Phenotyping of Human iPSC-derived Neurons: Patient-Driven Research

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PHENOTYPING OF HUMAN IPSC-DERIVED NEURONS
PHENOTYPING OF HUMAN IPSC-DERIVED NEURONS: PATIENT-DRIVEN RESEARCH
Copyright
Dedication
Contents
Contributors
I - Best practices and considerations when designing a new project
	1 - iPSC culture: best practices from sample procurement to reprogramming and differentiation
		Facility setup
			Tissue culture room design
			Tissue culture equipment
		Primary sample collection
			Somatic cells
			Quality control of somatic cells
		Reprogramming
			Pros and cons of each method
				Episomal vector transfection
				Sendai virus transduction
				mRNA reprogramming method
		iPSC line characterization
			Sterility
			Pluripotency
			Transgene elimination
			Identity
			Genetic stability
		Best practices prior to differentiation
			Cell banking
			Culturing conditions
		Differentiation
			Experimental design
				Cell line selection
				Differentiation protocol selection
			Best practices during differentiation
		References
	2 - Phenotypic assay development with iPSC-derived neurons: technical considerations from plating to analysis
		Introduction
		Establishing optimal conditions for phenotyping iPSC-derived neurons
			Differentiation protocol considerations
			Coating substrates
			High content imaging (HCI)
			Functional analysis
				Multi-electrode array (MEA) recording
				Calcium imaging
				Patch clamping
			Live imaging
			Fluorescent microplate assays
		Assay development for screening
		Conclusion
		References
	3 - Derivation of cortical interneurons from human pluripotent stem cells to model neurodevelopmental disorders
		Introduction
			Development of the human cortex
			Modeling human cortical interneuron development in vitro
			The development of protocols for cortical interneurons from human pluripotent stem cells (hPSCs) to model neurodevelopmenta ...
		A protocol for cortical interneuron derivation from human pluripotent stem cells (hPSCs)
			Equipment and supplies
			Reagents
			Preparation of reagents
				Accutase cell detachment solution
				B-27 supplement (50×) minus vitamin A
				Preparing matrigel
				Coating tissue culture plates with Matrigel
				Coating tissue culture plates with Matrigel-Laminin
				Small molecule preparation
			Media composition
			Protocol
				Specification of cortical interneuron progenitors from hPSCs
				Maintenance and expansion of cIN NPCs
				Cryopreservation of cIN neural progenitor cells
				Revival and maintenance of cryopreserved cIN neural progenitor cells
				Interneuron differentiation and maturation from cIN neural progenitor cells
		Enrichment and purification of cIN neural progenitor cells and neurons
			Enrichment for post-mitotic cINs with neural rosette selection reagent
			Purification of post-mitotic cINs with NCAM bead selection
			Critical steps and troubleshooting
		Cellular phenotyping of hPSC-derived cINs
			Using immunocytochemistry to benchmark hPSC-derived cINs and to assess NDD-related alterations of neurodevelopment
			Morphometric analysis of neurite extension and length
			Neuronal migration assay
			Measurement of synaptic puncta
		Alternate protocol for derivation of cIN NPCs from hPSCs
		Alternate protocol for differentiation of cIN NPCs into interneurons
		Acknowledgments
		References
	4 - Development of transcription factor-based strategies for neuronal differentiation from pluripotent stem cells
		Introduction
		Neuron differentiation driven by transcription factors
			Dopaminergic (DA) neurons
			Glutamatergic neurons
			GABAergic neurons
			Cholinergic motor neurons
				Retinal ganglion cells
			Glia: astrocytes, oligodendrocytes, and microglia
		Transcription factor-driven differentiation: considerations when designing a new protocol
			Design a cocktail of transcription factors
			Transcription factor delivery
				Genome integrating vectors
				Non-genome integrating viral vectors
				Synthetic mRNA
		Summary and future directions
		Acknowledgement
		References
	5 - Differentiation of Purkinje cells from pluripotent stem cells for disease phenotyping in vitro
		Development of the cerebellum
		Differentiation of pluripotent stem cells into Purkinje cells
			Cerebellar organoids derived from iPSCs and ESCs in 3D cultures
			Human iPSC- and ESC-derived Purkinje cell differentiation in 2D co-cultures with mouse cerebellar cells
			Functional characterization of human pluripotent stem cell-derived Purkinje cells in vitro and in vivo
			Challenges in the differentiation of human Purkinje cells in 2D- and 3D-cell cultures
		Disease phenotyping of Purkinje cells
			Purkinje cells in cerebellar ataxia
				Mouse Purkinje cell models of cerebellar ataxia
				Human iPSC-derived NPCs and Purkinje cells in cerebellar ataxia
			Purkinje cells in Tuberous Sclerosis Complex (TSC)
				Mouse Purkinje cell models of TSC
				TSC patient iPSC-derived Purkinje cells
		Future perspectives for stem cell-derived Purkinje cells in translational medicine
			Cell transplantation for treatment of cerebellar degeneration
			Drug screening with pluripotent stem cell-derived Purkinje cells
		Acknowledgments
		References
	6 - Brain organoids: models of cell type diversity, connectivity, and disease phenotypes
		Introduction
		Cerebral organoids
			Human corticogenesis overview
			Organoid differentiation overview
			Fidelity of hCO cell types and organization
		Other brain region specific organoids
		Neuronal activity and connectivity
			Synaptic activity
			Connectivity of neuronal organoids
		Non-neuronal cells
			Astrocytes
			Oligodendrocytes
			Microglia
			Vascularization/nutrient distribution
			Summary of non-neuronal cells
		Use of models in disease
			Microcephaly modeling with hCOs
			ASD modeling with hCOs
				Molecularly defined ASD
				Idiopathic ASD
			Limitations of hCO modeling for CNS disorders
		Reproducibility
			Sources of variability in organoid model systems
			Addressing reproducibility
		Conclusions and future directions
		References
II - The use of iPSC-derived neurons to study neurological disorders
	7 - Human models as new tools for drug development and precision medicine
		Introduction
		Drug development pipeline
		Human models as a screening tool for personalized precision medicine
			Monolayer models
			Organoids
			Organ-on-chip platforms
		Conclusion
		References
	8 - Use of cerebral organoids to model environmental and gene x environment interactions in the developing fetus an ...
		Introduction
		Maternal immune activation
			Cerebral organoids as a model system to study MIA and neuroinflammation
		Cerebral organoids as a model system to study infectious diseases that cause neurodevelopmental disorders
			Zika virus
			SARS-CoV-2
			Human immunodeficiency virus (HIV)
			Toxoplasmosis
			Cytomegalovirus (CMV)
			Herpes simplex virus (HSV)
		Cerebral organoids and cellular stress
			Heat shock
			Fetal alcohol syndrome
		Cerebral organoids to model neurodegenerative disorders
			Alzheimer\'s disease (AD)
				Cerebral organoids in familial AD
				Modeling sporadic AD
				Cerebral organoids for drug development in AD
			Modeling Parkinson Disease using organoid cultures
		Conclusion
		References
	9 - iPSC-derived models of autism: Tools for patient phenotyping and assay-based drug discovery
		Introduction
		Syndromic autisms
			Fragile X syndrome
			Rett syndrome
			FOXG1 deletion syndrome
			Tuberous sclerosis
			Pheland McDermid syndrome
			Prader-Willi and Angelman syndromes
			Timothy syndrome
		iPSC studies to model ASDs in vitro
			iPSC studies focused on syndromic and sporadic autisms
			iPSC studies focusing on sporadic non-syndromic autism
			Data collected by studies focused on iPSCs from idiopathic autism
				Gene expression profiling
				Concordances in gene expression profiles obtained from studies on iPSC-derived cells and post-mortem brain tissue from idio ...
				Morphological and electrophysiological properties in iPSC-derived neurons from patients with idiopathic autism
				Similar phenotypes between iPSC-derived neurons from patients with sporadic or syndromic autisms and idiopathic autism
		3D models of ASDs—a focus on organoids, spheroids, and assembloids
		The use of iPSCs to develop assays and novel therapies that can be translated to the clinic for ASD
			Limitations for using iPSC-derived neurons in drug screening platforms
				Quality control testing
				Automation challenges
				Cost
				Small “n”
				Epigenetic memory
				Well-to-well variability
				Variability within cell lines
				Variability across differentiation batches
				Disease modeling
				Screening of simple phenotypes
			The use of iPSC-derived neurons for personalized medicine
		Conclusions
		References
	10 - Probing the electrophysiological properties of patient-derived neurons across neurodevelopmental disorders
		Induced pluripotent stem cells and modeling brain disorders
		Progressing from gene discovery to functional gene groupings to pathophysiology
		Neuronal networks represent a logical level for the manifestation of NDDs
		Micro-electrode arrays as a scalable high-throughput functional assay
		Phenotyping NDD patient-derived neurons using MEA recordings
			Fragile X and Rett syndrome
			Kleefstra syndrome
		Neuronal networks as converging pathways?
		The way forward
		Acknowledgments
		References
	11 - Advantages and limitations of hiPSC-derived neurons for the study of neurodegeneration
		Introduction
		Biology of Alzheimer\'s disease
		Alzheimer\'s disease hiPSC models
			Familial AD (FAD)
			Sporadic Alzheimer\'s disease (SAD)
			Apolipoprotein E (APOE)
			Other AD risk factors
		Tauopathies: Alzheimer’s disease related dementias
		The challenge of aging in hiPSC models of age-related disease
		Modeling Alzheimer\'s disease with cerebral organoids
			Current challenges of 3D modeling and possible solutions
		Using hiPSC models for drug discovery
		Conclusions
		References
III - New technology, industry perspective, and transitioning to the clinic
	12 - Developing clinically translatable screens using iPSC-derived neural cells
		Introduction
		Is an iPSC-derived platform right for the application?
		What factors should be considered in developing iPSC-based assays?
			iPSC line selection
			Cell type selection
			Assay endpoint selection
		What factors should be considered when running an iPSC-based screen?
			Cell culture
				Undifferentiated iPSC culture and scale-up
				Differentiated iPSC culture and scale-up
				Cell plating, incubation, and long-term maintenance
				Media changes
			Assay optimization
				Assay formats
				Miniaturization
				Balancing throughput with assay stability
			Small molecule considerations
			Assay analytics
				Data analysis and hit determination
				Normalization methods and hit selection
		Summary
		References
	13 - Gene editing hPSCs for modeling neurological disorders
		Introduction to limitations of iPSC-derived neuronal models that can be improved with gene editing
			In vitro culture models are either artificially simplified or too complex for simple comparison
			Genetic background differences lead to high variability when comparing lines from different individuals
			Visualizing and analyzing human cells in in vivo transplant models is technology challenging
		Gene editing systems – past, present, and future
			“Version 1.0” – meganucleases
			“Version 2.0” – zinc finger nucleases and TAL effector nucleases
			“Version 3.0” – CRISPR/cas nucleases
			“Version 4.0” – genome editing systems for translational medicine
		Use of genetic modification to generate isogenic cell lines
			Gene editing systems for generating isogenic lines
			Isogenic hPSC lines for disease modeling
			Isogenic knock-out cell lines
		Safe harbor locus transgenic systems
			Promoters utilized at safe harbor loci
			Effector transgenes utilizing safe harbor loci
		Endogenous locus transgenes
			Endogenous locus gene replacement transgenes
			Endogenous locus fusion protein or peptide tag transgenes
			Bi-cistronic reporter transgenes
		Complex transgenic systems utilizing multiple gene editing events
		Future of genetic modification in hPSC-based neuronal research
		References
	14 - Cell therapy and biomanufacturing using hiPSC-derived neurons
		Introduction
		hiPSC-derived neurons to model specific neurological disorders
			Differentiating hiPSCs to NSCs
			Differentiating NSCs to specific neurons for disease modeling
				Alzheimer\'s disease (AD)
				Parkinson\'s disease (PD)
				Huntington disease (HD)
				Amyotrophic lateral sclerosis (ALS)
		Brief history of bio-manufacturing
			Historical perspective of the term “quality”
			Concept and methods applied to develop quality management
			Quality management applied to GLP
			The origins of good manufacturing practices (GMP)
			GMP applied to clinical-grade cell manufacturing
		Manufacturing hiPSCs and neuronal derivatives
			hiPSC reprogramming and differentiation process overview
			Informed consent
			Screening donor samples for contagious disease
			Tissue acquisition, cell isolation, and expansion
			Reprogramming method
			hiPSC banking and quality controls
			Specific differentiation and characterization of cellular subtypes
		Clinical trials with hiPSC-derived neurons
		Perspective and challenges for clinical translation
		References
	15 - Ethical considerations for the use of stem cell-derived therapies
		Overview of induced pluripotent stem cell (iPSC) therapies for neurological application
		Ethical and social issues
			Quality control (QC) and quality assurance (QA) issues
				Characterization of the therapeutic cell product
				Potency assays
				Tissue specificity
			Risks, benefits, and safety for participants and patients
			Informed consent and patient vulnerability
			Access/provision
		Ethical translation of promising stem cell-based neurological therapeutics
			Ethical translation in practice
		Conclusions
		References
Index
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	P
	Q
	R
	S
	T
	V
	W
	Z
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