Preface
Contents
Contributors
The piggyBac Transposon as a Platform Technology for Somatic Cell Reprogramming Stu
1 Introduction
2 Materials
2.1 Mouse Embryonic Fibroblast Preparation
2.1.1 Mice
2.1.2 MEF Medium
2.1.3 MEF Isolation
2.1.4 MEF Passage
2.1.5 Cell Counting
2.1.6 Preparation of Frozen MEF Stocks
2.2 Primary (1o) Reprogramming
2.2.1 Gelatin Coating
2.2.2 MEF Defrost
2.2.3 MEF Seeding for PB Transfection
2.2.4 PB Transfection
2.2.5 Mouse iPSC Culture Medium
2.2.6 Doxycycline Solution
2.2.7 Alkaline Phosphatase Staining
2.2.8 Surface Marker Analysis: Flow Cytometry
2.2.9 Whole-Well Fluorescence Microscopy Imaging
2.3 PB-iPSC Clone Screening/Validation for Secondary (2o) Reprogramming Application
2.3.1 Cells
2.3.2 PB-iPSC Colony Picking
2.3.3 PB-iPSC Expansion
2.3.4 Nanog-GFP Reporter Analysis by Flow Cytometry
2.3.5 PB-iPSC Freezing
2.3.6 Splinkerette PCR Screening
2.3.7 Southern Blotting
2.3.8 Preparation of Candidate PB-iPSC Lines
3 Methods
3.1 The PB Vector 1o Reprogramming System
3.2 Mouse Embryonic Fibroblast Preparation
3.2.1 Isolating MEFs
3.2.2 Passaging MEFs
3.2.3 Freezing MEFs
3.3 Primary (1o) Reprogramming of MEFs with PB Transposons
3.3.1 Defrosting MEFs (-d4)
3.3.2 Seeding NanogGFP-IRES-Puro/m2-rtTA MEFs (-d2)
3.3.3 piggyBac Transfection (-d1)
3.3.4 Dox Induction of Reprogramming Factors (d0)
3.3.5 Colony Quantification by Alkaline Phosphatase Staining (d8)
3.3.6 Harvest for Analysis (d8) and Extended Culture (d8-d18)
3.3.7 Whole-Well Fluorescence Microscopy Imaging (d18)
3.3.8 Harvest for Analysis (d18) and Assessing Transgene Independence (d18-d25)
Conducting Flow Cytometry for Reprogramming Markers (d18)
Seeding Cells for Transgene-Independent iPSC Culture (d18-d25)
3.4 Secondary (2o) Reprogramming Systems from Characterized 1o PB-iPSC Clones
3.4.1 PB-iPSC Colony Picking (d25)
3.4.2 Expansion of PB-iPSC Single Clones for Screening (d28)
3.4.3 Flow Cytometry Analysis of PB-iPSC Clones (d30)
3.4.4 Freezing PB-iPSC Clones in a 96-Well Plate (d30)
3.4.5 Verification of PB Insertions in 1o PB-iPSC Clones
3.4.6 Defrosting, Expansion, and Cryopreservation of Candidate Lines
3.4.7 Production of 2o MEFs Through Chimera or Tetraploid Complementation
3.4.8 2o MEF Isolation and Reprogramming
4 Notes
References
Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells Using Sendai Virus
1 Introduction
2 Materials
2.1 Equipment
2.2 Disposables
2.3 Chemicals
2.4 Reagent Setup
3 Methods
3.1 Isolation of Peripheral Blood Mononuclear Cells from Peripheral Blood
3.2 Generation of Human Induced Pluripotent Stem Cells Using Sendai Virus
4 Notes
4.1 Isolation of Pheripheral Blood Mononuclear Cells Not Working
4.2 Erythroblast Expansion Step Not Working
4.3 Sendai Virus Reprogramming Not Working
References
Sendai Virus-Based Reprogramming of Mesenchymal Stromal/Stem Cells from Umbilical Cord WhartonΒ΄s Jelly into Induced Pl
1 Introduction
2 Materials
2.1 Feeder Cells
2.2 Equipment
2.3 Plasticware and Other Disposables (See Note 2)
2.4 Reagents and Media
3 Methods
3.1 Conditioning Medium
3.2 Reprogramming
3.3 Cryopreservation (Vitrification) and Thawing of Vitrified Colonies
3.4 Adapting iPSC to Feeder-Free Conditions
3.5 Assessing Reprogramming Efficiency
3.6 Sendai Virus Detection PCR
4 Notes
References
Very Rapid and Efficient Generation of Induced Pluripotent Stem Cells from Mouse Pre-B Cells
1 Introduction
2 Materials
2.1 Mice
2.1.1 Cells and C/EBPΞ± Transgene
2.2 Media Components and Reagents
2.2.1 For Culture and Reprogramming of Mouse Primary CD19+ B Cells
2.2.2 For Culture of OP9 and S17 Stromal Cells
2.2.3 For Culture of Mouse Embryonic Fibroblasts (MEFs) and PlatE Cells
2.2.4 For Culture of iPS Cells
2.3 Equipment
2.4 Disposables
2.5 Solutions
2.5.1 B Cell Medium
2.5.2 Cell Reprogramming Medium
2.5.3 iPS Cell Medium
2.5.4 OP9/S17 Medium
2.5.5 Interleukin 7, 4, and 15 Stock Solutions
2.5.6 Leukemia Inhibitor Factor (LIF) Stock Solution
2.5.7 beta-Estradiol Stock Solution
2.5.8 Doxycycline Stock Solution
2.5.9 Labeling Buffer
2.6 Immunomagnetic Reagents for B Cell Separation
2.7 Preparation of Gelatin-Coated Dishes
3 Methods
3.1 B Cell Isolation
3.2 Reprogramming of B Cells
3.2.1 Retrovirus Production and B Cell Infection
3.2.2 Plating of B Cells on OP9 or S17 Stromal Cells
3.2.3 Isolating iPS Cell Colonies
3.3 Basic Characterization of iPS Cells
4 Notes
References
Generation of iPS Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors
1 Introduction
2 Materials
2.1 Cells
2.2 MNC Isolation
2.3 Cell Culture
2.4 Episomal Vectors and Nucleofection Kits
2.5 Plastics and Equipments
2.6 Immunohistochemistry and Flow Cytometry
3 Methods
3.1 Ficoll
3.2 MNC Culture
3.3 REF Feeder Preparation
3.4 Nucleofection
3.5 iPSC Generation
3.6 Picking iPSC Colonies
3.7 Long-Term Feeder-Free Culture
3.8 Freezing Down iPSCs
3.9 Thawing iPSCs
3.10 Selection of iPSC Clones Without Residual Episomal Plasmids
3.11 ALP Staining
3.12 Flow Cytometry
3.13 Confocal Imaging
3.14 Teratoma Assay
4 Notes
References
Enhancing Induced Pluripotent Stem Cell Generation by MicroRNA
1 Introduction
2 Materials
2.1 Preparation of Mouse Embryonic Fibroblasts (MEFs)
2.2 MicroRNA Enhanced iPSC Reprogramming
2.3 Derivation and Characterization of iPSC
2.4 Characterization of iPSC
2.5 Irradiating MEFs for Feeder Layer
3 Methods
3.1 Preparation of Mouse Embryonic Fibroblasts (MEFs)
3.2 MicroRNA Enhanced iPSC Reprogramming
3.3 Derivation of iPSC
3.4 Characterization of iPSC
3.5 Irradiating MEFs for Feeder Layers
4 Notes
References
Generation of Partially Reprogrammed Cells and Fully Reprogrammed iPS Cells by Plasmid Transfection
1 Introduction
2 Materials
2.1 Preparation of Mouse Embryonic Fibroblasts from Embryos
2.2 Generation of iPS Cells
2.3 Conversion of Partially Reprogrammed Cells into iPS Cells
2.4 Characterization of iPS Cells
2.4.1 Quantitative Real-Time PCR (Endo-Oct4, Endo-Sox2, and Nanog)
2.4.2 Chimera Formation Analysis
3 Methods
3.1 Preparation of Fibroblasts from OG2+/- and Wild-Type Embryos
3.2 Mitotic Inactivation of MEFs
3.3 Reprogramming into the Pluripotent State with Plasmid Vectors
3.3.1 Induction of Reprogramming by Transfection with Plasmid Vectors
3.3.2 Selecting the iPS Colonies and Partially Reprogrammed Cells
3.4 Conversion of Partially Reprogrammed Cells into Fully Reprogrammed iPS Cells
3.5 Characterization of iPS Cells
3.5.1 Quantitative Real-Time PCR
3.5.2 Chimera Formation
4 Notes
References
Using Oct4:MerCreMer Lineage Tracing to Monitor Endogenous Oct4 Expression During the Reprogrammin
1 Introduction
2 Materials
2.1 Reagents and Supplies
2.2 Antibodies
2.3 Cell Lines and Plasmid Vectors
2.4 Mouse Strains
2.5 Media
3 Methods
3.1 Culturing Fibroblasts from Oct4-MerCreMer mTmG e13.5 Embryos
3.2 Preparing PlatE Cells to Produce Retrovirus
3.3 Culturing Oct4-MerCreMer Fibroblasts for Reprogramming
3.4 Transfecting PlatE Cells
3.5 Retrovirus Infection
3.6 Tamoxifen Addition Time Points During Reprogramming
3.7 Fixing and Nanog Protein Detection
3.8 Expected Results
4 Notes
References
Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons
1 Introduction
2 Materials
2.1 PB Vector Preparation by Gateway Cloning
2.1.1 LR Recombination Reaction
2.1.2 DNA Preparation for Transfection
2.2 Routine Maintenance of Feeder-Free Human iPSCs
2.2.1 Cells
2.2.2 Ff-Human iPSC Culture Medium
2.2.3 Laminin-Coated Tissue Culture Plates
2.2.4 hiPSC Passage
2.2.5 Cell Counting
2.2.6 Preparation of Frozen Ff-hiPSC Stocks
2.3 Establishing Transgenic hiPSCs
2.3.1 Electroporation (EP)
2.3.2 Drug Selection
2.3.3 Population Expansion/Colony Picking and Expansion
2.4 Validation of hiPSCs with Inducible Transgene Expression
2.4.1 Doxycycline Treatment
2.4.2 Reporter Gene Analysis: Imaging
2.4.3 Reporter Gene Analysis: Luciferase Assay
2.4.4 Reporter Gene Analysis: Flow Cytometry
3 Methods
3.1 Gateway Construction of PB Vectors
3.1.1 Combining pENTR-GOI and PB-DEST Vectors by LR Clonase Recombination
3.1.2 DNA Preparation for hiPSC Electroporation
3.2 Feeder-Free Maintenance of Human iPSCs
3.2.1 Preparation of Laminin-Coated 6-Well Tissue Culture Plate
3.2.2 Harvesting Ff-hiPSCs
3.2.3 Preparation of Frozen Ff-hiPSC Stocks
3.3 Establishing of PB-Transgenic hiPSCs
3.3.1 Electroporation (EP)
3.3.2 Drug Selection
3.3.3 Isolation and Expansion of PB transgenic hiPSCs
3.4 Validation of Human iPSCs with Inducible Transgene Expression
3.4.1 Doxycycline Induction
3.4.2 Fluorescence Microscopy for Reporter Gene Expression
3.4.3 Luciferase Assay as an Example of GOI Induction
3.4.4 Flow Cytometry of hiPSC Populations and Clones
3.5 Optional Validation Protocols
4 Notes
References
Generation and Characterization of Rat iPSCs
1 Introduction
2 Materials
3 Methods
3.1 Construction of Lentiviral Vector
3.1.1 Reprogramming Factors
3.1.2 PCR Amplification
3.1.3 Construction of Lentiviral Vector
3.2 Cell Culture
3.2.1 Primary Rat Bone Marrow Cells (BMC) Culture
3.2.2 Primary Rat Fibroblasts (PEF) Culture
3.3 Virus Infection
3.4 Screening for Positive Cells After Reprogramming
3.4.1 Detection of the Infected Cell with Fluorescence and Alkaline Phosphatase (AP) Staining
3.4.2 Picking Positive Cells After Reprogramming
3.5 Characterization of Rat iPS Cells
3.5.1 Alkaline Phosphatase (AP) Staining
3.5.2 Detection of Genes Highly Expressed in Stem Cells
3.5.3 Immunostaining for Pluripotent Markers Nanog and SSEA-1
3.5.4 Determination of the Endogenous and Exogenous Gene Expression
3.5.5 Stem Cell-Specific Promoter Demethylation Detection
3.5.6 Determination of the Telomerase Activity
3.5.7 Determination of the In Vitro Differentiation Potential by Embryonic Body (EB) Formation
3.5.8 Determination of the In Vivo Differentiation Potential by Teratoma Formation
4 Notes
References
Generation of Induced Pluripotent Stem Cells in Rabbits
1 Introduction
2 Materials
2.1 Rabbit Fibroblast (rbF) Derivation
2.2 Retrovirus Production
2.3 Feeder Cell Preparation
2.4 Fibroblast Infection
2.5 RbiPSC Clone Isolation and Culture
2.6 Test of Transgene and Endogenous Gene Expression by RT-PCR
2.7 Analysis of Pluripotency Markers by Immunofluorescence Microscopy and Flow Cytometry
2.8 Analysis of the Expression of Pluripotency Genes by RT-qPCR
2.9 Teratoma Formation
2.10 Karyotyping
2.11 Freezing and Thawing rbFs, MEFs or rbiPSCs
3 Methods
3.1 Rabbit Fibroblast Derivation
3.2 Retrovirus Production and Titration
3.3 Feeder Cell Preparation
3.4 Fibroblast Infection
3.5 RbiPSC Clone Isolation and Culture
3.6 Test of Transgene and Endogenous Gene Expression by RT-PCR
3.7 Analysis of Pluripotency Markers by Immunofluorescence Microscopy and Flow Cytometry
3.8 Analysis of the Expression of Pluripotency Genes by RT-qPCR
3.9 Teratoma Formation
3.10 Karyotyping
3.11 Freezing and Thawing rbFs, MEFs or rbiPSCs
4 Notes
References
Cynomolgus Monkey Induced Pluripotent Stem Cells Generated By Using Allogeneic Genes
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Mouse Embryonic Fibroblast (MEF) Cells
3.2 Preparation of Plat-A Cells
3.3 Preparation of Cynomolgus Monkey Somatic Cells
3.4 Production of Retroviral Vectors
3.5 Generation of iPS Cells
3.6 Passage and Culture of iPS Cells
3.7 Vitrification and Thawing of iPS Cells
4 Notes
References
Induced Pluripotent Stem Cells from Nonhuman Primates
1 Introduction
2 Materials
2.1 Cell Culture Medium
2.2 Enzymes
2.3 Materials for Reprogramming
2.4 Extracellular Matrix Components
2.5 Cells for Preparing Feeder Layers
3 Methods
3.1 Preparation of NHP Skin Fibroblasts
3.2 Reprogramming Using Retroviruses Encoding Oct4, Sox2, Klf4, and c-Myc
3.3 Routine Maintenance of iPS Cells
3.4 Investigation of Pluripotency by Teratoma Formation
4 Notes
References
Computational Biology Methods for Characterization of Pluripotent Cells
1 Introduction
2 Materials
3 Methods
3.1 Install Bioconductor
3.2 Design of the Transcriptomics Experiment to Assess Pluripotency
3.3 Read the Data
3.4 Inspect the Raw Data
3.5 Normalize the Data
3.6 Define the Indexes of the replicates of Each Population
3.7 Perform the Principal Component Analysis (PCA)
3.8 Draw the Bidimensional Principal Component Analysis (PCA)
3.9 Perform the Hierarchical Clustering of Samples
3.10 Annotate the Arrays
3.11 Extract the Official Gene Symbols
3.12 Visualize the Expression of Some Pluripotent Markers
3.13 Calculate the Mean Values of Each Population
3.14 Calculate the Correlation Between the Mean Representatives of Each Population
3.15 Draw the Pairwise Scatterplots
3.16 Final Assessment of Pluripotency Based on Computational Analysis of Transcriptomics Data
4 Notes
References
cGMP-Compliant Expansion of Human iPSC Cultures as Adherent Monolayers
1 Introduction
2 Materials
2.1 Reagents
2.2 Formulating Hypertonic Citrate
2.3 Preparing Recombinant Vitronectin Solution (500mug/mL Stock)
2.4 Preparing Essential 8 Medium
3 Methods
3.1 Maintenance of Adherent Monolayer iPSC Cultures
3.2 Coating Tissue Culture Flasks with Recombinant Vitronectin
3.3 Dissociation of Adherent Monolayer iPSC Cultures Without Direct Colony Contact by the Operator and Minimal Shear Stress
3.4 Quenching the Chelation Reaction and Collecting the Dissociated Colonies
3.5 Distribution and Expansion of Dissociated Colonies
4 Expected Results
5 Notes
References
Analysis of the Mitochondrial DNA and Its Replicative Capacity in Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Culture of Human-Induced Pluripotent Stem Cells
2.2 Oxygen Consumption Measurement
2.3 ATP Assay
2.4 Lactate Assay
2.5 mtDNA Copy Number
2.6 Immunoprecipitation of Methylated DNA (MeDIP)
2.7 Analysis of Gene Expression of mtDNA Replication Factors and mtDNA-Encoded Genes
2.7.1 cDNA Synthesis
2.7.2 Real-Time Quantitative PCR
2.8 Analysis of mtDNA Variants by Next-Generation Sequencing
2.8.1 Long PCR
2.8.2 Purification of PCR Products
2.8.3 Quantification of Double-Stranded DNA
2.8.4 Shearing of Long PCR Amplicons
2.8.5 Sample Purification
2.8.6 Size Selection of the DNA Library
2.8.7 Quantification of the DNA Libraries
2.8.8 DNA Template Amplification Through Emulsion PCR
2.8.9 Enrichment of Template-Positive Ion Sphere Particles
2.8.10 Assessment of the Quality of the Enriched Ion Sphere Particles
2.8.11 Generation of the DNA Libraries for Ion Torrent PGM Sequencing
2.8.12 Sequencing of Libraries with the Ion Torrent PGM
Analysis of Sequence Outputs
2.9 High-Resolution Melting Analysis
3 Methods
3.1 Manual Passage of Human-Induced Pluripotent Stem (iPS) Cells (See Note 1)
3.2 Transition from Manual to Enzymatic Passage
3.3 Enzymatic Passage of iPS Cells
3.4 Spontaneous Differentiation of iPS
3.5 To Measure Oxygen Consumption in iPS Cells
3.6 To Measure ATP Content in iPS Cells
3.7 To Measure Lactate Release by iPS Cells
3.8 Preparation for Standard Curve Using Quantitative PCR
3.9 Determining mtDNA Copy Number
3.10 MeDIP
3.11 Analysis of Gene Expression of mtDNA Replication Factors and mtDNA-Encoded Genes
3.12 Next-Generation Sequencing
3.12.1 Long PCR
3.12.2 Purification of the PCR Products
3.12.3 Quantification of Double-Stranded DNA
3.12.4 Shearing of Long PCR Amplicons (See Note 34)
3.12.5 End Repair of the Sheared DNA
3.12.6 Sample Purification
3.12.7 Adaptor Ligation
3.12.8 Size Selection
3.12.9 Amplification of the 200-bp DNA Libraries
3.12.10 Quantification of the DNA Libraries
3.12.11 Preparation of the IKA DT-20 Solution and Ion Sphere Particles
3.12.12 Preparation of the Aqueous Master Mix for the Emulsion PCR
3.12.13 Generation of the Emulsion
3.12.14 Emulsion PCR
3.12.15 Recovery of the Ion Sphere Particles
3.12.16 Enrichment of the Template-Positive Ion Sphere Particles
3.12.17 Assessment of the Quality of the Enriched Ion Sphere Particles
3.12.18 Initialization of the Ion Torrent PGM Sequencer
3.12.19 Preparation of the Positively Enriched Ion Sphere Particles for Sequencing
3.12.20 Testing of a New Ion Chip
3.12.21 Loading the Ion Sphere Particles on the Ion Chip (See Note 37)
3.12.22 Analysis of Sequence Outputs (See Note 38)
3.13 To Determine Variant Load by High-Resolution Melting Analysis
4 Notes
References
Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines
1 Introduction
2 Materials
2.1 Selection of Cell-Binding Peptides from a Peptide Phage Display Library
2.2 Amplification of Phage Particles from Lysates or Phage Plaques and Quantitation by Titration
2.3 Sequencing of Recovered Phage
2.4 Validation of Phage Binding by Immunofluorescence
2.5 Validation of Peptide Specific Phage Binding by Peptide Competition
2.6 Cell Labeling with Peptide Targeted Qdot Complexes
3 Methods
3.1 Selection of Cell-Binding Peptides from a Peptide Phage Display Library: Round 1
3.1.1 Subtraction of Nonspecific Binders by Pre-adsorption Against hDFa Cells
3.1.2 Selection of Library
3.1.3 Harvesting Phage Particles from Target Cells
3.1.4 Quantitation of Phage Particles in Lysate by Titration
3.1.5 Amplification of Recovered Phage Particles
3.2 Selection of Cell-Binding Peptides from a Peptide Phage Display Library: Round 2 and 3
3.3 Sequencing of Phage DNA from Phage Plaques
3.3.1 Plaque Amplification
3.3.2 Rapid Amplification of Templates by PCR and Sequencing
3.3.3 Peptide Sequence Analysis
3.3.4 Small-Scale Amplification of Phage Particles
3.3.5 Large-Scale Amplification of Phage Particles
3.4 Validation of Phage Binding by Immunofluorescence
3.5 Validation of Peptide Specific Phage Binding by Peptide Competition
3.6 Cell Labeling with Peptide Targeted Qdot Complexes
4 Notes
References
piggyBac Transposon Mediated Reprogramming and Flow Cytometry Analysis of CD44 and ICAM1 Cell-Surface Marker Changes
1 Introduction
2 Materials
2.1 Reprogramming Materials
2.2 FACS Analysis Materials
3 Methods
3.1 Reprogramming of MEF with PB Transposon
3.2 Harvesting Samples for FACS Time Course Analysis
3.3 Antibody Staining of Samples for FACS Time Course Analysis
3.4 Control Samples Required for FACS Time Course Analysis
3.5 FACS Time Course Analysis of Reprogramming Samples
4 Notes
References
Generation and In Vitro Expansion of Hepatic Progenitor Cells from Human iPS Cells
1 Introduction
2 Materials
2.1 Preparation of Mitomycin C-Treated MEF Feeder Cells
2.2 Maintenance and Differentiation of Human iPS Cells into Hepatic Lineage Cells
2.3 Isolation of Hepatic Progenitor Cells from Human iPS Cell-Derived Hepatic Lineage Cells
2.4 In Vitro Long-Term Culture of Hepatic Progenitor Cells
2.5 Characterization of Human iPS Cell-Derived Hepatic Progenitor Cells
2.5.1 Analyses of Hepatocytic Differentiation Steps Derived from Human iPS Cells Using Real-Time (RT) PCR
2.5.2 Analyses of Hepatocytic Marker Gene Expression Using Immunofluorescence Staining
2.5.3 In Vitro Differentiation of Human iPS Cell-Derived Hepatic Progenitor Cells into Mature Hepatocytes
3 Methods
3.1 Preparation of Mitomycin C-Treated MEF Feeder Cells
3.2 Culture and Differentiation of Human iPS Cells into Hepatic Cell Lineages
3.2.1 Culture of Human iPS Cells
3.2.2 Differentiation of Human iPS Cell Culture into Hepatocytic Cells
3.3 Isolation of Hepatic Progenitor Cells from Human iPS Cell-Derived Hepatic Lineage Cells
3.4 In Vitro Long-Term Culture of Hepatic Progenitor Cells
3.5 Characterization of Human iPS Cell-Derived Hepatic Progenitor Cells
3.5.1 Analyses of Hepatocytic Differentiation Steps Derived from iPS Cells Using RT PCR
3.5.2 Analyses of Hepatocytic Marker Gene Expression Using Immunofluorescence Staining
3.5.3 In Vitro Differentiation of Human iPS Cell-Derived Hepatic Progenitor Cells into Mature Hepatocytes
4 Notes
References
Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 iPS Cell-Freezing Medium
2.3 Medium and Growth Factors
3 Methods
3.1 Aseptic Techniques and Biohazard Procedures
3.2 Culture Conditions for iPS Cells
3.2.1 iPS Cell Culture
3.2.2 Passaging iPS Cells
3.2.3 Freezing iPS Cells
3.3 Induction of Differentiation of iPS Cells into Endothelial Cells (ECs)
3.3.1 Materials
3.3.2 Differentiation and Sorting of ECs
4 Notes
References
Derivation of Neural Stem Cells from Mouse Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Feeder Fibroblast, iPSC, and EB Culture Components
2.2 Neural Induction Medium and Neurosphere Culture Components
2.3 Medium Preparation
3 Methods
3.1 Maintenance of iPSCs
3.1.1 Preparation of Plates with Inactivated MEFs
3.1.2 Maintenance of iPSCs in the Undifferentiated State
3.2 Differentiation of iPSCs
3.2.1 Embryoid Body Formation
3.2.2 Enrichment for Neural Precursors with Neural Induction
3.2.3 Expansion of NSCs
3.2.4 Neurosphere Culture
3.2.5 Freezing iPSC-Derived NSCs
4 Notes
References
Production of Retinal Cells from Confluent Human iPS Cells
1 Introduction
2 Materials
2.1 Culture of hiPS Cells
2.2 Generation of NR-Like Structures from hiPS Cells
2.3 Generation of hiPS-Derived RPE Cells
2.4 RNA Extraction and TaqMan Assay
2.5 Inclusion
2.6 Immunostaining
3 Methods
3.1 Maintenance and Expansion of hiPS Cells
3.2 Generation of NR-Like Structures from hiPS Cells
3.3 Maturation of NR-Like Structures by Floating Cultures
3.4 Generation of hiPS-Derived RPE
3.5 RNA Extraction and Analysis of Retinal Differentiation Using TaqMan Assay
3.6 Immunohistochemistry Analysis
4 Notes
References
Differentiation of iPSC to Mesenchymal Stem-Like Cells and Their Characterization
1 Introduction
2 Materials
2.1 Induction of iPSC to MSC-Like Cells
2.2 Initial Flow Analysis
2.3 Osteogenic Induction
2.4 Adipogenic Induction
2.5 Chondrogenic Induction
3 Methods
3.1 Induction of iPSC to MSC-Like Cells
3.1.1 Differentiation of iPSC
3.1.2 Serial Passaging to Select MSC-Like Cells
3.2 Characterization of Cells Generated
3.2.1 Initial Flow Cytometric Analysis During Differentiation
3.2.2 Comprehensive Flow Cytometric Analysis During Differentiation
3.2.3 Tri-lineage Differentiation
4 Notes
References
Hepatic Differentiation from Human Ips Cells Using M15 Cells
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Mitomycin C Treated M15 Cells (MMC-M15 Cells)
3.2 Preparation of Gelatin-Coat Plates
3.3 Preparation of MMC Treated M15 Feeder Plates
3.4 Plating of Human iPS Cells (See Note 10)
4 Notes
References
Determination of Functional Activity of Human iPSC-Derived Hepatocytes by Measurement of CYP Metabolism
1 Introduction
2 Materials
2.1 Human Induced Pluripotent Stem Cell (iPSC) Lines
2.2 Matrigel
2.3 iPSC Culture Medium
2.4 iPSC Differentiation Medium
2.5 Hepatocyte Culture Medium (HCM)
2.6 Promega CYP Assay Kits
2.7 Assay Plate
2.8 Luminometer
2.9 Quantitative Real-Time PCR
3 Methods
3.1 Matrigel
3.1.1 Preparation of the MG Aliquots
3.1.2 Preparation of Matrigel Plates
3.2 Human iPSC Maintenance
3.3 Human iPSC Differentiation
3.4 CYP Measurement
3.4.1 Detection Reagent Reconstitution
3.4.2 Preparation of P450-Glo Luminogenic Substrates
3.4.3 Detection of CYP in Adherent Hepatocytes
3.4.4 Measurement of CYP Activity in Adherent Hepatocytes
3.4.5 Quantification of CYP mRNA Expression in Adherent Hepatocytes
4 Notes
References
Induced Pluripotent Stem Cells: Generation, Characterization, and Differentiation-Methods and Protocols
1 Introduction
2 Materials
3 Methods
3.1 Dissection of the Adult Mouse Ciliary Body and CEC Isolation
3.2 iPSC Generation
4 Notes
References
Mesoderm Differentiation from hiPS Cells
1 Introduction
2 Materials
2.1 hiPS Cell Maintenance
2.2 Formation of EB
2.3 Induction of Mesoderm Cells
2.4 Purification of Mesoderm Cells by FACS
2.5 Culture of Mesoderm Cells
2.6 CFU-F Assay
2.7 Bone Cell Differentiation from Mesoderm Cells
2.8 Cartilage Cell Differentiation from Mesoderm Cells
3 Methods
3.1 In Vitro hiPS Cell Differentiation
3.1.1 Formation of EB
3.1.2 Induction of Mesoderm Cells
3.1.3 Purification of Mesoderm Cells by FACS
3.2 Maintenance, Expansion, and Validation of Mesoderm Cells
3.2.1 Culture of Mesoderm Cells
3.2.2 CFU-F Assay
3.3 Differentiation into Descendants of Mesoderm Cells
3.3.1 Induction of Bone Cells
3.3.2 Alizarin Red Staining
3.3.3 Induction of Cartilage Cells
3.3.4 Alcian Blue Staining
4 Notes
References
Enhancing Human Cardiomyocyte Differentiation from Induced Pluripotent Stem Cells with Trichostatin A
1 Introduction
2 Materials
2.1 Cells
2.2 Cell Culture Medium and Solutions
2.3 Chemicals
2.4 Plasticware
3 Methods
3.1 Fibroblast Expansion, Inactivation, and Feeder Layer Preparation
3.2 Human iPS Cell Culture and Expansion
3.3 Cardiomyocyte Differentiation of Human iPS Cells
4 Notes
References
Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7
1 Introduction
2 Materials
2.1 Reagents, Medium, and Supplies Needed for Expansion of Human ES/iPS Cells
2.2 Reagents, Medium, and Supplies Needed for Expansion and Lentivirus Production in 293T Cells
2.3 Reagents, Medium, and Supplies Needed for EB Differentiation of Human ES/iPS Cells
2.4 Reagents, Medium, and Supplies Needed for Cell Sorting Using FACS
2.5 Reagents, Medium, and Supplies Needed for Expansion and Differentiation of Sorted Myogenic Progenitors
3 Methods
3.1 Matrigel Coating of Plates and Flasks
3.2 Thawing and Expansion of Human Pluripotent Stem Cells in mTeSR Medium
3.2.1 Thawing hES or iPS Cells
3.2.2 Expansion of hES/iPS Cells
3.2.3 Enzymatic Passaging Using Accumax
3.3 Lentivirus Production
3.3.1 Required Plasmids
3.3.2 Lentiviral Production Protocol
3.4 Infection of hES/iPS Cells with the Inducible PAX7 Lentiviral System
3.5 Testing the Infection Efficiency
3.6 EB Differentiation of hES/iPS Cells
3.7 PAX7 Induction and Purification of Myogenic Precursors
3.8 Expansion and Differentiation of Myogenic Precursors
3.8.1 Expansion of PAX7+-Myogenic Precursors
3.8.2 Terminal Differentiation of Expanding Myogenic Progenitors into Myotubes
4 Notes
References
Chondrogenic and Osteogenic Induction from iPS Cells
1 Introduction
2 Materials
2.1 Human-Induced Pluripotent Stem Cells
2.2 Medium, Buffers, and Reagents
2.3 Western Blot Antibody
2.4 Immunohistochemistry (IHC) Antibody
2.5 Staining Solution and Kit
3 Methods
3.1 hiPSC Culture and Differentiation into Embryoid Bodies
3.2 Induction of In Vitro Chondrogenic Differentiation and Analysis
3.2.1 Chondrogenic Pellet Culture with hiPSCs
3.2.2 Chondrogenic Differentiation of hiPSCs in Alginate Gel
3.2.3 Biochemical Assays for DNA and GAG Quantification
3.2.4 Western Blot Analysis for Chondrogenic Protein
3.2.5 Immunohistochemistry (IHC)
3.3 Induction of In Vitro Osteogenic Differentiation and Analysis
3.3.1 Osteoblast Culture of hiPSCs
3.3.2 Alkaline Phosphatase (ALP) Staining
3.3.3 Alizarin Red Staining
3.3.4 Western Blot Analysis for Osteogenic Protein
3.3.5 Immunohistochemistry (IHC)
3.4 In Vivo Implantation Procedure
3.4.1 Osteochondral Defect Model for In Vivo Cartilage Regeneration
3.4.2 Calvarial Defect Model
3.5 Macroscopic Observation and Histology
4 Notes
References
Generation of iPS Cells from Granulosa Cells
1 Introduction
2 Materials
2.1 Isolation and Culture of Granulosa Cells (See Note 1)
2.2 Preparation of Feeder Cells
2.3 Induction of iPS Cells
2.4 Culture, Passage, and Cryopreservation of iPS Cells
3 Methods
3.1 Generation of Mouse iPS Cells from Granulosa Cells
3.1.1 Isolation of Mouse Granulosa Cells
3.1.2 Packaging of Retroviral Vectors
3.1.3 Induction of Mouse iPS Cells from Granulosa Cells
3.1.4 Culture and Passage of mGCiPS
3.1.5 Cryopreservation of mGCiPS Cells
3.1.6 Thawing of mGCiPS Cells
3.2 Generation of iPS Cells from Pig Granulosa Cells
3.2.1 Isolation and Culture of Pig Granulosa Cells
3.2.2 Packaging of Retroviral Vectors
3.2.3 Induction of Pig iPS Cells from Granulosa Cells
3.2.4 Culture and Passage of pGCiPS
3.2.5 Cryopreservation and Thawing of pGCiPS Cells
3.3 Anticipated Results
4 Notes
References
The Characteristics of Murine 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 Western Blotting
3 Methods
3.1 Cell Culture
3.2 siRNA Application for Knockdown
3.3 Real-Time PCR Analysis
3.4 Western Blotting
4 Notes
References
Hepatic Differentiation from Murine and Human iPS Cells Using Nanofiber Scaffolds
1 Introduction
2 Materials
2.1 Murine iPS Cell Differentiation
2.2 Human iPS Cell Differentiation
3 Methods
3.1 Murine iPS Cell: Plating
3.2 Murine iPS Cell: Differentiation
3.3 Human iPS Cell: Preconditioning
3.4 Human iPS Cell: Plate Preparation
3.5 Human iPS Cell: Plating
3.6 Human iPS Cell: Differentiation
3.7 Human iPS Cell: Differentiation (2 Step Methods, Alternative for Responsive Cell Line)
4 Notes
References
Erratum to: Generation of iPS Cells from Granulosa Cells
Index