Induced Pluripotent Stem Cells and Human Disease: Methods and Protocols

Preface
Contents
Contributors
Analysis of Mitochondrial Dysfunction by Microplate Reader in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Disorder
	1 Introduction
	2 Materials
		2.1 Cell Culture
			2.1.1 Cell Culture Plasticware, Equipment and Software
			2.1.2 Cell Dissociation and Plate Coating Reagents
			2.1.3 hiPSC Culture Medium on MEF Feeders (Composition for 500 mL)
			2.1.4 Neural Precursor Culture Medium (Composition for 500 mL)
			2.1.5 Neuronal Culture Medium (Composition for 500 mL)
		2.2 Analysis of Mitochondrial Metabolism Functional Parameters by Microplate Reader
			2.2.1 Fluorescence Analysis for ΔΨm, ROS, Mitochondrial Ca2+ and Superoxide
			2.2.2 Luminescence Analysis for ATP/ADP Levels
			2.2.3 Bradford Protein Assay
	3 Methods
		3.1 Neuronal Differentiation
			3.1.1 hiPSC Culture and Differentiation into Neural Precursors
			3.1.2 Differentiation of hiPSC-Derived NPs into Neurons
		3.2 Analysis of Mitochondrial Parameters in hiPSC-Derived Neurons
			3.2.1 Analysis of Mitochondrial Membrane Potential with TMRE in Microplate Reader
			3.2.2 Analysis of Mitochondrial Calcium with Fluo-3 AM in Microplate Reader
			3.2.3 Analysis of Reactive Oxygen Species with DCF in Microplate Reader
			3.2.4 Analysis of Mitochondrial Superoxide with MitoSox in Microplate Reader
			3.2.5 Analysis of ATP/ADP Levels Via Bioluminescence Assay in Microplate Reader
	4 Notes
	References
Generation and Hematopoietic Differentiation of Mesenchymal Stromal/Stem Cell-Derived Induced Pluripotent Stem Cell Lines for
	1 Introduction
	2 Materials
		2.1 Cell Culture
			2.1.1 Media, Buffers, Growth Factors and Supplements
			2.1.2 Kits
			2.1.3 Preparation of Stock Solutions, Buffers, and Media
				Stock Solutions and Buffers
				Media
			2.1.4 Cells
		2.2 FACS and Immunofluorescence
			2.2.1 Primary Antibodies
			2.2.2 Secondary Antibodies
	3 Methods
		3.1 Thawing and Culture of MSCs
		3.2 Immunophenotyping of MSCs
		3.3 Preparation and Lentiviral Transduction of MSCs
		3.4 Culture and Selection of iPSCs
		3.5 Colony Picking, Culture, and Freezing-Thawing of iPSCs
			3.5.1 Colony Picking and Propagation of iPSCs
			3.5.2 Passaging of iPSCs
			3.5.3 Freezing of iPSCs
			3.5.4 Thawing of iPSCs
		3.6 Basic Characterization of iPSCs
			3.6.1 Flow Cytometry
			3.6.2 Immunofluorescent Staining
		3.7 Hematopoietic Differentiation of iPSCs
			3.7.1 Culture of Op9 Cells
			3.7.2 Op9/iPSC Cocultures
			3.7.3 Colony Assays
	4 Notes
	References
Establishment of Human Induced Pluripotent Stem Cells from Multiple Sclerosis Patients
	1 Introduction
	2 Materials
		2.1 Cell Culture Materials
		2.2 Agarose Gel
		2.3 Cell Culture Media
		2.4 Materials for Immunostaining
	3 Methods
		3.1 Obtaining PBMCs from MS Patients
		3.2 Expansion and Mitotic Inactivation of Mouse Embryonic Fibroblasts
		3.3 Isolation of PBMCs from Whole Blood
		3.4 Expansion of PBMCs Prior to Reprogramming
		3.5 Generation of Feeder-Dependent hiPSCs from Expanded PBMCs
		3.6 Alkaline Phosphatase Live Staining
		3.7 Clonal Expansion and Manual Passaging of hiPSCs
		3.8 Enzymatic Passaging of hiPSCs
		3.9 Cryopreservation of iPSCs
		3.10 RNA Isolation and cDNA Synthesis
		3.11 RT-PCR for the Analysis of Removal of Sendai Transgene Expression
		3.12 PCR for Mycoplasma Analysis
		3.13 Analysis of Pluripotency Genes by RT-qPCR
		3.14 Immunocytochemistry for Pluripotency Markers
		3.15 In Vitro Differentiation of hiPSCs to Three Germ Layers
	4 Notes
	References
Monitoring Axonal Degeneration in Human Pluripotent Stem Cell Models of Hereditary Spastic Paraplegias
	1 Introduction
	2 Materials
		2.1 Stock Solution
		2.2 Medium
		2.3 Transfection Reagents
		2.4 Tissue Culture Supplies
		2.5 Antibodies
		2.6 Primers (Forward, Reverse)
		2.7 Software
	3 Methods
		3.1 Generation of HSP iPSC Models
		3.2 Validation of HSP iPSCs
			3.2.1 Gene Expression Based iPSCs Validation with RT-PCR/qPCR
			3.2.2 Protein Expression Based iPSCs Validation
			3.2.3 In Vivo Based iPSC Validation with the Teratoma Assay
		3.3 Differentiation of Cortical PNs from iPSCs
			3.3.1 Day 0-4 iPSCs Culture in Suspension
			3.3.2 Days 4-7 Neural Specification
			3.3.3 Days 7-14 Formation of Neuroepithelial Cells
			3.3.4 Days 15-27 Specification of Cortical PNs
			3.3.5 Day 28: Plating Cortical PNs
		3.4 Examining the Axonal Outgrowth Defects
		3.5 Monitoring Axonal Transport Defects
			3.5.1 Visualize and Perform Mitochondrial Transport (Fig. 2a)
			3.5.2 Analysis of Mitochondrial Transport Using ImageJ (Fig. 2b)
		3.6 Examining the pNF-H Release
	4 Notes
	References
Efficient Generation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells for Disease Modeling and Disease Gene
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Media, Buffers and Solutions
	3 Methods
		3.1 iPSC Culture
		3.2 Stage I: Differentiation of Definitive Endodermal (DE) Cells
		3.3 Stage II: Differentiation of Hepatic Precursors
		3.4 Stage III: Differentiation of Mature Functional Hepatocytes
		3.5 Immunocytochemistry (ICC) Based Characterization of the Generated Cells
	4 Notes
	References
Autophagy Dysfunction as a Phenotypic Readout in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Diseases
	1 Introduction
	2 Materials
		2.1 Cell Culture
			2.1.1 Cell Culture Plasticware, Solutions, Coating Reagents and Equipment
			2.1.2 Cell Dissociation Reagents
			2.1.3 Cell Freezing Medium
			2.1.4 MEF Culture and Inactivation Medium (Composition for 500 mL)
			2.1.5 hiPSC Culture Medium on MEF Feeders (Composition for 500 mL)
			2.1.6 Neural Precursor Differentiation Medium (Composition for 500 mL)
			2.1.7 Neural Precursor Culture Medium (Composition for 500 mL)
			2.1.8 Neuronal Culture and Differentiation Medium (Composition for 500 mL)
			2.1.9 Autophagy Modulators and Treatments
		2.2 Western Blotting
			2.2.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
			2.2.2 Immunoblotting
		2.3 Immunofluorescence
	3 Methods
		3.1 hiPSC Culture and Neuronal Differentiation
			3.1.1 Culturing and Freezing of Inactivated MEFs for hiPSC Feeder Layer
			3.1.2 Culturing hiPSCs on MEF Feeder Layer
			3.1.3 Differentiation of hiPSCs into Neural Precursors
			3.1.4 Culturing and Freezing of hiPSC-Derived Neural Precursors
			3.1.5 Differentiation of hiPSC-Derived NPs into Neurons
		3.2 Autophagy Analysis in hiPSC-Derived Neurons
			3.2.1 Western Blotting for Analyzing Autophagosomes and Autophagy Substrate
			3.2.2 Immunofluorescence for Analyzing Autophagy Markers
			3.2.3 Analysis of Autophagosome Flux with Bafilomycin A1
			3.2.4 Other Markers and Assays for Analyzing Autophagy
		3.3 Troubleshooting
	4 Notes
	References
Derivation of Induced Pluripotent Stem Cell (iPSC) Lines from Patient-Specific Peripheral Blood Mononuclear Cells (PBMC) Using
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Media Composition
		2.3 Real-Time PCR
		2.4 Immunofluorescence
	3 Methods
		3.1 Isolation of PBMCs
		3.2 Cryopreservation of PBMCs
		3.3 Thawing and Expansion of PBMCs
		3.4 Preparation of Matrigel Coated Plates
		3.5 Day 0: Transfection of Cells Using the Neon Transfection System
		3.6 Days 3-21: Introduction of ReproTeSR Medium
		3.7 Days 21-30: Replacing ReproTeSR Medium to Stem Flex Medium
		3.8 Passaging of iPSC Clones: Manual Subculture Through Colony Cutting (Recommended for Early Passage p0-p5)
		3.9 Enzymatic Passaging of iPSC Colonies (Recommended Once the Culture Is Stable)
		3.10 Freezing of iPSCs
		3.11 Confirmation of Authenticity and Purity of iPSCs by Indirect Immunofluorescence
		3.12 RNA Isolation, cDNA Synthesis, Semiquantitative PCR, and Real-Time Quantitative PCR
		3.13 Karyotyping and Short Tandem Repeat (STR) Genotyping
		3.14 Assessing the Tri-Lineage Potential of hiPSCs by Spontaneous Differentiation into Embryoid Bodies
	4 Notes
	References
Differentiating Induced Pluripotent Stem Cells Toward Mesenchymal Stem/Stromal Cells
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Preparation of a Plate for EB Generation
		2.2 EB Generation
		2.3 Changing EB Medium
		2.4 Harvesting EBs
		2.5 Differentiating EBs to MSCs
		2.6 Flow Cytometry Analysis
		2.7 Preparing Cells for Tri-Lineage Differentiation
		2.8 Adipogenic and Osteogenic Differentiation
		2.9 Chondrogenic Differentiation
		2.10 Equipment
	3 Methods
		3.1 Preparation of a Plate for EB Generation
		3.2 EB Generation (Day 0)
		3.3 Changing EB Medium (Day 1)
		3.4 Harvesting EBs (Day 2)
		3.5 Differentiating EBs to MSCs (Day 5)
		3.6 FACS
		3.7 Preparing Cells for Tri-Lineage Differentiation
		3.8 Adipogenic and Osteogenic Differentiation
		3.9 Chondrogenic Differentiation
	4 Notes
	References
A High-Efficiency Method for the Production of Endothelial Cells from Human Induced Pluripotent Stem Cells
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Human iPSC Passaging
		2.2 Plating Human iPSCs for Differentiation
		2.3 Mesoderm Induction
		2.4 Endothelial Specification
		2.5 Human iPSC-EC Harvest and Magnetic Automated Cell Sorting (MACS)
		2.6 Plating CD31+ iPSC-ECs Post-MACS Enrichment
		2.7 Expansion of Human iPSC-ECs
		2.8 Cryopreservation of Human iPSC-ECs
		2.9 Equipment
	3 Methods
		3.1 Human iPSC Passaging (Days -5 to -3)
		3.2 Plating Human iPSCs for Differentiation (Day 0)
		3.3 Mesoderm Induction (Days 1-3)
		3.4 Endothelial Cell Induction (Days 4-6)
		3.5 Human iPSC-EC Harvest and Magnetic Automated Cell Sorting (MACS)
		3.6 Plating CD31+ iPSC-ECs Post-MACS Enrichment
		3.7 Expansion of iPSC-ECs
		3.8 Cryopreservation of Human iPSC-ECs
	4 Notes
	References
Image-Based Quantitation of Kainic Acid-Induced Excitotoxicity as a Model of Neurodegeneration in Human iPSC-Derived Neurons
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Media and Reagents
		2.3 Other Specialty Materials
		2.4 Data Processing and Analysis
	3 Methods
		3.1 Coating of Cultureware
		3.2 Generation of Neural Stem Cells (NSCs)
			3.2.1 Neural Induction of iPSCs
			3.2.2 Passaging of P0 NSCs
			3.2.3 Following Passages of NSCs
			3.2.4 Cryopreserving NSCs
			3.2.5 Thawing NSCs
		3.3 Neuronal Differentiation of NSCs
		3.4 Kainic Acid-Induced Excitotoxicity Assay
		3.5 Data Acquisition
			3.5.1 Using Live Cell Imaging
			3.5.2 Using Live Cell Staining
			3.5.3 Using Immunofluorescence
		3.6 Data Processing
			3.6.1 Manual Counting of Neurite Degeneration
			3.6.2 Automated Quantification
	4 Notes
	References
Amyloid β (Aβ) ELISA of Human iPSC-Derived Neuronal Cultures
	1 Introduction
	2 Materials
		2.1 iPSC-Derived Neuronal Cultures
		2.2 ELISA
	3 Methods
		3.1 Sample Preparation for Aβ Measurement
		3.2 Sandwich Aβ ELISAs
	4 Notes
	References
Creating Cell Model 2.0 Using Patient Samples Carrying a Pathogenic Mitochondrial DNA Mutation: iPSC Approach for LHON
	1 Introduction
	2 Materials
		2.1 Skin Biopsy and Primary Fibroblast Culture
		2.2 Primary Fibroblast Cell
		2.3 iPSC Transfection
		2.4 Plasmids for Transfection
		2.5 iPSCs Cell Culture
		2.6 Alkaline Phosphatase (AP) Stain Kit (Systems Biosciences)
		2.7 RNA Isolation and cDNA Synthesis
		2.8 PSC Cardiomyocyte Differentiation Kit (ThermoFisher Scientific)
		2.9 Retinal Ganglion Cell Media
	3 Methods
		3.1 Obtaining Primary Fibroblasts by Skin Biopsy
		3.2 Reprogramming of Primary Fibroblast
		3.3 Manual Picking and Expansion of iPSC Clones
		3.4 Pluripotency Confirmation of iPSC Clones
		3.5 Analysis of Endogenous Pluripotency of iPSC
		3.6 Differentiation of iPSCs into Cardiomyocytes
		3.7 Generation of Retinal Ganglion Cells
		3.8 Confirmation of Retinal Ganglion Cells
	4 Notes
	References
CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells
	1 Introduction
	2 Materials
		2.1 Plasmids
		2.2 Cells
		2.3 Software
		2.4 Reagents and Consumables
		2.5 Equipment
	3 Methods
		3.1 Puromycin (PURO) Concentration Titration in Cas9+MNhiPSC
		3.2 Lentiviral CRISPR gRNA Library Titration in Cas9+MNhiPSC
		3.3 CRISPR gRNA Library Screens in Cas9+MNhiPSC
		3.4 Sequencing Libraries Preparation and Deep Sequencing
	4 Notes
	References
Generation and Encapsulation of Human iPSC-Derived Vascular Smooth Muscle Cells for Proangiogenic Therapy
	1 Introduction
	2 Materials
		2.1 Cell Culture Equipment and Consumables
		2.2 Feeder-Free Human iPSC Culture
		2.3 VSMC Differentiation
		2.4 Cell Encapsulation
	3 Methods
		3.1 Feeder-Free Human iPSC Culture (See Notes 1-5)
		3.2 VSMC Differentiation Protocol (See Notes 6-15)
			3.2.1 Embryoid Body (EB) Formation (See Notes 6 and 7)
			3.2.2 EB Differentiation in Suspension (See Notes 8 and 9)
			3.2.3 EB Differentiation on Gelatin Plate (See Notes 10 and 11)
			3.2.4 Differentiation on Matrigel Plate/G0 Stage of Differentiation (See Note 12)
			3.2.5 G1 Stage of Differentiation/P0 Stage (See Notes 13-15)
		3.3 Cell Encapsulation Strategies (See Notes 16-26)
			3.3.1 Hydrated Collagen Scaffold (See Notes 16-21)
			3.3.2 In Situ Collagen Hydrogel (See Notes 22-26)
	4 Notes
	References
Methods to Induce Small-Scale Differentiation of iPS Cells into Dopaminergic Neurons and to Detect Disease Phenotypes
	1 Introduction
	2 Materials
		2.1 Maintenance of Human iPSCs
		2.2 Induction into Midbrain Dopaminergic Neurons
		2.3 Image-Based Analysis
	3 Methods
		3.1 iPSC Maintenance
		3.2 Neural Induction into Midbrain Dopaminergic Neurons
		3.3 Image-Based Phenotype Detection and Evaluation of the Drug Effects
	4 Notes
	References
Modeling Early Neural Crest Development via Induction from hiPSC-Derived Neural Plate Border-like Cells
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Cell Culture Plastics
		2.3 General Cell Culture Reagents
		2.4 For hiPSC Medium
		2.5 For NBC Medium
		2.6 For NCC Medium
	3 Methods
		3.1 Preparation of Matrigel-Coated Dishes (Fig. 2)
		3.2 hiPSC Cell Culture
			3.2.1 hiPSCs Thawing and Maintenance (Fig. 3)
			3.2.2 For hiPSC Passaging (Fig. 4)
			3.2.3 For hiPSC Cryopreservation (Fig. 5)
		3.3 NBC and NCC Differentiation
			3.3.1 hiPSCs Harvesting and Seeding in Preparation for Differentiation (Fig. 6)
			3.3.2 NBC Differentiation (Time to Results: 4 Days) (Fig. 7)
		3.4 NCC Differentiation (Time to Results: 7 Days for EMT; 15 Days for Fully Converted NCCs) (Fig. 8)
	4 Notes
	References
Generation of Human Induced Pluripotent Stem Cells from Renal Epithelial Cells
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Medium Configuration
	3 Methods
		3.1 Isolation and Proliferation of Renal Epithelial Cells
		3.2 Reprogramming of Renal Epithelial Cells
	4 Notes
	References
A Protocol for Stepwise Differentiation of Induced Pluripotent Stem Cells into Retinal Pigment Epithelium
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Culture Dishes and Consumables
		2.3 Preparation of Culture Media
		2.4 Preparation of Solutions
			2.4.1 Mitomycin C
			2.4.2 CTK
			2.4.3 0.1% BSA/PBS Solution
			2.4.4 bFGF Solution for iPSC Maintenance
			2.4.5 bFGF Solution for iPSC-RPE Maintenance
			2.4.6 Y-27632 Solution
			2.4.7 SB Solution
			2.4.8 CKI Solution
		2.5 Coating of Culture Dishes
			2.5.1 Gelatin-Coated Dish
			2.5.2 CELLstart-Coated Dish
	3 Methods
		3.1 Preparation of MEF Feeder Cultures
		3.2 Maintenance and Passage of iPSCs
		3.3 Differentiation of iPSCs to Retinal Endothelium
	4 Notes
	References
Genome Editing of Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes to Model Genetic Ocular Disease
	1 Introduction
	2 Materials
	3 Methods
		3.1 Synthesis of Single Guide (sg) RNA
		3.2 Screening sgRNAs
		3.3 Electroporation of iPSCs
		3.4 Low-Density Seeding and Picking Colonies
		3.5 Screening Colonies
		3.6 Thawing Frozen Clones from a 96-Well Plate
	4 Notes
	References
Generation of Cardiomyocytes and Endothelial Cells from Human iPSCs by Chemical Modulation of Wnt Signaling
	1 Introduction
	2 Materials
		2.1 Preparation of Matrigel-Coated Plates
		2.2 Preparation of Cardiomyocyte Differentiation Media
		2.3 Preparation of Endothelial Differentiation Media
		2.4 Preparation of Gelatin-Coated Plates
		2.5 Preparation of Solutions for Immunofluorescence Staining
		2.6 Preparation of Solutions for Flow Cytometry
	3 Methods
		3.1 Chemically Defined Cardiomyocyte Differentiation
			3.1.1 Cardiomyocyte Differentiation from Human iPSCs
			3.1.2 Passage Human iPSC-CMs
		3.2 Chemically Defined Endothelial Cell Differentiation
		3.3 Immunofluorescence Staining
		3.4 Flow Cytometry
	4 Notes
	References
Immunoassay for Quantitative Detection of Antibody Transcytosis Across the Blood-Brain Barrier In Vitro
	1 Introduction
	2 Materials
	3 Methods
		3.1 Test Plate to Evaluate Detection Antibodies for Linear Detection and Cross-Reactivity
			3.1.1 Sample Preparation
			3.1.2 Loading of Wes Plate
		3.2 Analysis of the Data
		3.3 Quantification of Transport in BBB Transwell Samples
	4 Notes
	References
Generation of Cortical, Dopaminergic, Motor, and Sensory Neurons from Human Pluripotent Stem Cells
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 List of Antibodies and qPCR Primers
		2.3 In-House Recipes for N2 and Collagenase IV Solution
		2.4 Preparation of Basal Media
		2.5 Preparation of Neuronal Specification Media
	3 Methods
		3.1 Maintenance of iPSCs
		3.2 Accutase-Based Single-Cell Dissociation
		3.3 Generation of Cortical Neurons from hiPSCs
		3.4 Generation of Midbrain Dopaminergic Neurons from hiPSCs
		3.5 Generation of Spinal Motor Neurons from hiPSCs/hESCs
		3.6 Generation of Sensory Neurons from hiPSCs/hESCs
	4 Notes
	References
CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent S
	1 Introduction
	2 Materials
		2.1 Reagents and Materials
			2.1.1 Molecular Biology Reagents
			2.1.2 CRISPR/Cas Reagents
			2.1.3 iPSC Culture Reagents
		2.2 Laboratory Consumables
		2.3 Specific Equipment
	3 Methods
		3.1 Design and Generation of AAVS1-Fluorescent Biosensor Homology-Directed Repair Template
			3.1.1 Gibson Assembly of GEFB with AAVS1-Donor Vector
			3.1.2 Quality Control for AAVS1-Fluorescent Biosensor Plasmid
		3.2 iPSC Editing Using CRISPR-Cas and Homology-Directed Repair
			3.2.1 Assembly of CRISPR/Cas Ribonucleoprotein
			3.2.2 Nucleofection of iPSC and Puromycin Selection
			3.2.3 Cryopreservation of Cell Cultures
			3.2.4 Isolation and Expansion of AAVS1-GEFB Monoclonal iPSC Line
		3.3 Quality Control of Monoclonal iPSC Lines
		3.4 Testing Fluorescent Biosensor Model Functionality
	4 Notes
	References
Cancer Stem Cell Initiation by Tumor-Derived Extracellular Vesicles
	1 Introduction
	2 Materials
	3 Methods
		3.1 Conditioned Medium Collection
		3.2 Isolate EVs from CM
		3.3 Mouse iPSC/ESC Managing
		3.4 Conversion of Mouse iPSCs/ESCs into CSCs
		3.5 miPS-LLCev Cells Preparation for Injection
		3.6 Expected Results
		3.7 Conclusion
	4 Notes
	References
Genome Editing Using Cas9-gRNA Ribonucleoprotein in Human Pluripotent Stem Cells for Disease Modeling
	1 Introduction
	2 Materials
		2.1 Media
		2.2 Reagents
	3 Methods
		3.1 Standard PSCs Passage
		3.2 PSCs Passage Previous to Transfection
		3.3 Cas9-sgRNA Complex Transfection
		3.4 Analysis of sgRNA Efficiency: Cleavage Assay with GeneArt Genomic Cleavage Detection Kit (See Note 9)
		3.5 Clonal Selection
		3.6 Expansion of All Clones
		3.7 Mutation Analysis
		3.8 Expansion of Desired Clones
		3.9 Off-Target Analysis
		3.10 Karyotyping
		3.11 Long-Term Cryostorage
	4 Notes
	References
Index