Phenotyping of Human iPSC-derived Neurons: Patient-Driven Research examines the steps in a preclinical pipeline that utilizes iPSC-derived neuronal technology to better understand neurological disorders and identify novel therapeutics, also providing considerations and best practices. By presenting example projects that identify phenotypes and mechanisms relevant to autism spectrum disorder and epilepsy, this book allows readers to understand what considerations are important to assess at the start of project design. Sections address reproducibility issues and advances in technology at each stage of the pipeline and provide suggestions for improvement. From patient sample collection and proper controls to neuronal differentiation, phenotyping, screening, and considerations for moving to the clinic, these detailed descriptions of each stage of the pipeline will help everyone, regardless of stage in the pipeline.
In recent years, drug discovery in the neurosciences has struggled to identify novel therapeutics for patients with varying indications, including epilepsy, chronic pain, and psychosis. Current treatment options for such patients are decades old and offer little relief with many side effects. One explanation for this lull in novel therapeutics is a lack of novel target identification for neurological disorders (and target identification requires exemplar preclinical data). To improve on the preclinical work that often relies on rodent modeling, the field has begun utilizing patient-derived induced pluripotent stem cells (iPSCs) to differentiate neurons in vitro for preclinical characterization of neurological disease and target identification.
Author(s): Elizabeth D. Buttermore
Publisher: Academic Press
Year: 2022
Language: English
Pages: 372
City: London
Front Cover
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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