Preface
Contents
Contributors
Human Induced Pluripotent Stem Cell (iPSC) Handling Protocols: Maintenance, Expansion, and Cryopreservation
1 Introduction
2 Materials
2.1 iPSC Lines
2.2 Cell Culture Reagents
2.3 Preparation of Rho Kinase (ROCK) Inhibitor Stock Solutions
2.4 Preparation of Geltrex Stock Solution
2.5 Plate Coating
2.6 FACS Antibodies
3 Methods
3.1 Thawing and Seeding iPSCs
3.2 Passaging iPSCs for Maintenance and Expansion
3.3 Cryopreservation of iPSCs
3.4 Flow Cytometry Analysis and Sort of iPSCs
3.4.1 Cell Dissociation for Flow Cytometry Analysis and FACS
3.4.2 Staining Protocol
4 Notes
References
Generation of Induced Pluripotent Stem Cells from Human Bone Marrow-Derived Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Consumables
3 Methods
3.1 Isolation of hBM-MSCs
3.2 Flow Cytometry
3.3 Immunofluorescence Staining
3.4 Reprogramming
3.5 Isolation and Expansion of the Colonies
3.6 Characterization of the Colonies
3.6.1 Stem Cell Markers
3.6.2 Flow Cytometry
3.6.3 Gene Expression Analysis
3.6.4 In Vitro Embryonic Body Formation
4 Notes
References
Analysis of Clonal Composition in Human iPSC and ESC and Derived 2D and 3D Differentiated Cultures
1 Introduction
2 Materials
3 Methods
3.1 Prerequisites
3.1.1 Install Git
3.1.2 Installing Perl Modules
3.1.3 Installing R Packages
3.1.4 Download Reference Genome in FASTA Format
3.2 FASTQ Alignment
3.3 PCDH Profiling and Data Plotting
3.4 UCSC Browser Visualization
3.5 Cell Authentication
3.5.1 SNV Calling
3.5.2 Coverage Extraction
3.5.3 Selection of Shared Sequenced Regions Between Samples
3.5.4 Generation of a Correlation Matrix
3.5.5 Similarity Plot Based on SNV Profiles
4 Notes
References
Culturing Human Pluripotent Stem Cells on Micropatterned Silicon Surfaces
1 Materials
1.1 Cell Culture
1.2 Immunofluorescence
1.2.1 General Reagents
1.2.2 Primary Antibodies
1.2.3 Secondary Antibodies
1.2.4 Nuclear Staining
1.2.5 Mounting Medium
2 Methods
2.1 Fabrication of Micropatterned Silicon Substrates
2.2 Coating of Micropatterned Silicon Substrates with Matrigel
2.3 Plating the hPSCs on Silicon Substrates
2.4 Performing Immunofluorescence Using Silicon Substrates
3 Notes
References
Porcine iPSC Generation: Testing Different Protocols to a Successful Application
1 Introduction
2 Materials
2.1 General Lab Equipment
2.2 Swine Fibroblast Isolation
2.3 Fibroblast dedifferentiation: iPSC Generation
2.3.1 Pretreatments to Aid Reprogramming
5β²-AZA-2β²deoxycytidine Demethylation
Bona Fide iPS Cell Extracts
2.3.2 Reprogramming Methods
RNA Reprogramming
Episomal Nucleofection Reprogramming
Sendai Virus Reprogramming
Retroviral Reprogramming
2.3.3 Reprogramming Media
HES Medium-1 L
N2B27 Medium-250 mL
HES Swine Medium (Own Not Published Data)
2.4 Reprogramming Characterization
2.4.1 iPSC Clone Purification
2.4.2 Alkaline Phosphatase Staining
2.4.3 Chromosome Spread Analysis and G-Banding
3 Methods
3.1 Swine Fibroblast Isolation by Enzymatic Digestion
3.1.1 Cell Source
3.1.2 Skin Fibroblasts Isolation
3.2 Fibroblast Dedifferentiation: iPSC Generation
3.2.1 Pretreatments to Aid Reprogramming
5β²-AZA-2β²deoxycytidine Demethylation (See Note 2)
Bona Fide iPSC Extracts (See Note 3)
3.2.2 Reprogramming Methods
RNA Reprogramming (Nonintegrative) (See Note 4)
Episomal Nucleofection Reprogramming (Nonintegrative) (See Note 5)
Sendai Virus Reprogramming (Non-integrative) (See Note 6)
Retroviral Reprogramming (Integrative) (See Note 7)
3.3 Reprogramming Characterization
3.3.1 iPSC Clone Purification
3.3.2 Alkaline Phosphatase (AP) Staining (See Note 9)
3.3.3 Chromosome Spread Analysis and G-Banding (See Note 9)
4 Notes
References
Efficient High-Density hiPSCs Expansion in Simple Dialysis Device
1 Introduction
2 Materials
2.1 Device Design
2.2 hiPSCs Aggregates Forming
2.3 High-Density Expansion in the Simple Dialysis Culture System
2.4 Cell Number Determination
2.5 Glucose and Lactate Measurement
2.6 Evaluation of the Growth Factors Accumulation by ELISA
2.7 Evaluation of Pluripotency by FACS
3 Methods
3.1 Simple Dialysis Culture Device Construction
3.2 hiPSCs Aggregates Forming
3.3 High-Density hiPSCs Expansion
3.4 The Cell Number Determination
3.5 Glucose and Lactate Concentration Measurement
3.6 Evaluation of Growth Factors Accumulation in the Culture Compartment
3.7 Characterization of Pluripotency by FACS
4 Notes
References
Expanding the Differentiation Potential of Already-Established Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Cells
2.2 Vectors and RNA Sequences
2.3 Culture Media and Transduction Reagents and Equipment
3 Methods
3.1 Inducible miR-203 Overexpression in PSCs
3.2 Viral Production and Transduction with Viral Particles
3.2.1 Production of Murine Stem Cell Virus (MSCV) Particles
3.2.2 Infection of Mouse iPSCs with MSCVs Retroviruses
3.3 Transduction of PSCs with miR-203 Mimics
3.4 Monitoring and Quantification
3.4.1 Use of 2-Cell-like Stage Reporters
3.4.2 Generation of Embryoid Bodies from PSCs
4 Notes
References
Generation of Quiescent Cardiac Fibroblasts Derived from Human Induced Pluripotent Stem Cells
1 Introduction
2 Materials
3 Methods
3.1 Cardiac Progenitor Differentiation with GSK3 Inhibitor and Wnt Inhibitor
3.2 Differentiation of Cardiac Progenitors into Proepicardial Cells
3.3 Differentiation of Quiescent Cardiac Fibroblasts from iPSC-Derived Epicardial Cells
4 Notes
References
Inductive Coculture Differentiation of Induced Pluripotent Stem Cells into Cardiomyocytes
1 Introduction
2 Materials
2.1 iPS Cell Lines
2.2 iPS Cell Culture Media and Reagents
2.3 GiWi-Differentiation Media and Reagents
2.4 Immunofluorescence Reagents and Imaging Equipment
3 Methods
3.1 Matrigel Coating for iPS Cell Culture
3.2 iPS Cell Culture
3.3 Cardiac Differentiation with GSK3 and Wnt Inhibitors (GiWi Protocol)
3.4 Inductive Coculture Differentiation of iPS Cells with iCMs
3.5 Qualitative Assays for Cardiomyocytes Using Microscopy
3.6 Quantitative Assay for Cardiomyocytes Using Flow Cytometry
4 Notes
References
3D Microwell Platform for Cardiomyocyte Differentiation of Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Equipment and Reagents for hPSC Expansion and Passage
2.2 Equipment and Reagents for 3D Differentiation of hPSCs into Cardiomyocytes
2.3 Equipment and Reagents for Flow Cytometry Analysis of Cardiac Cells
2.4 Equipment and Reagents for Immunostaining
2.5 Equipment and Reagents for Analysis of Calcium Transients
3 Methods
3.1 hPSC Thawing, Expansion, and Passage
3.2 3D Differentiation of hPSC in Cardiomyocytes
3.2.1 Cell Seeding and Predifferentiation Culture
3.2.2 3D Differentiation of hiPSCs in Cardiomyocytes
3.2.3 Harvesting of hPSC-Derived CMs at the End of Culture
3.3 Flow Cytometry Analysis of Cardiac Cell Markers
3.3.1 Intracellular Markers (cTNT and CALP)
3.3.2 Surface Markers (CD90 and CD31)
3.4 Immunostaining Analysis
3.4.1 Preparation of Gelatin Blocks and the Respective Slices
3.4.2 Immunostaining
3.5 Analysis of Calcium Transients in 3D Aggregates of CMs
4 Notes
References
Scalable Generation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
1 Introduction
2 Materials
2.1 Reagents, Chemicals, and Growth Factors
2.2 Antibodies Primary and Secondary
2.3 Equipment and Instruments
3 Methods
3.1 Stock Solution and Aliquot Preparation
3.2 Culture Human Induced Pluripotent Stem Cells
3.3 Assembling Bioreactor Parts
3.4 Inoculation of hiPSCs and Differentiation into EB-CMs
3.5 Dissociation of Cardiomyocytes
3.6 Flow Cytometry Analysis of Single Cell of hiPSC-CMs
3.7 Immunostaining of EB-CMs
3.8 Fluorescent Labeling and Puromycin Selection of hiPSC-CMs
3.9 Nucleofection
3.10 Differentiation of DSR Cell Lines into hiPSC-CMs, Purification, and Characterization
4 Notes
References
Application of Human Induced Pluripotent Stem Cell Technology for Cardiovascular Regenerative Pharmacology
1 Introduction
1.1 hiPSC-Derived Cardiovascular Cell Culture Platforms
2 Materials
2.1 Two-Dimensional (2D) Monolayer Maintenance of hiPSCs
2.2 CM Differentiation
2.3 Two-Dimensional (2D) Monolayer Endothelial Differentiation of hiPSCs
2.4 Scalability of hiPSC Derivatives in Bioreactors
2.5 Generation of 3D Spheroids from hiPSCs or Their Derivatives
2.6 Two-Dimensional (2D) Monolayer Co-Culture of hiPSC-CMs and -ECs
2.7 Acute and Chronic Models of Doxorubicin-Induced Cardiomyocyte Toxicity
2.8 Cell Cycle/Proliferation Assays
2.9 Methyltiazoletetrazolium (MTT) Cell Viability Assay
3 Methods
3.1 hiPSC Culture
3.2 Cardiomyocyte Differentiation of hiPSCs
3.3 hiPSC-EC Differentiation
3.3.1 FACS of Day 12 hiPSC-ECs
3.3.2 Passage of hiPSC-ECs
3.4 Scalability of hiPSC Derivatives in Bioreactors
3.4.1 Preparation of hiPSC-ECs
3.4.2 The Micro-Matrix Bioreactor
3.4.3 Drug Treatment
3.4.4 Completion of the Drug Treatment Regime
3.4.5 Viability Assessment of Collected hiPSC-ECs
3.5 Generation of 3D Spheroids from hiPSCs or Their Derivatives
3.5.1 Immunostaining of 3D Spheroids
3.6 Two-Dimensional (2D) Monolayer Co-Culture of hPSC-CMs and -ECs
3.7 Acute and Chronic Models of Doxorubicin-Induced Cardiomyocyte Toxicity
3.8 Cell Cycle/Proliferation Assays
3.8.1 5-Bromo-2β²-Deoxyuridine (BrdU) Cell Proliferation Assay
BrdU Labeling
Fixing and Immunostaining of BrdU Labeled Samples
Automated High-Content Image Analysis of BrdU/ Immunofluorescent Stained Plates
3.8.2 Methyltiazoletetrazolium (MTT) Cell Viability Assay
4 Notes
References
Efficient and Safe Method of Generating Induced Pluripotent Stem Cells from Human Skin Fibroblasts and Subsequent Differentiat
1 Introduction
2 Materials
2.1 Equipment
2.2 Medium and Growth Factors
2.3 Medium Preparation
3 Methods
3.1 Reprogramming of Human SF into iPSCs
3.1.1 Transfection with DNA
3.1.2 Transfection with mRNA
3.2 Passaging iPSCs (Feeder Free)
3.3 Freezing of iPSCs
3.4 Characterization of iPSCs
3.4.1 Staining the iPSCs
3.4.2 Trilineage Differentiation
3.4.3 Western Blot Analysis
3.4.4 Quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR)-Array
3.4.5 Flow Cytometry Analysis
3.5 Differentiation of iPSCs into Cardiomyocytes
3.6 Applications
4 Notes
References
An Optical-Flow-Based Method to Quantify Dynamic Behavior of Human Pluripotent Stem Cell-Derived Cardiomyocytes in Disease Mod
1 Introduction
2 Materials
2.1 Maintenance, Differentiation and Adrenaline Treatment of Human PSC into Cardiomyocytes (hiPSC-CMs)
2.2 Phase-Contrast Imaging Acquisition System
2.3 Image Processing and Optical-Flow Analysis
3 Methods
3.1 Differentiation of Human PSCs into Cardiomyocytes
3.2 Image Acquisition of Human PSC-Derived CMs
3.3 Image Processing
3.4 PIV Analysis
3.5 Post Processing and Data Extraction for Mapping Contractile Cycles of CMs
3.6 An Example: Assessing the Effect of Adrenaline on Contractile Behavior of PSC-Derived CMs Using the Optical-Flow-Based Ana...
3.7 Conclusion
4 Notes
References
Production of Innervated Skeletal Muscle Fibers Using Human Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Preparation of Small Molecules and Growth Factors
2.2 Preparation of Matrigel-Coated Cell Dishes
2.3 Preparation of Cell Culture Media
2.3.1 Basal Differentiation Medium
2.3.2 Differentiation Medium Step 1
2.3.3 Differentiation Medium Step 2
2.3.4 Differentiation Medium Step 3
2.3.5 Differentiation Medium Step 4
2.3.6 Differentiation Medium Step 5
2.4 Cell Freezing and Thawing
3 Methods
4 Notes
References
Generation of Human Neural Progenitors from Blood Samples by Interrupted Reprogramming
1 Introduction
2 Materials
2.1 Establishment of NPC Cultures
2.2 Immunostaining
2.3 RT-PCR
2.4 Ca2+ Imaging
3 Methods
3.1 Establishment of NPC Cultures
3.1.1 Day 8-p2
3.2 Characterization of NPC Cultures by Immunocytochemistry
3.3 Characterization of NPC Cultures by RT-PCR
3.4 Characterization of NPC Cultures by Ca2+ Imaging (see Note 25)
4 Notes
References
Robust and Highly Efficient Protocol for Differentiation of Human Pluripotent Stem Cells into Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 hPSC Culture
2.2 hPSC Differentiation into MSCs
2.3 Flow Cytometry
2.4 Trilineage Differentiation and Assessment
2.5 General Consumables and Equipment
3 Methods
3.1 Culture and Maintenance of hPSCs
3.2 Induction of Embryoid Body (EB) Formation (Day 1 and 2)
3.3 Enhancement of EB Expansion and Differentiation Potential by Treatment with RA (Day 3-7)
3.4 Plating hPSC-Derived EBs on Matrigel-Coated Dishes and Differentiation Initiation (Day 8 - Day 12-17)
3.5 Differentiation and Amplification of hPSC-Derived EB into MSCs by Cell Dissociation and bFGF Treatment (Day 12-17 - Day 18...
3.6 Examination of MSC Phenotypes
3.6.1 MSC-Like Morphology
3.6.2 Expression of MSC Markers by Flow Cytometry
3.6.3 Multipotency of the MSCs (Trilineage Differentiation)
Differentiation of MSCs into Adipocytes
Differentiation of MSCs into Chondrocytes
Differentiation of MSCs into Osteocytes
3.7 Expansion, Freezing, and Thawing hPSC-Derived MSCs
3.7.1 Expansion of MSCs
3.7.2 Freezing of MSCs
3.7.3 Thawing of MSCs
4 Notes
References
The Development of Tissue Engineering Scaffolds Using Matrix from iPS-Reprogrammed Fibroblasts
1 Introduction
2 Materials
2.1 Cell Expansion and ECM Production
2.2 ECM Preparation
2.3 ECM-Activated Collagen Slurry Preparation
2.4 ECM-Activated Scaffolds Fabrication
2.5 Scaffold Preparation Before Use
2.6 ECM-Activated Scaffold Seeding
3 Methods
3.1 Post-iPSF Expansion and ECM Production
3.2 Post-iPSF ECM Preparation
3.3 Post-iPSF ECM-Activated Collagen Slurry Preparation (Post-iPSF Coll)
3.4 ECM-Activated Scaffold Fabrication
3.5 Post-iPSF Coll Scaffold Preparation Before Use
3.6 ECM-Activated Scaffold Seeding
4 Notes
References
3D Organoid Culture Using Skin Keratinocytes Derived from Human Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Cells
2.2 Cell Culture Reagents
2.3 Differentiation into Keratinocytes from iPSCs
2.4 Construction of 3D Skin Organoids
2.5 Immunohistochemistry
3 Methods
3.1 NB1RGB Cell Culture
3.2 HEKn Cell Culture
3.3 iPSCs Cell Culture
3.4 Differentiation into Keratinocytes from iPSCs (Fig. 1)
3.5 Construction of 3D Skin Organoids (Fig. 2)
3.6 Immunohistochemistry (Fig. 3)
4 Notes
References
Pluripotent Stem Cell Differentiation Toward Functional Basal Stratified Epithelial Cells
1 Introduction
2 Materials
3 Methods
3.1 Coating
3.2 Single Cell Seeding
3.3 Simple Epithelium Induction
3.4 Passaging Differentiated Cells
3.5 2D Stratification of iKCs
4 Notes
References
Serum-Free Production of Three-Dimensional Hepatospheres from Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Human Pluripotent Stem Cell (hPSC) Culture
2.2 Generation of Self-Aggregated Spheres (Stage 1)
2.3 Differentiation of 3D Aggregates into Definitive Endoderm (Stage 2)
2.4 Differentiation of 3D Aggregates into Hepatoblast (Stage 3)
2.5 Differentiation 3D Aggregates into Hepatocyte (Stage 4)
2.6 Maintenance and Long-Term Culture of 3D Heps (Stage 5)
3 Methods
3.1 Preparation of Agarose Microplate Molds
3.2 Preparation of polyHEMA-Coated Plates
3.3 Stage 1: Formation of hPSCs Aggregates in Agarose Microwell Plates
3.4 Stage 2: Differentiating Self-Aggregated Spheres to 3D Definitive Endoderm
3.5 Stage 3: Generation of 3D Hepatoblasts
3.6 Stage 4: Maturation of 3D Hepatoblasts to Generate 3D Heps
3.7 Stage 5: Long-Term Culture of 3D Heps
3.8 Measuring CYP Activity of Long-Term Cultured 3D Heps
3.9 Assessment of Protein Production Using Enzyme-Linked Immunosorbent Assay (ELISA)
3.10 Immunocytochemistry
4 Notes
References
The Differentiation of Human Induced Pluripotent Stem Cells into Podocytes In Vitro
1 Introduction
2 Materials
2.1 Equipment
2.2 iPSC Differentiation Reagents
2.3 Immunofluorescence Staining Reagents
3 Methods
3.1 Culture
3.1.1 Growing iPSCs on Geltrex
3.1.2 Differentiation of iPSCs into Podocytes
3.2 Immunofluorescent Staining of iPSC-Derived Podocytes
3.2.1 Plating Podocytes
3.2.2 Immunofluorescence Staining
4 Notes
References
A Method for Encapsulation and Transplantation into Diabetic Mice of Human Induced Pluripotent Stem Cells (hiPSC)-Derived Panc
1 Introduction
2 Materials
2.1 Equipment
2.2 Disposables
2.3 Chemicals
2.4 Reagent Preparation
2.5 Mice
3 Methods
3.1 Matrigel Coating
3.2 hiPSC Plating
3.3 In Vitro Differentiation in Planar Conditions
3.4 Encapsulation in Alginate Beads
3.4.1 Cell Preparation
3.4.2 Cell Count and Viability
3.4.3 Encapsulation in Alginate Beads
3.5 In Vitro Differentiation of Encapsulated Pancreatic Progenitors
3.6 DT-Induced Diabetic Mouse Model
3.6.1 DT Administration
3.6.2 Method for Glucose Measurement
3.6.3 Procedure for the Administration of Insulin Pellets
3.7 Xenotransplantation in Diabetic Mice
3.7.1 Beads Preparation
3.7.2 Mouse Preparation and Transplantation
3.7.3 Beads Collection
4 Notes
References
Highly Efficient Differentiation of Human Pluripotent Stem Cells into Pancreatic Progenitors Co-expressing PDX1 and NKX6.1
1 Introduction
2 Materials
2.1 Human Pluripotent Stem Cell (hPSC) Culture
2.2 hPSC Differentiation into Definitive Endoderm (DE)
2.3 hPSC Differentiation into Primitive Gut Tube (PGT)
2.4 hPSC Differentiation into Pancreatic Progenitors (PPs)
2.5 Immunofluorescence
2.6 Flow Cytometry
2.7 General Consumables and Equipment
3 Methods
3.1 Culture and Maintenance of hPSCs
3.2 Induction of Definitive Endoderm (DE) (Stage 1)
3.3 Dissociation of Definitive Endoderm and Induction of Primitive Gut Tube (PGT) (Stage 2)
3.4 Differentiation to Posterior Foregut (Pancreatic Progenitor 1) (Stage 3)
3.5 Differentiation to Pancreatic Progenitors (PP2) (Stage 4)
3.6 Assessment of Pancreatic Differentiation Efficiency
3.6.1 Expression of Pancreatic Markers PDX1 and NKX6.1 by Immunofluorescence
3.6.2 Expression of Pancreatic Markers PDX1 and NKX6.1 by Flow Cytometry
4 Notes
References
Derivation of Three-Dimensional Human Induced Pluripotent Stem Cell-Derived Vocal Fold Mucosa for Clinical and Pharmacological
1 Introduction
2 Materials
2.1 hiPS Cell Culture
2.2 Differentiation of hiPS Cells into Definitive Endoderm (Days 1-3)
2.3 Differentiation of Definitive Endoderm into Anterior Foregut Endoderm (Days 4-7)
2.4 Differentiation of Anterior Foregut Endoderm into Vocal Fold Basal Progenitors (Days 8-12)
2.5 Dissociation of Vocal Basal Progenitors (VBP) and Reseeding of the Cells on Collagen-Fibroblast Constructs (Day 10)
2.6 Differentiation of Vocal Fold Basal Progenitors into VF Stratified Epithelium (Days 12-32)
2.7 VF Fibroblast Cell Cultures for Collagen-Fibroblast Constructs and FAD Conditional Medium Preparation
2.8 Collagen-Fibroblast Constructs Preparation (Day 9)
2.9 Conditional FAD Medium Preparation (Day 11)
3 Methods
3.1 Maintenance of hiPS Cells
3.2 Definitive Endoderm (DE) and Anterior Foregut Endoderm (AFE) Differentiation (Days 1-7)
3.3 VBP Differentiation (Days 8-10) and Collagen-Fibroblast Construct Preparation (Day 9)
3.4 Preparation of FAD and Conditional FAD Media (Day 11)
3.5 Inducing of Stratification of VBP
4 Notes
References
Generation of Human Induced Pluripotent Stem Cells Using Endothelial Progenitor Cells Derived from Umbilical Cord Blood and Ad
1 Introduction
2 Materials
3 Methods
3.1 Isolation of Mononuclear Cells (MNCs) from Blood Samples Using Ficoll Gradient Separation
3.2 Derivation of Endothelial Progenitor Cells (EPCs) from Mononuclear Cells (MNCs)
3.3 Generation of iPSCs from EPCs Using Stemgent StemRNA-SR Reprogramming Kit
4 Notes
References
Development of a Blood-Brain Barrier Permeability Assay Using Human Induced Pluripotent Stem Cell Derived Brain Endothel
1 Introduction
2 Materials
2.1 iPSC Culture and BECs Differentiation
2.2 BEC Cell Plating on Transwell Inserts
2.3 BBB Transport Assay
2.4 Measurement of Transendothelial Electrical Resistance (TEER)
3 Methods
3.1 Preparation of Matrigel Plates for BEC Differentiation
3.2 BEC Differentiation, Days 0-11
3.3 BEC Sub Culturing on Transwell Inserts (Day 11)
3.4 TEER Measurements Using CellZscope (Day 12)
3.5 Experimental BBB Transport-Top to Bottom (A > B)
4 Notes
References
Revised ``hPSC-Sac Method´´ for Simple and Efficient Differentiation of Human Pluripotent Stem Cells to Hematopoieti
1 Introduction
2 Materials
2.1 Culturing of C3H10T1/2 Feeder Cells
2.2 Differentiation of Multipotent HPCs from hPSCs via hPSC-sacs
2.3 Characterization of HPCs Generated from hPSCs In Vitro
3 Methods
3.1 Culturing of C3H10T1/2 and Their Preparation for Hematopoietic Differentiation
3.1.1 Thawing of C3H10T1/2 Cells
3.1.2 Maintenance of C3H10T1/2 Cells
3.1.3 Preparation of Feeder Cells for Hematopoietic Differentiation
3.2 Differentiation of HPCs via a hPSC-sac
3.2.1 Determination of hPSC Density Prior to Differentiation
3.2.2 Preparation of hPSCs for Differentiation
Differentiation of hPSCs on a MEF-Coated 6-cm Culture Dish
Differentiation hESCs on LM511-E8- or Matrigel-Coated 6-Well Plates
3.2.3 Differentiation of hPSCs into hPSC-sacs Containing HPCs
3.2.4 Collection of HPCs from hPSC-sacs
3.3 Characterization of hPSC-Derived HPCs
3.3.1 Flow Cytometric Analysis of Surface Molecules on HPCs
3.4 Perspective
4 Notes
References
Efficient Generation of iPSC-Derived Hematoendothelial Progenitors and Specification Toward T cell Lineage
1 Introduction
2 Materials
2.1 Culture of iPSCs
2.2 Culture of OP9-DL1 Cells
2.3 Differentiation of iPSCs to T cells
2.4 Flow Cytometric Analysis of Surface Markers
3 Methods
3.1 Culture of iPSCs
3.1.1 Thawing
3.1.2 Passaging
3.1.3 Cryopreservation
3.2 Culture of OP9-DL1 Cells
3.2.1 Thawing
3.2.2 Passaging
3.2.3 Cryopreservation
3.3 Generation of HEPs from iPSCs (Days -3 to 5)
3.4 Differentiation of HEPs Toward T cells Using an OP9-DL1 Co-culture System
3.4.1 Preparation of OP9-DL1 Feeder Dishes
3.4.2 Co-culture of HEPs and OP9-DL1 Cells
3.4.3 Transfer of the Differentiated Cells to the New OP9-DL1 Dish (Days 6, 12, and 18)
3.5 Generation of CD8 Single-Positive T cells by T cell Receptor (TCR) Stimulation
3.5.1 TCR Stimulation Using T cell TransAct (Day 24)
3.5.2 Change of TCR Stimulation Medium
3.6 Flow Cytometric Analysis of Surface Markers
4 Notes
References
Derivation and Characterization of Mesenchymal Stem Cells from iPS Cells
1 Introduction
2 Materials
2.1 For Derivation of MSCs from iPSCs
2.2 For Characterization of iPSC-MSCs
3 Methods
3.1 Coat Cell Culture Dish with Matrigel
3.2 Thaw and Culture Human iPS Cells
3.3 Passage Human iPS Cells
3.4 Derive MSCs from iPSCs
3.5 Confirm the Complete Differentiation of iPSCs into MSCs with qRT-PCR Analysis
3.6 Osteogenic Differentiation of iPSC-MSCs
3.7 Adipogenic Differentiation of iPSC-MSCs
3.8 Chondrogenic Differentiation of iPSC-MSCs
3.9 Colony-Forming Unit-Fibroblasts (CFU-F) Assay
3.10 Transwell Cell Migration and Invasion Assay
4 Notes
References
Human Pluripotent Stem Cell Differentiation to Microglia
1 Introduction
2 Materials
2.1 Mesoderm Induction Medium (d0-d4)
2.2 Hematopoietic Differentiation Medium (d4-d6)
2.3 Myeloid Differentiation Medium (d6-d14)
2.4 Microglia Progenitor Medium (d14-d30)
2.5 Microglia Maturation Medium (After FACS Sort for CD14/CX3CR1)
2.6 Microglia Activation
2.7 Antibodies
2.8 Reconstitution Instructions
3 Methods
3.1 Day -3 to 3
3.2 Day 4-10
3.3 Day 10-14
3.4 Day 14-30
3.5 Day 30-52
3.6 Maturation
3.7 Microglia Activation
4 Notes
References
Human Induced Pluripotent Stem Cell-Derived Microglia (hiPSC-Microglia)
1 Introduction
2 Materials
3 Methods
3.1 Differentiation of iPSCs to Hematopoietic Progenitor Cells (HPCs)
3.2 Differentiation of HPCs to Microglia
4 Notes
References
Isolation and Culture of Human-Induced Pluripotent Stem Cell-Derived Cerebral Organoid Cells
1 Introduction
2 Materials
2.1 Reagent and Medium
2.2 Supplies
2.3 Preparation of Reagents, Culture Dishes/Multiple Well Plates, and Cell Culture Medium in Advance
2.3.1 DNase I Stock Solution (5 mg/ml)
2.3.2 Ara-C Stock Solution (5 mM)
2.3.3 Matrigel Stock Solution (2 mg/ml) (See Note 1)
2.3.4 Coating Culture Dishes/Plates with Matrigel Working Solution (80 ΞΌg/ml) (See Notes 1 and 2)
2.3.5 Cell Culture Medium (See Note 3)
3 Methods
3.1 Collect and Wash Cerebral Organoids Derived from Human iPSCs
3.2 Tissue Dissociation
3.3 Cell Trituration
3.4 Cell Plating and Culturing
4 Notes
References
A Simple Method for Generating, Clearing, and Imaging Pre-vascularized 3D Adipospheres Derived from Human iPS Cells
1 Introduction
2 Materials
2.1 Reagent Setup
2.2 Equipment Setup
3 Methods
3.1 Generation of Spheroids and Induction of Differentiation
3.2 Generation of Pre-vascularized Spheroids and Induction of Differentiation
3.3 Fixation of Spheroids/Adipospheres
3.4 EdU Staining
3.5 ORO-Immunofluorescence Staining of Adipospheres
3.6 Immunofluorescence Staining of Spheroids/Adipospheres for Clearing
3.7 Clearing
3.8 Mounting
3.9 Imaging
3.10 Anticipated Results
4 Notes
References
A Method for In Vitro Fabrication of Hybrid Bone/Cartilage Tissue Using Mouse Induced Pluripotent Stem Cells
1 Introduction
2 Materials
3 Methods
3.1 Maintenance Culture of Mouse iPSCs
3.2 Fabrication of Mouse iPSC Spheres
3.3 Hybrid Bone/Cartilage Tissue Differentiation from Mouse iPSCs
3.3.1 Osteogenic (Os) Induction of 3D-iPSC Constructs (See Fig. 1)
3.3.2 Osteo-Chondrogenic (Os-Chon) Induction of 3D-iPSC Constructs (See Fig. 2)
4 Notes
References
Differentiation of Human Induced Pluripotent Stem Cells (hiPSC) into Endothelial-Type Cells and Establishment of an In Vitro B
1 Introduction
2 Materials
2.1 HiPSC Culture
2.1.1 Reagents
2.1.2 Solutions
2.2 Preparation of Primary Astrocyte Culture
2.2.1 Reagents
2.2.2 Preparation of Solutions
2.3 HiPSC Differentiation and Establishment of Two-Chamber In Vitro BBB Model
2.3.1 Reagents
2.3.2 Preparation of Solutions
2.4 Supplies
3 Methodology
3.1 Feeder-Free hiPSC Culture Protocol
3.1.1 Matrigel-Coated Plate
3.1.2 Thawing hiPSC Cells
3.1.3 Passaging hiPSC Cells (See Note 5)
3.2 Preparation of Rat Primary Astrocyte Culture
3.2.1 PDL-Coated Plate
3.2.2 Procedure of Primary Culture of Rat Cortical Astrocytes
3.2.3 Dissociation for Collecting Astrocytes
3.3 Differentiation of hiPSC into iPSC-Endothelial Cells (iPSC-EC) and Establishment of the Two-Chamber In Vitro BBB Model
3.3.1 Differentiation of hiPSC into iPSC-EC
3.3.2 The Establishment of the Two-Chamber In Vitro BBB Model (Fig. 1)
Coating Transwell Insert
Preparation of hiPSC-ECs
4 Notes
References
CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 General Molecular Biology Reagents
2.2 sgRNA In Vitro Transcription (IVT)
2.3 hiPSC Culture
2.4 Transfection Reagents
3 Methods
3.1 PCR Screening Strategy and sgRNA Design
3.2 sgRNA Generation by IVT
3.3 Determining the On-Target Activity of the sgRNAs
3.4 ssODN Design
3.5 Targeted Introduction of Specific Mutations in hiPSCs-Electroporation Procedure
3.6 Targeted Introduction of Specific Mutations in hiPSCs-Lipofection Procedure
3.7 Screening the Bulk Population of Transfected hiPSCs
3.8 Clonal Isolation of Transfected hiPSCs by Single Cell Deposition
3.9 Replicating Clonal hiPSCs Cultured in 96-Well Plates
3.10 Screening the hiPSC Clones
3.11 Cryopreservation of Clonal hiPSCs from 96-Well Plates
3.12 Thawing Targeted Clonal hiPSCs
4 Notes
References
CRISPR/Cas9-Mediated Gene Knockout and Knockin Human iPSCs
1 Introduction
2 Materials
2.1 General Reagents
2.2 Equipment
2.3 Reagents for Transfection
2.4 Electroporation of Human Embryonic Stem Cells (hESCs) or iPSCs with CRISPR Reagents and Single-Cell Cloning
3 Methods
3.1 Guide Design and Cas9 Delivery
3.2 Transfection of the Cas9-RNP Complex
3.3 Mutation Detection with T7EI
3.4 Editing hiPSC by Electroporation
3.5 Single-Cell Cloning of Edited Cells
3.6 Culturing Single-Cell Colonies
4 Notes
References
Generation of Monoclonal iPSC Lines with Stable Cas9 Expression and High Cas9 Activity
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cells
2.3 Software
2.4 Reagents and Consumables
3 Methods
3.1 Lentivirus Package
3.2 Transduction of Human iPSCs with Lentiviral Cas9-BSD
3.3 Blasticidin (BSD) Selection of Transduced iPSCs
3.4 Subcloning to Generate Single Cell-Derived iPSC Colonies
3.5 Picking Up Single Cell-Derived iPSC Colonies
3.6 Transduction of Cas9 Expressing Monoclonal iPSC with BFP-GFP Reporter
3.7 Analysis of BFP and GFP Signal by FACS
4 Notes
References
CRISPR/Cas9-Mediated Genome Editing to Generate Clonal iPSC Lines
1 Introduction
2 Materials
2.1 Cloning sgRNA into Cas9-Expressing Plasmid
2.2 iPSC Culture and Passaging
2.3 Transfection of CRISPR/Cas9 Components into iPSCs
2.4 Single-Cell Sorting by FACS
2.5 Genotyping and Cryopreservation of iPSC Colonies
3 Methods
3.1 Identifying sgRNA Targeting Sequences
3.2 Cloning sgRNA into Cas9-Expressing Plasmid
3.3 Transformation of Ligation Reaction
3.4 Coating Culture Plates with Matrigel
3.5 Thawing hiPSCs
3.6 Passaging hiPSCs
3.7 Nucleofection of hiPSCs
3.8 Single-Cell Sorting by FACS
3.9 Genotyping of Surviving Colonies
3.10 Cryopreservation of hiPSCs in 96-Well Plate Format
3.11 Verification of iPSC Pluripotency and Quality
4 Notes
References
CRISPR/Cas9 Ribonucleoprotein Complex-Mediated Efficient B2M Knockout in Human Induced Pluripotent Stem Cells (iPSCs)
1 Introduction
2 Materials
2.1 Culture of iPSCs
2.2 Generation of sgRNA Using In Vitro Transcription (IVT)
2.3 Transfection of CRISPR/Cas9 into iPSCs by Nucleofection
2.4 Enrichment of HLA-I-Null iPSCs by Fluorescence-Activated Cell Sorting (FACS)
2.5 Clonal Isolation by Limiting Dilution
2.6 Clonal Validation by Off-Target Analysis
3 Methods
3.1 Culture of iPSCs
3.1.1 Thawing
3.1.2 Passaging
3.1.3 Cryopreservation
3.2 Generation of sgRNA Using In Vitro Transcription (IVT)
3.3 Transfection of CRISPR/Cas9 into iPSCs by Nucleofection
3.3.1 Nucleofection
3.3.2 Evaluation of the B2M Knockout iPSCs
3.4 Enrichment of HLA-I-Null iPSCs by Fluorescence-Activated Cell Sorting (FACS)
3.5 Clonal Isolation by Limiting Dilution
3.6 Clonal Validation
3.6.1 Subculture of iPSC Clones
3.6.2 Off-Target Analysis
Genomic DNA Extraction Using QuickExtract DNA Extraction Solution
Identification of Potential Off-Target Regions Using an Online Tool
Validation of Potential Off-Target Regions by Sanger Sequencing
4 Notes
References
Embryonal Carcinoma and Glioblastoma Cell Lines Derived from Monkey Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Culture of iPSCs (See Note 1)
2.2 Embryoid Body (EB) Formation and Differentiation of iPSCs
2.3 Oncogenic Gene Induction by Retroviral Vectors
2.4 In Vitro Tumor Formation (Soft Agar Colony Formation Assay)
2.5 In Vivo Tumor Formation
2.6 Histological Analysis and Immunohistochemistry
3 Methods
3.1 Culture of iPSCs
3.1.1 Preparation of Feeder Cells
3.1.2 Culture of iPSCs
3.1.3 Passage of iPSCs (See Note 8)
3.1.4 Stock of iPSCs
3.2 Embryoid Body (EB) Formation and Differentiation of iPSCs
3.2.1 EB Formation with Aggrewell400
3.2.2 Hematopoietic Differentiation
3.2.3 Neural Progenitor Cell (NPC) Differentiation
3.3 Oncogenic Gene Induction by Retroviral Vectors
3.3.1 Viral Preparation
3.3.2 Viral Infection
3.4 In Vitro Tumor Formation (Soft Agar Colony Formation Assay) (See Note 11)
3.4.1 Preparation of Agar
3.4.2 Cell Seeding
3.5 In Vivo Tumor Formation
3.6 Histological Analysis by Immunohistochemistry
4 Notes
References
Methods for Isolation and Reprogramming of Various Somatic Cell Sources into iPSCs
Abbreviations
1 Introduction
2 Materials
2.1 Dermal Fibroblasts Isolation
2.2 Blood Cells (CD34+) Isolation
2.3 Keratinocytes Isolation
2.4 Isolation of Epithelial Cells Present in the Urine
3 Methods
3.1 Isolation of Somatic Cells
3.1.1 Dermal Fibroblasts
3.1.2 Blood Cells (CD34+)
3.1.3 Human Keratinocytes
3.1.4 Epithelial Cells Present in the Urine
3.2 Reprogramming Methods
3.2.1 Sendai Virus
3.2.2 Adenoviral Vectors and Infection
4 Notes
References
The Characteristics of Human iPS Cells and siRNA Transfection Under Hypoxia
1 Introduction
2 Materials
2.1 Cell Culture
2.2 siRNA Application for Knockdown
2.3 Real-Time PCR Analysis
2.4 STAT3 Inhibition Analyses
3 Methods
3.1 Cell Culture
3.2 siRNA Application for Knockdown
3.3 Real-Time PCR Analysis
3.4 STAT3 Inhibition Analyses
4 Notes
References
mRNA-Based Reprogramming Under Xeno-Free and Feeder-Free Conditions
1 Introduction
2 Materials
2.1 Cells
2.2 Plasticware and Other Disposables
2.3 Reagents and Media
3 Methods
3.1 Preparation of NM-RNA Reprogramming Cocktail
3.2 Preparation of MSC Culture Medium
3.3 Preparation of MSCs
3.4 Reprogramming
3.5 Isolating iPSCs Clones
3.6 Expanding iPSC Colonies
3.7 Quality Assurance (QA) of iPSC Clones
4 Notes
References
Non-modified RNA-Based Reprogramming of Human Dermal Fibroblasts into Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 Plasticware and Other Disposables
2.3 Cell Culture Media
2.4 Cell Culture Solutions
2.5 Enzymes
2.6 Materials for Reprogramming
2.7 Extracellular Matrix Components
3 Methods
3.1 Isolation, Culture, and Expansion of Fibroblasts from Human Dermis (Fig. 1)
3.2 Reprogramming Steps (Fig. 2)
3.2.1 Day 0: Cell Seeding
3.2.2 Day 1: Transfection (See Note 12)
3.2.3 Day 2-4: Repeat and Complete the Transfection
3.3 Day 5-10: Media Replacements
3.3.1 Colony Formation
4 Notes
References
Efficient Induction of Primate iPS Cells Using a Combination of RNA Transfection and Chemical Compounds
1 Introduction
2 Materials
2.1 RNA
2.2 Cell Culture
2.3 Immunocytochemistry
3 Methods
3.1 Preparation of Dominant-Negative P53 mRNA and d2EGFP mRNA
3.2 Preparation of Conditioned Medium
3.3 Induction of iPS Cells
3.4 Immunofluorescence Analyses of iPS Cells
4 Notes
References
Generation and Cultivation of Transgene-Free Macaque and Baboon iPSCs Under Chemically Defined Conditions
1 Introduction
2 Materials
2.1 Equipment
2.2 Disposables
2.3 Cell Culture Media and Reagents
3 Methods
3.1 Isolation and Cultivation of NHP Skin and Gingival Fibroblasts
3.1.1 Preparations
3.1.2 NHP Fibroblast Isolation
3.1.3 NHP Fibroblast Culture Conditions
3.1.4 Cell Passaging
3.1.5 NHP Fibroblast Cryopreservation
3.2 Reprogramming NHP Fibroblasts
3.2.1 Cell Transfection
3.2.2 Colony Picking
3.2.3 Assessing Reprogramming Efficiency
3.3 NHP-iPSC Maintenance
3.3.1 NHP-iPSC Culture Conditions
3.3.2 Cell Passaging
3.3.3 NHP-iPSC Cryopreservation
3.3.4 Thawing Cryopreserved NHP-iPSCs
3.4 Basic Characterization of NHP-iPSC Lines
3.4.1 Episomal Detection PCR
3.4.2 Mycoplasma Testing
3.4.3 Pluripotent Stem Cell Marker Detection Using NHP-Validated Antibodies
4 Notes
References
Generation of Marmoset Monkey iPSCs with Self-Replicating VEE-mRNAs in Feeder-Free Conditions
1 Introduction
2 Materials
2.1 Equipment
2.2 Disposables
2.3 Reagents and Supplements
2.4 Solutions
2.5 VEE-mRNAs
3 Methods
3.1 Culture of cjFFs
3.2 Transfection of cjFFs with VEE-OKSiM (Table 2)
3.3 Primary Culture and Generation of Intermediate Primary Colonies (First Step of Reprogramming)
3.4 Generation of iPSCs (Second Step of Reprogramming)
3.5 Long-Term Culture of iPSCs
3.6 Cryopreservation of iPSCs
3.7 Thawing Procedure
4 Notes
References
Differentiation of Human Induced Pluripotent Stem Cells into Definitive Endoderm Using Simple Dialysis Culture Device
1 Introduction
2 Materials
2.1 Preparation of Dialysis Culture Device
2.2 Preparation and Maintenance of Adherent Monolayer hiPSC Cultures
2.3 Formation of hiPSC Aggregates
2.4 Endodermal Differentiation of hiPSC Aggregates in Simple Dialysis Culture System
2.5 Harvesting Endodermal Aggregates
2.6 Glucose and Lactate Measurement
2.7 Quantitative Reverse Transcription PCR
2.8 Medium Component
3 Methods
3.1 Preparation of Dialysis Culture Device
3.2 Preparation and Maintenance of Adherent Monolayer hiPSC Cultures
3.3 Formation of hiPSC Aggregates
3.4 Endodermal Differentiation of hiPSC Aggregates in Simple Dialysis Culture System
3.5 Cell Number Determination
3.6 Glucose and Lactate Measurement
3.7 Quantitative Reverse Transcription PCR
4 Notes
References
Detecting and Modulating ER Stress to Improve Generation of Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Retrovirus Production
2.2 Somatic Cell Reprogramming
2.3 Fluorescent Assays to Measure ER Stress
2.4 Luminescent Assay to Measure ER Stress
2.5 Chemical Alleviation of ER stress
3 Methods
3.1 Preparation of OSKM and ER Stress Sensors Retroviruses
3.2 Detecting ER Stress During Cell Reprogramming
3.2.1 Measuring ER Stress by Fluorescent Assay
3.2.2 Measuring ER Stress by Luminescent Assay
3.3 Improving iPSC Generation by ER Stress Alleviation
4 Notes
References
Gene Editing in Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible CRISPR-Cas9 System
1 Introduction
2 Materials
2.1 Plasmids
2.2 Feeder-Free Human iPSC Culture
2.3 Transfection of iPSCs
2.4 Cell Sorting
2.5 Junction PCR
2.6 Western Blotting
2.7 Immunofluorescence
2.8 Designing gRNA
2.9 Cloning of gRNAs
2.10 Lentivirus Production and Transduction
2.11 Cas9 Expression and Gene Editing
2.12 Other Required Materials
3 Methods
3.1 Human iPSC Culture and Maintenance
3.2 Transfection and Selection of Stably Transfected iPSCs
3.3 Single Cell Sorting and Subcloning of iPSCs
3.4 Junction PCR
3.5 Western Blotting
3.6 Screening of Clones by Immunofluorescence
3.7 Designing gRNA and Cloning
3.8 Transformation, Colony Picking and Screening
3.9 Lentivirus Production
3.10 Transduction of iPSCs and Sorting of the Transduced Cells
3.11 Flow Sorting the Transduced Cells
3.12 Inducing Gene Editing
3.13 T7 Endonuclease I (T7EI) Assay
4 Notes
References
Derivation of Clinical-Grade Induced Pluripotent Stem Cell Lines from Erythroid Progenitor Cells in Xenofree Conditions
1 Introduction
2 Materials
2.1 Cells and Episomal Reprogramming Vectors
2.2 Peripheral Blood Mononuclear Cells (PBMNC) Isolation and Cryopreservation
2.3 Cell Culture
2.4 Electroporation Equipment and Reagents
2.5 Antibodies and Buffer for Flow Cytometry
2.6 Other Requirements
3 Methods
3.1 PBMNC Isolation by Ficoll-Paque Premium Density Gradient Centrifugation and Their Cryopreservation
3.2 Expansion of Erythroid Progenitor Cells
3.2.1 Thawing of PBMNC (Day 0 of Erythroid Progenitor Expansion)
3.2.2 Day 2 of Erythroid Progenitor Expansion
3.2.3 Day 4 of Erythroid Progenitor Expansion
3.2.4 Day 6 of Erythroid Progenitor Expansion
3.2.5 Day 8 of Erythroid Progenitor Expansion
3.3 Flow Cytometry of Erythroid Progenitor Cells
3.3.1 Day 9 of Erythroid Progenitor Expansion
3.4 Coating of Tissue Culture Plates with Biolaminin 521 CTG
3.5 Electroporation of Erythroid Progenitor Cells
3.5.1 Day 9 of Erythroid Progenitor Expansion (See Note 6)
3.6 Maintenance of the Reprogramming Plates
3.6.1 Days +1 and +2 of Reprogramming
3.6.2 Day +3 of Reprogramming (See Note 11)
3.6.3 Day +5 of Reprogramming
3.6.4 Day +7 of Reprogramming
3.6.5 Days +8 to +22 of Reprogramming
3.7 Picking of the iPSC Clones
3.8 Maintenance and Passaging of iPSC Clones
3.9 Cryopreservation of iPSC Lines
3.10 Thawing and Expansion of iPSC Lines
4 Notes
References
Generation of Murine Induced Pluripotent Stem Cells through Transposon-Mediated Reprogramming
1 Introduction
2 Materials
2.1 Mice
2.2 MEF Isolation
2.3 Expression Vectors
2.4 Media and Solutions
2.5 Culture Media
2.5.1 MEF Culture Medium
2.5.2 iPS Cell Culture Medium
2.5.3 Wash Solution
2.5.4 Freezing Medium
2.5.5 0.1% Gelatin Solution
2.6 Disposable Sterile Plastic and Tissue Culture Plastic
2.7 Dissection Material
2.8 Chemicals
2.9 Lab Equipment
2.10 Feeder Cells
2.11 Cell Freezing
2.12 Plasmid Construction and Preparation
2.13 Alkaline Phosphatase Assay
2.14 Embryoid Body (EB) Formation
2.15 Immunohistochemistry for Determining the Pluripotency Markers
2.16 Analysis of the Expression of Pluripotency Genes by RT-qPCR
3 Methods
3.1 Isolation of Mouse Embryonic Fibroblasts (MEFs)
3.2 MEF Dissociation and Passage
3.3 Freezing of MEF
3.4 Thawing of MEF
3.5 Preparation of Feeder MEF
3.6 Electroporation of the MEFs with Reprogramming Transposon Systems
3.7 Picking of iPS Colonies
3.8 Characterization of iPSC
3.8.1 Alkaline Phosphatase Staining
3.8.2 Determination of the In Vitro Differentiation Potential by Embryoid Body (EB) Formation
3.8.3 Determination of the In Vivo Differentiation Potential
4 Notes
References
Human Pluripotent Stem Cells for High-Throughput Drug Screening and Characterization of Small Molecules
1 Introduction
2 Materials
3 Methods
3.1 Equipment
3.2 Maintenance of Pluripotent Cells Using Automated Cell Culture
3.3 Coating 384-Well Plates and Cell Seeding
3.4 Automated Media Change in 384-Well Plates
3.5 Compound Administration and Viability Assessment
4 Notes
References
Correction to: Human Pluripotent Stem Cells for High-Throughput Drug Screening and Characterization of Small Molecules
Index