Stem Cell-Based Neural Model Systems for Brain Disorders

Preface
Contents
Contributors
Chapter 1: Generation of Cerebral Cortical Neurons from Human Pluripotent Stem Cells in 3D Culture
1 Introduction
2 Materials
2.1 hPSC Maintenance
2.2 Cerebral Cortical Neuron Differentiation from hPSCs
3 Methods
3.1 hPSC Maintenance
3.1.1 Thaw hPSCs from a Cell Bank
3.1.2 hPSC Culture and Passage
3.2 Differentiation of Forebrain Cortical Neurons in 3D Suspension Culture (Fig. 1)
3.2.1 Neural Induction
3.2.2 Differentiation and Expansion of Cortical Progenitors
3.2.3 Differentiation and Maturation of Cortical Neurons
4 Notes
References
Chapter 2: Generation of Homogeneous Populations of Cortical Interneurons from Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Cells
2.2 hPSC Culture Media
2.3 Differentiation Media Preparation
3 Methods
3.1 hPSC Culture, Passaging, and Cryopreservation
3.2 Generation of cINs
4 Notes
References
Chapter 3: Generation and Co-culture of Cortical Glutamatergic and GABAergic-Induced Neuronal Cells
1 Introduction
2 Materials
2.1 Reagents Necessary for hPSC Culture
2.2 Reagents Necessary for Lentivirus Production
2.3 Reagents Necessary for Mouse Glia Preparation
2.4 Reagents Necessary for iN Induction
2.5 Reagents Necessary for Ngn2, A/D Selection and Co-culture
2.6 Reagents Necessary for Maturation of Mixed Cultures
3 Methods
3.1 hPSC Culture
3.2 Lentivirus (LV) Production
3.3 Mouse Glia Preparation
3.3.1 Dissection
3.3.2 Dissociation
3.4 Ngn2 iN Differentiation
3.4.1 Day 1: Infection
3.4.2 Day 0: Induction
3.4.3 Day 1-4: Selection
3.4.4 Day 5: Collecting Cells
3.5 A/D iN Differentiation
3.5.1 Day -1: Infection
3.5.2 Day 0: Induction
3.5.3 Day 1-4: Selection
3.5.4 Day 5: Collecting Cells
3.6 Maturation of Mixed Cultures
3.6.1 Plating
3.6.2 Establishing the Cultures
3.6.3 Maturation
4 Notes
References
Chapter 4: Transcription Factor-Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Transfection of PiggyBac Plasmids and Generation of the PiggyBac-6F Lines
2.2 Lentivirus Production
2.3 Cell Culture
2.4 Animals
2.5 Equipment
2.6 Immunofluore-scence Analysis
3 Methods
3.1 Generation of PiggyBac-6F (PB6F) Lines
3.2 Lentivirus Production
3.3 Glia Isolation and Culture
3.4 MEF Isolation and Culture
3.5 iN Cell Induction and Culture
3.6 iN Cell Characterization by Immunofluorescence Analysis
4 Notes
References
Chapter 5: Directed Differentiation of Human iPSCs into Microglia-Like Cells Using Defined Transcription Factors
1 Introduction
2 Materials
2.1 Lentivirus Generation
2.1.1 Cell Lines
2.1.2 Media/Solutions
2.1.3 Reagents
2.1.4 Consumables
2.2 Human iPSC Culture
2.2.1 Cell Lines
2.2.2 Media/Solutions
2.2.3 Consumables
2.3 Differentiation of iMG Cells from hiPSCs
2.3.1 Cell Lines
2.3.2 Media, Solutions, and Reagents
2.3.3 Compositions of the Differentiation Media
2.4 Validation of the iMG Cells
2.4.1 Flow Cytometry
2.4.2 Immunocytochemistry
3 Methods
3.1 Generation of Lentivirus for Gene Delivery
3.1.1 HEK293FT Seeding (Day -1)
3.1.2 Transfection (Day 0)
3.1.3 First Medium Collection (Day 1.5)
3.1.4 Second Medium Collection and Concentration (Day 3)
3.2 Generation of hiPSC Lines Carrying the Doxycycline-Inducible SPI1 and CEBPA Transgenes (see Note 4)
3.2.1 hiPSC Recovery
3.2.2 Regular Passaging
3.2.3 Generation of hiPSC Lines Carrying the Doxycycline-Inducible SPI1 and CEBPA Transgenes (See Note 6)
3.3 Differentiation of Microglia-Like Cells from hiPSCs
3.3.1 hiPSC Seeding (Day -1)
3.3.2 hiPSC-to-iMG Induction (Day 0)
3.3.3 hiPSC-to-iMG Induction and Selection (Day 1)
3.3.4 hiPSC-to-iMG Differentiation (Days 2 and 3)
3.3.5 hiPSC-to-iMG Differentiation and Maturation (Day 4 to the End of Differentiation)
3.4 Verification of the iMG Cells
3.4.1 Flow Cytometry
3.4.2 Immunocytochemistry
4 Notes
References
Chapter 6: The Generation and Functional Characterization of Human Microglia-Like Cells Derived from iPS and Embryonic Stem Ce...
1 Introduction
2 Materials
2.1 Cultureware
2.2 Equipment
2.3 iPSC Lines
2.4 Reagents
2.5 Media
2.6 Flow Cytometry
2.6.1 Reagents
2.6.2 Consumables
2.7 Immunohistochemistry
2.7.1 Reagents
3 Methods
3.1 Generation of Hematopoietic Precursor Cells (HPCs)
3.2 Validation of Hematopoietic Precursor Cells (HPCs)
3.3 Microglia Differentiation
3.4 Microglia Maturation
3.5 Validation of Microglia-Like Cells
4 Notes
References
Chapter 7: Modeling Cellular Crosstalk of Neuroinflammation Axis by Tri-cultures of iPSC-Derived Human Microglia, Astrocytes, ...
1 Introduction
2 Materials
3 Methods
3.1 Tri-culture Preparation and Maintenance
4 Notes
References
Chapter 8: Generation of Oligodendrocytes from Human Pluripotent and Embryonic Stem Cells
1 Introduction
2 Materials
3 Methods
3.1 Prepare for iPSC/ESC Differentiation into Neural Progenitor Cells (NPC) (Day 1)
3.2 Neural Progenitor Cell Generation (Days 0-7)
3.3 Oligodendrocyte Progenitor Cell Generation (Days 8-14)
3.4 OPC-Neural Co-culture
3.5 Oligodendrocyte Maturation (Days 15-28)
4 Notes
References
Chapter 9: Characterizing the Neuron-Glial Interactions by the Co-cultures of Human iPSC-Derived Oligodendroglia and Neurons
1 Introduction
2 Materials
2.1 ES Cell Medium
2.2 iN Culture Medium
2.3 NPC Induction Medium
2.4 OPC Induction Medium
2.5 Ols Induction Medium
2.6 Co-culture Medium
2.7 Other Chemicals, Kits, and Reagents
2.8 qPCR Primers and Probes
2.9 Antibodies
2.10 Apparatus
3 Methods
3.1 iN Induction
3.2 NPC Induction
3.3 iOPC Generation (~7 days) and Validation (~7-14 days)
3.4 iOls Maturation
3.5 iN-iOPC Co-culturing
3.5.1 Immediate Co-culture
3.5.2 Delayed Co-culture
3.6 Summary
4 Notes
References
Chapter 10: Defined Differentiation of Human Pluripotent Stem Cells to Brain Microvascular Endothelial-Like Cells for Modeling...
1 Introduction
2 Materials
2.1 Small Molecule and Protein Aliquots
2.2 ECM Components and Plates
2.3 Media and Differentiation Materials
2.4 Immunocytochemistry
2.5 Accumulation Assay
2.6 Trans Endothelial Electrical Resistance (TEER)
3 Methods
3.1 Differentiation of hPSCs to BMEC-Like Cells
3.2 Immunocytochemical Analysis
3.3 Rhodamine 123 Accumulation Assay
3.4 Trans Endothelial Electrical Resistance (TEER)
3.5 BMEC-Like Cell Cryopreservation
3.6 BMEC-Like Cell Thawing
4 Notes
References
Chapter 11: Modeling the Blood-Brain Barrier Using Human-Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Cells
2.2 Media
2.2.1 StemFlexTM Basal Medium
2.2.2 Freezing Media iPSCs, NPCs, and Pericytes
2.2.3 NPC (Neural Progenitor Cell) Media
2.2.4 Astrocyte Media
2.2.5 Freezing Media for Astrocytes
2.2.6 DeSR1
2.2.7 DeSR2
2.2.8 hECSR
2.2.9 Freezing Media for Endothelial Cells
2.2.10 N2B27
2.2.11 iBBB Media
2.3 Cell Culture and Differentiation
2.4 Transwell Assays
2.5 Immunostaining and Imaging
3 Methods
3.1 Coating Cell Culture Plates
3.2 Differentiation of Human iPSCs into Astrocytes
3.3 Differentiation of Human iPSC into Brain Microvascular Endothelial Cells
3.3.1 Without ETV2-Inducible Activation
3.3.2 With ETV2-Inducible Activation
3.4 Differentiation of Human iPSCs into Pericytes
3.5 Barrier Model
3.6 Transwell Permeability Assay
3.7 iBBB Culture
3.8 Fixation and Staining of Cultures
3.9 Anticipated Results
4 Notes
5 Troubleshooting
References
Chapter 12: A Three-Dimensional Primary Cortical Culture System Compatible with Transgenic Disease Models, Virally Mediated Fl...
1 Introduction
2 Materials
2.1 Agarose Injection Molds
2.2 Tissue Dissociation
2.3 Adeno-Associated Viral Transduction
2.4 Live Confocal Microscopy
2.5 Immunohistochemistry
2.6 Clearing Solutions
3 Methods
3.1 Agarose-Molded Multi-well Plate (Fig. 2)
3.2 Neonatal Cortical Tissue Collection
3.3 Tissue Dissociation and Plate Seeding
3.4 Adeno-Associated Viral Transduction (Fig. 3)
3.5 Live Confocal Imaging
3.6 Immunohistochemistry
4 Notes
References
Chapter 13: Method to Generate Dorsal Forebrain Brain Organoids from Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Reagents Necessary for hPSC Culture
2.3 Brain Organoid Generation
2.3.1 Neural Induction Medium
2.3.2 Neural Differentiation Medium
2.3.3 Neural Maturation Medium
2.4 Cryosectioning
2.5 Antigen Retrieval
2.6 Immunohistochemistry
3 Methods
3.1 hPSC Culture
3.2 EB Generation
3.3 Neural Induction (Vontinued from Day 0)
3.4 Patterning and Differentiation
3.5 Maturation
3.6 Cryosectioning
3.7 Immunohistochemistry
3.8 Antigen Retrieval
3.9 Live Single-Cell Dissociation for scRNAseq
4 Notes
References
Chapter 14: A 3D Bioengineered Neural Tissue Model Generated from Human iPSC-Derived Neural Precursor Cells
1 Introduction
2 Materials
2.1 NPC Thawing
2.2 NPC Passaging and Expansion
2.3 Scaffold Coating
2.4 Scaffold Seeding
2.5 Collagen Filling
2.6 Neural Induction and Long-Term Maintenance
3 Methods
3.1 NPC Thawing
3.2 NPC Passaging and Expansion
3.3 Scaffold Coating
3.4 Scaffold Seeding
3.5 Collagen Filling
3.6 Neural Induction and Long-Term Maintenance
4 Notes
References
Chapter 15: FACS-Based Sequencing Approach to Evaluate Cell Type to Genotype Associations Using Cerebral Organoids
1 Introduction
2 Materials
2.1 iPSC Cell Culture
2.2 Nucleofection
2.3 Cell Counter
2.4 Cerebral Organoid Dissociation
2.5 Cell Staining
2.6 DNA Extraction and Library Preparation
3 Methods
3.1 Nucleofection of Cas9 and Multi-guide RNA
3.2 Cerebral Organoid Dissociation
3.3 Staining and FACS
3.4 DNA Extraction and Library Preparation for Sequencing
3.5 Sequence Analyses
4 Notes
References
Chapter 16: Dynamic Measurement of Endosome-Lysosome Fusion in Neurons Using High-Content Imaging
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture
3.2 Cell Treatment
3.2.1 One Day Before Imaging: Lysosome Labeling
3.2.2 The Day of Imaging: Endosome Labeling and Bafilomycin A1 Treatment
3.3 Time-Lapse Imaging Using Opera Phenix High-Content Screening System
3.3.1 Define the Global Experiment Settings
3.3.2 Select the Wells, Fields, and Planes to Be Measured
3.3.3 Select Channels
3.3.4 Set Time Series
3.3.5 Save the Experiment for Future Use or Modification
3.3.6 Run Experiments
3.4 Imaging Analysis: Build Up a Late Endosome-Lysosome Fusion Analysis Protocol
3.5 Run Analysis for the Whole Experiment
3.6 Data Analysis and Graph
4 Notes
References
Chapter 17: Live-Imaging Detection of Multivesicular Body-Plasma Membrane Fusion and Exosome Release in Cultured Primary Neuro...
1 Introduction
2 Materials
2.1 Primary Culture of Mouse Hippocampal Neurons
2.2 Transfecting Primary Neurons
2.3 Visualizing MVB-PM/Exosome Release
3 Methods
3.1 Primary Culture of Mouse Hippocampal Neurons
3.2 Transfecting Primary Hippocampal Neurons
3.3 Visualizing MVB-PM/Exosome Release Using Total Internal Reflection Fluorescence (TIRF) Microscopy
3.4 Quantifying MVB-PM/Exosome Release
4 Notes
References
Chapter 18: Assays of Monitoring and Measuring Autophagic Flux for iPSC-Derived Human Neurons and Other Brain Cell Types
1 Introduction
2 Materials
2.1 Chemical Modulation of Autophagy
2.2 Cell Lysis and Western Blotting
2.3 Antibodies and Detection Reagents
2.4 Human RFP-GFP-LC3B Reporter Plasmid Cloning
2.5 Stable Transduction of Cell Line Using Lentivirus
2.6 Chemical Modulation of Autophagy
2.7 Sample Preparation and Acquisition
3 Methods
3.1 Chemical Modulation of Autophagy in Human iPSC Neurons
3.2 Cell Lysis and Western Blotting (Fig. 1)
3.3 Antibodies and Imaging
3.4 Human RFP-GFP-LC3B Reporter Plasmid Cloning
3.5 Stable Transduction of Cell Line Using Lentivirus
3.6 Chemical Modulation of Autophagy (Fig. 2)
4 Notes
References
Chapter 19: Measuring Neuronal Network Activity Using Human Induced Neuronal Cells
1 Introduction
2 Materials
2.1 Cells
2.2 Plating and Culturing Reagents
3 Methods
3.1 Chip Preparation (Fig. 1)
3.2 Cell Plating on Chips
3.3 Chip Recording: Activity Assay
3.4 Chip Recording: Network Scan
3.5 Cleaning Chips After Use
3.6 Analysis Parameters
4 Notes
References
Chapter 20: A Simple Ca2+-Imaging Approach of Network-Activity Analyses for Human Neurons
1 Introduction
2 Materials
2.1 Equipment
2.2 Plasmids
2.3 Reagents Setup
2.4 Imaging Microscope
3 Methods
3.1 Mouse Glia Culture
3.2 iN Generation from Human ES Cells
3.3 Calcium Imaging
3.4 Analysis
3.4.1 Quantification of Network Activity
3.4.2 Analysis of Single-Neuron Dynamics
4 Notes
References
Chapter 21: Whole Cell Patch Clamp Electrophysiology in Human Neuronal Cells
1 Introduction
2 Materials
2.1 Solutions
2.2 Equipment
2.3 Additional Materials
3 Methods
3.1 Recording Protocols (Samples Provided Using Molecular Devices Multiclamp 700B and pClamp 10 Software)
3.2 Setup
3.3 Obtaining the Whole Cell Patch Configuration
3.4 Patch-Clamp Recordings
4 Notes
References
Chapter 22: Assaying Chemical Long-Term Potentiation in Human iPSC-Derived Neuronal Networks
1 Introduction
2 Materials
2.1 Co-culture of iDopa Neurons and iCell Astrocytes on MEAs
2.2 Co-culture hiPSC-Derived Cortical Neurons and Primary Human Astrocytes on MEAs
2.3 cLTP Induction and Pharmacological and Other Blocking Reagents
3 Methods
3.1 Co-culture of iCell Dopaminergic Neurons (iDopa) and iCell Astrocytes (iAstro) on MEA Plates
3.1.1 Prepare 0.1% PEI Solution
3.1.2 Coating MEA Plates
3.1.3 Thawing and Plating iDopa and iAstro on MEA
3.1.4 Maintaining iDopa and iAstro Co-cultures on MEAs
3.2 Co-culture of hiPSC-Derived Cortical Neurons and Primary Human Astrocytes on MEAs
3.2.1 Differentiation of hiPSC-Derived Cortical Neurons
3.2.2 Replating and Maintaining hiPSC-Derived Cortical Neurons on MEAs
3.3 cLTP Induction and Manipulations to Investigate Underlying Molecular Mechanisms Using Pharmacological and Other Blocking R...
3.3.1 cLTP Induction
3.3.2 Conditioned Medium Experiments
3.4 MEA Recording and Data Analysis
3.4.1 MEA Plate Recording
3.4.2 MEA Data Analysis
3.4.3 Assign Wells to Treatment and Control Groups to Achieve a Comparable Distribution of Baseline Activity
4 Notes
References
Index