Brain Organoid Research

Dedication
Preface to the Series
Preface
Contents
Contributors
Chapter 1: Self-Organizing Brain Organoids with Ventricles Amenable to Injection and Electroporation
	1 Introduction: Neural Organoids for Modelling Human Brain Development
		1.1 Self-Organizing Cerebral Organoids to Model Tissue Architecture
	2 Materials
		2.1 Cell Lines and Cell Culture Reagents
		2.2 Injection Setup
	3 Methods
		3.1 Generation of Cerebral Organoids with Large Ventricles
		3.2 Injection and Electroporation of Cortical Lobes
		3.3 Downstream Analysis of Organoids
	4 Conclusions
	References
Chapter 2: Profiling Cell Type-Specific Gene Regulatory Regions in Human Cortical Organoids
	1 Introduction
		1.1 Apical Radial Glia Are Key to Understand Healthy and Diseased Cortical Development
		1.2 ATAC-Seq Allows Identification of Open Chromatin with Low Input Cell Numbers
	2 Materials and Equipment
		2.1 iPSC Culture and Cortical Organoid Generation
		2.2 Preparing Organoids for FACS Sorting
		2.3 Fluorescence-Activated Cell Sorting
		2.4 RNA Extraction, cDNA Synthesis, and RT-qPCR Analysis
		2.5 ATAC-seq Reaction and Library Amplification
		2.6 ATAC-seq Library Size Selection and Sequencing
		2.7 Mouse Neural Stem Cell Isolation and Culture
		2.8 Luciferase Assay
	3 Methods
		3.1 iPSC Culture and Cortical Organoid Generation
		3.2 Labeling of Neural Cell Populations for FACS Sorting
			3.2.1 DiI Labeling
			3.2.2 Single-Cell Suspension
			3.2.3 Fixation and Antibody Labeling
		3.3 Fluorescence-Activated Cell Sorting
		3.4 RNA Extraction, cDNA Synthesis, and RT-qPCR Analysis
		3.5 ATAC-Seq Reaction and Library Amplification
		3.6 ATAC-Seq Library Size Selection and Sequencing
		3.7 Mouse Neural Stem Cell Isolation and Culture
		3.8 Luciferase Assay
	4 Notes/Troubleshooting
		4.1 DiI Labeling
		4.2 Dissociation of Organoids
		4.3 FACS: Gating Single Cells
		4.4 ATAC-seq: Transposition Reaction
		4.5 ATAC-seq: Size Selection
		4.6 mNSCs: Isolation
		4.7 Luciferase Assay: Native Chromatin Context
		4.8 Luciferase Assay: Variability
	5 Conclusions
	References
Chapter 3: Visualization of 3D Organoids Through the Latest Advancements in Microscopy
	1 Introduction
	2 Advancements in 3D Imaging of Organoids
	3 Light-Sheet Microscopy for Organoid Imaging
	4 Methods for Specimen Preparation
		4.1 Chemical Clearing Techniques for 3D Imaging
		4.2 Fluorophore Labeling Techniques for Signal Enhancement
		4.3 Genetically Encoded Fluorescent Tags
		4.4 Multicolor Labeling via Nontargeted or Targeted Fluorescent Tags
	References
Chapter 4: Generation and Isolation of Retinal Ganglion Cells and Photoreceptors from Human iPSC-Derived Retinal Organoids by ...
	1 Introduction: Recapitulating Retinal Development In Vitro
	2 Materials
	3 Methods
		3.1 Generation of Human iPSC-Derived Retinal Organoids
		3.2 Selection of Specific Retinal Subtypes from Retinal Organoids Using MAC-Sorting
			3.2.1 Selection of Photoreceptor Cells Using CD73 Cell Surface Marker
			3.2.2 Selection of Retinal Ganglion Cells Using CD90 Cell Surface Marker
		3.3 Cryopreservation In Vitro of Selected Retinal Cells
			3.3.1 Cryopreservation of Whole Retinal Organoids
			3.3.2 Characterization of Cryopreserved CD73-Positive Photoreceptor Cells
			3.3.3 Characterization of Cryopreserved CD90-Positive Retinal Ganglion Cells
	4 Conclusion
	References
Chapter 5: Micromanipulator-Assisted Subretinal Transplantation of Human Photoreceptor Reporter Cell Suspensions into Mice
	1 Introduction
	2 Materials
		2.1 Dissociation
		2.2 Sorting
		2.3 Transplantation
	3 Methods
		3.1 Dissociation
		3.2 Sorting
		3.3 Transplantation
	4 Notes
	5 Conclusion
	References
Chapter 6: Generation of Three-Dimensional Retinal Tissue with Physiologically Competent, Light-Sensitive Photoreceptors from ...
	1 Introduction
	2 Materials
		2.1 Cell Lines
		2.2 3D Retinal Organoid Media
		2.3 Other Reagents
	3 Methods
		3.1 Matrigel Preparation and Plate Coating
			3.1.1 Prepare Matrigel Stock (See Note 1)
			3.1.2 Plate Coating for hiPSC Culture (See Note 3)
		3.2 Thawing hiPS Cells
		3.3 Passaging hiPS Cells
		3.4 Freezing hiPS Cells Using mFreSR
		3.5 Generation of Neural Aggregates
		3.6 Differentiation of Neural Aggregates to Retinal Fate
		3.7 Advanced Differentiation for Promoting Functional Photoreceptors
	4 Characterization of Human Retinal Organoids by Immunofluorescence
		4.1 Preparing Retinal Organoid Samples for Immunofluorescence Staining
		4.2 Differentiation of Neural Aggregates to Retinal Fate: D7-D42
		4.3 Differentiation of Retinal Organoids: Development of Retinal Layers (D42-D120)
		4.4 Advanced Differentiation of Retinal Organoids: Appearance of a Bipolar Cell Layer and Functionally Active Photoreceptors (...
	5 Notes
	6 Conclusions
	References
Chapter 7: Quantitative Assessment of Fluorescent Reporter Expression in 3D Retinal Organoids
	1 Introduction
	2 Materials
		2.1 Cell Lines and Organoid Differentiation
		2.2 Materials for Fluorescent Reporter Quantification
		2.3 Materials for Fluorescence-Based Assessment of Oxidative Stress
	3 Methods
		3.1 Plate Preparation
		3.2 Plate Reading
		3.3 Size Normalization
		3.4 Statistical Methods
		3.5 Application Example: Longitudinal Tracking of Genetically Encoded Fluorescent Reporters
		3.6 Application Example: Quantification of Reactive Oxygen Species by DHE Staining
		3.7 Application Example: Quantification of Mitochondrial Membrane Depolarization by JC-1 Staining
	4 Notes
	5 Conclusions
	References
Chapter 8: A Three-Dimensional Organoid Culture System to Model Invasive Patterns of Patient-Derived Glioma Stem Cells
	1 Introduction
		1.1 Role of Glioma Stem Cells in Glioblastoma
		1.2 Traditional Methods to Study GSCs
		1.3 Recent Models Maintain the Heterogeneity of GBM
		1.4 Using Human Brain Organoids to Study GSC Invasion Patterns
	2 Materials
		2.1 Medium Components
	3 Methods
		3.1 Maintenance and Culturing of Human iPSCs
		3.2 Generation of Human Brain Organoids
		3.3 Culturing and Maintenance of Patient-Derived GSCs
		3.4 Establishment of Different Assay Conditions to Model Patient-Derived GSCs in Brain Organoids
			3.4.1 Assay 1: Coculturing of GSCs with iPSCs During Brain Organoid Differentiation
			3.4.2 Assay 2: Coculturing of GSCs with Brain Organoids
			3.4.3 Assay3: Fusion of GSCs Spheres to Organoids
		3.5 Tissue Clearing of Cocultured Organoids
		3.6 Immunostaining of Organoids and Cocultured Organoids
		3.7 Immunofluorescence Microscopy and Time-Lapse Imaging of Organoids
			3.7.1 Time-Lapse Imaging of Cocultured Organoids
			3.7.2 Imaging of Tissue-Cleared Organoids
			3.7.3 Super-Resolution Imaging of GSCs Interaction with Neurons
		3.8 Culturing Organoid Slices at the Interphase of the Air-Liquid Surface to Generate Neuronal Outgrowth
		3.9 Immunostaining of GSCS: Neuronal Outgrowth Interactions
	4 Notes and Troubleshooting
	References
Chapter 9: Modeling Brain Tumors Using Genetically Edited Brain Organoids
	1 Introduction
		1.1 Brain Tumor
		1.2 Models to Study Brain Tumor
	2 Materials
		2.1 Plasmid Construction
		2.2 Preparation of Lentivirus Particles
		2.3 Animal Preparations
	3 Methods
		3.1 Cell Culture
		3.2 Culture of Cerebral Organoids
		3.3 Induce Gene Mutation/Amplification of Organoids
		3.4 Preparation of Organoid for Immunostaining
		3.5 RNA Sequencing
		3.6 Renal Subcapsular Engrafting and Intracranial Injection
		3.7 Drug Testing on Modeling Brain Tumor Cerebral Organoids
	4 Conclusion and Limitation
	References
Chapter 10: Brain Organoids as a Model to Study Zika Virus and SARS-CoV-2 Infections
	1 Introduction
	2 Materials
		2.1 Brain Organoid Preparation
			2.1.1 Equipment
			2.1.2 Plasticware
			2.1.3 Reagents/Cells
		2.2 Viral Preparation
			2.2.1 Equipment
			2.2.2 Plasticware
			2.2.3 Reagents/Cells
	3 Methods
		3.1 Brain Organoid Culture
		3.2 Brain Organoid Infection
		3.3 Viral Detection
		3.4 Plaque Assay
			3.4.1 Cell Maintenance Medium
		3.5 Viral Propagation
	4 Notes
	References
Chapter 11: Infection of 3D Brain Organoids with Human Pathogenic Viruses Under Biosafety Level-3 Conditions with Subsequent I...
	1 Introduction: Neurotropism of Human Pathogenic Viruses
		1.1 Models to Study Viral Neurotropism
	2 Material and Methods
		2.1 Specific Media Compositions and Buffer Preparations
			2.1.1 Maintenance Medium
			2.1.2 TEM Fixation Solution
			2.1.3 Lead Citrate (Reynolds)
			2.1.4 Sol D
			2.1.5 Protein Lysis Buffer
			2.1.6 Western Blot Loading Buffer
			2.1.7 hiPSC Expansion and Maintenance Medium
			2.1.8 Neural Induction Medium
			2.1.9 Organoid Differentiation Medium Composition
		2.2 Viral Strains, Cell Lines, Instruments, and Reagents
			2.2.1 Viral Strains
			2.2.2 Cell Lines
			2.2.3 Laboratory Instruments and Softwares
			2.2.4 Laboratory Chemicals and Reagents
			2.2.5 Primary and Secondary Antibodies
	3 Methods
		3.1 Inoculation of Cell Culture Allows Generation of Clinical Virus Isolates
		3.2 Transmission Electron Microscopy to Verify Viral Infection
		3.3 Determination of Viral Titer: Endpoint Dilution Assay
		3.4 Generation of Human Brain Organoids
		3.5 Organoid Dissociation
		3.6 Inactivation of Complete 3D Brain Organoids by Cell Lysis for RNA and Protein Expression Analysis
		3.7 Inactivation of 3D Brain Organoids via Fixation to Perform Confocal Microscopy
		3.8 Immunostaining of Fixed Brain Organoids
		3.9 Confocal Microscopy
	4 Conclusion
	References
Index