 Materials -- 8.3 Methods -- 8.3.1 Neural Induction -- 8.3.2 NSC Expansion -- 8.3.3 Cryo-Preservation of NSCs -- 8.3.4 Recovery of Cryo-Preserved NSCs -- 8.3.5 Characterization of NSCs -- 8.4 Notes -- References -- Chapter 9 Directed Differentiation of Human NSC/NPC into Dopaminergic Neurons -- 9.1 Introduction -- 9.2 Materials -- 9.2.1 Cell Culture Reagents -- 9.2.2 Antibodies -- 9.2.3 Cell Culture Media -- 9.3 Methods -- 9.3.1 Coating Cell Culture Dishes with Substrates -- 9.3.2 Generation of NSCs from Human Pluripotent Stem Cells -- 9.3.3 Enzymatic Passage of NSCs -- 9.3.4 Banking of NSCs -- 9.3.5 Recovering NSC from Frozen Stocks -- 9.3.6 Characterization by Immunocytochemistry (Double Labeling of β III-Tubulin and Tyrosine Hydroxylase: TH) -- 9.4 Notes -- References -- Chapter 10 In vitro Differentiation of Pluripotent Stem Cells towards either Forebrain GABAergic or Midbrain Dopaminergic Neurons -- 10.1 Introduction -- 10.2 Materials -- 10.2.1 Equipment -- 10.2.2 PSC Culture -- 10.2.3 Forebrain GABAergic Differentiation -- 10.2.4 Midbrain Dopaminergic Differentiation -- 10.3 Methods -- 10.3.1 PSC Maintenance -- 10.3.2 GABAergic Differentiation -- 10.3.3 Dopaminergic Differentiation -- 10.4 Notes -- 10.5 Typical Results -- References -- Chapter 11 Generation of Astrocytes from Human Pluripotent Stem Cells using a Defined System -- 11.1 Introduction -- 11.2 Materials and Methods -- 11.2.1 Antibodies -- 11.2.2 Cell Culture Media -- 11.2.3 Coating Cell Culture Dishes with Substrates -- 11.2.4 Thawing and Expansion of ESC/IPSC-derived NSC.

11.2.5 Enzymatic Passage of NSC -- 11.2.6 Cryopreservation of NSC -- 11.2.7 Astrocyte Differentiation of NSC in Defined Medium -- 11.2.8 Expansion and Cryopreservation of Astrocytes -- 11.3 Notes -- References -- Chapter 12 Directed Differentiation of Human PSC into Oligodendrocytes -- 12.1 Introduction -- 12.2 Materials -- 12.2.1 Oligodendrocyte Differentiation -- 12.2.2 Coating Mixture for Adherent Oligodendrocyte Cultures -- 12.2.3 Immunocytochemical Characterization of Differentiating Oligodendrocytes -- 12.2.4 Flow Cytometric (FACS) Characterization of Differentiating Oligodendrocytes -- 12.2.5 Genetic Characterization of Differentiating Oligodendrocytes -- 12.3 Methods -- 12.3.1 Human Pluripotent Stem Cell Culturing -- 12.3.2 Oligodendrocyte Differentiation -- 12.3.3 Immunocytochemical and Flow Cytometric Characterization of Differentiating Oligodendrocytes -- 12.3.4 Genetic Characterization of Differentiating Oligodendrocytes -- 12.4 Notes -- Acknowledgments -- References -- Chapter 13 Directed Differentiation towards Human Neural Retinal Cells -- 13.1 Introduction -- 13.2 Materials -- 13.2.1 Culture of Undifferentiated Human Embryonic Stem Cells -- 13.2.2 Generation of Retinal Cells from Undifferentiated Human ESCs -- 13.2.3 Coating for Adherent Culture of Cells -- 13.3 Methods -- 13.3.1 Substrate for Adherent Culture of Cells -- 13.3.2 Culture of Undifferentiated Human Embryonic Stem Cells -- 13.3.3 Generation of Retinal Cells from Undifferentiated Human ES Cells -- 13.3.4 Passaging of hESC-Derived Retinal Cells -- 13.3.5 Analysis of Retinal Differentiation using Quantitative Real-Time PCR -- 13.3.6 Analysis of Retinal Differentiation using Fluorescent Immunohistochemistry -- References.

Chapter 14 Directed Differentiation of Photoreceptors and Retinal Pigment Epithelium from Adult Mouse Retinal Stem Cells -- 14.1 Introduction -- 14.2 Materials -- 14.2.1 Reagents for RSC Dissection -- 14.2.2 Reagents for RSC-Derived Photoreceptor Differentiation -- 14.2.3 Reagents for RSC-Derived RPE Differentiation -- 14.3 Methods -- 14.3.1 Mouse RSC Dissection Protocol -- 14.3.2 RSC-Derived Photoreceptor Differentiation -- 14.3.3 RSC-derived RPE -- 14.4 Notes -- Acknowledgments -- References -- Chapter 15 Induction of Schwann Cells from Rat Bone Marrow Mesenchymal Stem Cells -- 15.1 Introduction -- 15.2 Materials -- 15.2.1 Preparation of Adult Rat BMSCs -- 15.2.2 Preparation of Adult Monkey BMSCs -- 15.2.3 Preparation of Adult Human BMSCs -- 15.2.4 In vitro induction of Schwann cells from BMSCs -- 15.2.5 Immunocytochemistry -- 15.2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) -- 15.3 Method -- 15.3.1 Preparation of Adult Rat BMSCs -- 15.3.2 Preparation of Adult Monkey BMSCs -- 15.3.3 Preparation of Adult Human BMSCs -- 15.3.4 In vitro Induction of Schwann Cells from BMSCs -- 15.3.5 Immunocytochemistry -- 15.3.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) -- 15.4 Notes -- References -- Chapter 16 Neural Cell Viability Assays -- 16.1 Introduction -- 16.2 Materials -- 16.2.1 Complete Media -- 16.3 Methods -- 16.3.1 Controls -- 16.3.2 Plating Primary Rat Hippocampus or Cortex Neurons -- 16.3.3 Maintenance of Primary Rat Hippocampus or Cortex Neuronal Cultures -- 16.3.4 Plating Neuroscreen-1TM Cells -- 16.3.5 Viability Assays -- 16.4 Notes -- References -- Chapter 17 Genetic Modification of Pluripotent Stem Cell Derived Human Neural Progenitor Cells -- 17.1 Introduction -- 17.2 Materials -- 17.2.1 Human Neural Progenitor Cells -- 17.2.2 Cell Culture Reagents and Supplies.

17.2.3 Transfection Reagents.