Embryonic Stem Cell Protocols

Preface
Contents
Contributors
Dissecting Molecular Phenotypes Through FACS-Based Pooled CRISPR Screens
1 Introduction
2 Materials
2.1 Cloning and PCR
2.2 Cell Culture
2.3 Antibody Staining and Flow-FISH
2.4 Genomic DNA Extraction
2.5 Equipment
3 Methods
3.1 SgRNA Library Design
3.2 SgRNA Library Cloning
3.3 Lentiviral Transduction of the sgRNA Library
3.4 Phenotypic Enrichment
3.5 NGS Library Preparation and Deep Sequencing
3.6 Data Analysis
4 Notes
References
Feeder-Free Human Embryonic Stem Cell Culture Under Defined Culture Conditions
1 Introduction
2 Materials
2.1 Cell Line
2.2 Cell Culture Materials and Reagents
2.3 Preparation of Media and Cell Culture
2.4 Preparation of ROCK Inhibitor
3 Methods
3.1 Preparation of Matrigel and Coating of Plates
3.2 Thawing hESCs by Feeder-Independent mTeSR1 Protocol
3.2.1 Preparation of mTeSR1
3.2.2 Thawing hESCs
3.3 Passaging hESCs by Versene
3.4 Freezing hESCs by mFreSR Serum-Free Cryopreservation Medium
3.5 Preparation of Embryoid Bodies for Differentiation of hESCs
4 Notes
References
Rat-Induced Pluripotent Stem Cells-Derived Cardiac Myocytes in a Cell Culture Dish
1 Introduction
2 Materials
2.1 0.1% Gelatin-Coated Plates
2.2 Medium for Cell Culture
2.3 Constituents for Hanging Drop Method
2.4 Constituents for Immunocytochemistry
2.5 Constituents for cDNA Synthesis and RT-PCR
2.6 Constituents for Western Blotting
2.7 Constituents for Flow Cytometry
3 Methods
3.1 Preparation of 0.1% Gelatin-Coated Plates
3.2 Formation of EBs Using the Hanging Drop Method (First Day)
3.3 Preparation of EBs Suspension (Second Day)
3.4 Preparation of EB Culture Plates (Sixth Day)
3.5 Maintenance of EB Culture (Seventh Day and Alternate Days)
3.6 Observation of EBs
3.7 Characterization of Cardiac Myocyte Markers Using Immunocytochemistry
3.8 Characterization of Cardiac Myocytes Using RT-PCR
3.9 Characterization of Cardiac Myocytes Using Western Blotting
3.10 Characterization of Cardiac Myocytes Using Flow Cytometry
4 Notes
References
Transient Induction and Characterization of Mouse Epiblast-Like Cells from Mouse Embryonic Stem Cells
1 Introduction
2 Materials
2.1 mESC Culture
2.2 mEpiLC Culture
3 Methods
3.1 mESC Differentiation into mEpiLCs
3.2 mEpiLCs Characterization
3.2.1 Morphology
3.2.2 Pluripotency Genes and Protein Expression
4 Notes
References
High Content Image Analysis of Spatiotemporal Proliferation and Differentiation Patterns in 3D Embryoid Body Differentiation M
1 Introduction
2 Materials
2.1 Mouse Embryonic Stem Cell Lines
2.2 Media and Reagents
2.2.1 ESCl/EB Culture Media
2.2.2 Reagents for EdU Labeling Assay
2.2.3 Reagents for Alkaline Phosphatase Activity Assay
2.2.4 Reagents for Immunofluorescence Analysis of Oct4 and Gata4 Expression
2.3 Equipment and Laboratory Plasticware
2.4 Software
3 Methods
3.1 Maintenance of Undifferentiated Mouse ESCs in Feeder-Free Culture System
3.2 Preparation of 3D EB Body Differentiation Model: Formation and Differentiation of EBs
3.2.1 Generation of EBs Using Hanging Drop Methods
3.2.2 Suspension Culture of Differentiating EBs
3.3 Analysis of Growth Dynamics and Proliferation Pattern in EBs
3.3.1 Assessment of EB Growth Rate
3.3.2 Assessment of Cell Proliferation Patterns Using the EdU Labeling Assay
3.4 Analysis of Differentiation Pattern in EBs
3.4.1 Detection of Alkaline Phosphatase Activity in Differentiating EBs
3.4.2 Immunofluorescence Analysis of Oct4 and Gata4 Expression Patterns in Differentiating EBs
3.4.3 Preparation of EB Specimens for Confocal Microscope Scanning (Clearing and Mounting)
3.5 Confocal Microscopy Image Acquisition and Processing
3.5.1 Confocal Microscope Scanning of Differentiating EBs
3.5.2 Building of 3D Projections for Analysis of EB Architecture
3.6 Quantification and High Content Imaging Analysis
3.6.1 The 2D Automated Cell Counter Tool in ImageJ/Fiji
3.6.2 3D Objects Counter in the Fiji/ImageJ
4 Notes
References
Derivation of Multipotent Neural Progenitors from Human Embryonic Stem Cells for Cell Therapy and Biomedical Applications
1 Introduction
2 Materials
2.1 Reagents Grade and Quality Specifications
2.2 Equipment and Consumables
2.3 Cell Culture
2.4 Additional Reagents for Spontaneous lt-NES Differentiation
2.5 Additional Reagents for lt-NES Differentiation to Dopaminergic Neurons
2.6 Additional Reagents for lt-NES Differentiation to Motoneurons
2.7 Coating Cell Culture Plates with VTN-N
2.8 Coating Cell Culture Plates with Laminin 521
2.9 Preparation of N2 Base Differentiation Medium (N2 Medium)
2.10 Growth Factors/Molecules Stock Concentration
3 Methods
3.1 HESCs Maintenance, Passaging, Freezing
3.2 Embryoid Body Formation (Day 0)
3.3 Neural Induction (Day 5)
3.4 Lt-NES Derivation (Day 8/10)
3.5 Lt-NES Maintenance
3.6 Lt-NES Spontaneous Differentiation
3.7 Lt-NES-Directed Differentiation Toward Dopaminergic Neurons
3.8 Lt-NES-Directed Differentiation Toward Motoneurons
4 Notes
References
Feeder-Dependent/Independent Mouse Embryonic Stem Cell Culture Protocol
1 Introduction
2 Materials
2.1 Mouse
2.2 Preparation of Media
2.3 Preparation of Mitomycin C
2.4 Preparation of LIF
3 Methods
3.1 MEF Culture
3.1.1 Isolation
3.1.2 Culture
3.1.3 Treatment
3.2 Mouse Embryonic Stem Cell Culture on MEFs
3.2.1 Thawing and Culturing mESCs on MEFs
3.2.2 Passaging and Freezing mESCs on MEFs
3.3 Feeder-Free Protocol of Mouse Embryonic Stem Cell Culture and Differentiation
3.4 Embryoid Body (EB) Formation from mESCs in Suspension Culture
4 Notes
References
ChIP-qPCR for Polycomb Group Proteins During Neuronal Differentiation of Human Pluripotent Stem Cells
Abbreviations
1 Introduction
2 Materials
2.1 Cell Culture Reagents Preparation
2.2 Chromatin Immunoprecipitation Reagents/Buffers
2.3 Sonication and DNA Extraction
2.4 Real-Time PCR
2.5 Instruments
3 Methods
3.1 Differentiation of hESC and hiPSC into Neuronal Lineage
3.2 Harvesting Differentiated Cells for ChIP Sonication and Preparation of the Sample
3.2.1 Harvesting the Cells for ChIP Assay
3.2.2 Sonication Using Probe Sonicator
3.3 Chromatin Immunoprecipitation
3.4 Results
4 Notes
References
Directed Differentiation of Human Pluripotent Stem Cells into Inner Ear Organoids
1 Introduction
2 Materials
2.1 Reagent Setup
2.2 Preparation of Culture Media
2.3 Equipment
3 Methods
3.1 hPSC Maintenance and Passaging in Feeder-Free Condition
3.1.1 Maintenance
3.1.2 Passaging
3.2 Generating Human Inner Ear Organoids
3.2.1 Day -2: hPSC Aggregation
3.2.2 Day 0: Transfer Aggregates to Differentiation E6
3.2.3 Day 3: bFGF and LDN-193189 Treatment
3.2.4 Day 5: Medium Change and CHIR-99021 Treatment
3.2.5 Day 8: Medium Change
3.2.6 Day 11: Transfer to OMM Containing Matrigel and CHIR
3.2.7 Day 13 and 15: Medium Change with OMM + CHIR
3.2.8 Day 18 and Thereafter: Transition to the Long-Term Culture
4 Notes
References
Glycolytic Profiling of Mouse Embryonic Stem Cells (mESCs)
1 Introduction
2 Materials
2.1 Mouse Embryonic Stem Cell (mESC) Culture and Seahorse XFe24 Live-Cell Metabolic Assay Normalization by Cell Count
2.2 Seahorse XFe24 Live-Cell Metabolic Assay
3 Methods
3.1 Seahorse XF Glycolysis Stress Test
3.1.1 Assay Preparation (Performed the Day Before) (See Notes 1 and 2)
3.1.2 Seahorse Assay Protocol
3.1.3 Normalization of the Seahorse Assay by Cell Count (see Note 8)
4 Notes
References
Replating Protocol for Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Abbreviations
1 Introduction
2 Materials
2.1 Differentiation of hiPSC-CMs
2.2 Coating Tissue Culture Plates with ECM Proteins
2.3 Preparation of Dissociation Reagents
2.4 Preparation of Reseeding Reagents
3 Methods
3.1 Differentiation of hiPSC-CMs
3.2 Replating of hiPSC-CMs by Dissociation with Collagenase A + B
3.3 Replating of hiPSC-CMs by Dissociation with Collagenase II
3.4 Replating of hiPSC-CMs by Dissociation with TrypLE Express
3.5 Replating of hiPSC-CMs by Dissociation with EDTA
4 Notes
References
Hematopoietic Cell Isolation by Antibody-Free Flow Cytometry in the Zebrafish Embryo
1 Introduction
2 Materials
2.1 Isolation of HECs
2.2 Isolation of Erythrocytes
2.3 Isolation of Neutrophils
3 Methods
3.1 Isolation of HECs
3.2 Isolation of Erythrocytes
3.3 Isolation of Neutrophils
4 Notes
References
Human Trophectoderm Spheroid Derived from Human Embryonic Stem Cells
1 Introduction
2 Materials
2.1 Equipment Required for Cell Culture and Spheroid Formation
2.2 Materials Required for hESC Culture
2.3 Materials Required for BAP-EB Formation
2.4 Materials Required for Suspension Culture of BAP-EB
3 Methods
3.1 Preparation of AggreWell Plate
3.2 Preparation of Single-Cell Suspension of hESC
3.3 Formation of EB from hESC
3.4 Differentiation of BAP-EB in Ultra-Low Attachment Plates
4 Notes
References
Embryoid Bodies-Based Multilineage Differentiation of Human Embryonic Stem Cells Grown on Feeder-Free Conditions
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Generation of Suspended Aggregates
2.3 Plating of Cells in Adherent Conditions
2.4 Antibodies
3 Methods
3.1 Generation of Suspended Aggregates
3.2 Medium Change
3.3 Plating Cells in Adherent Conditions
4 Notes
References
A Simple, Rapid, and Cost-Effective Method for Loss-of-Function Assays in Pluripotent Cells
1 Introduction
2 Materials
3 Methods
3.1 Maintenance of Mouse Embryonic Stem Cells
3.2 Design and Cloning of shRNA Expressing Vectors
3.3 Validation of Positive Clones
3.4 Generation of Lentiviral Particles for Infection
3.5 Infection of mESCs
3.6 Quantification of Gene Silencing Efficiency and Phenotype Analysis
3.7 Generation of Concentrated Lentiviral Particles
3.8 Infection of mESCs in Suspension with Concentrated Lentiviral Particles
4 Notes
References
Embryoid Body Formation from Mouse and Human Pluripotent Stem Cells for Transplantation to Study Brain Microenvironment and Ce
1 Introduction
2 Materials
2.1 Supplies
2.2 Stock Solutions for Mouse Embryonic Stem Cell Culture
2.3 Media for Mouse Embryonic Stem Cell Culture
2.4 Media for Human Pluripotent Stem Cell Culture
2.5 Stock Solutions for Embryoid Body-Derived Cell Transplantation and Tissue Processing
3 Methods
3.1 Mouse Embryonic Fibroblast Cell Culture
3.2 Mouse Embryonic Stem Cells Culture
3.2.1 First Stage of Mouse Embryonic Stem Cell Culture: Expansion Phase
3.2.2 Second Stage of Mouse Embryonic Stem Cell Culture: Embryoid Body Formation
3.2.3 Third Stage of Mouse Embryonic Stem Cell Culture: Neural Precursor Cells Selection
3.2.4 Fourth Stage of Mouse Embryonic Stem Cell Culture: Neural Precursor Cells Expansion
3.2.5 Fifth Stage of Mouse Embryonic Stem Cell Culture: Neural Differentiation
3.3 Human Pluripotent Stem Cell Culture
3.3.1 First Step of Human Pluripotent Stem Cell Culture: Expansion Phase on MEFs
3.3.2 Second Step of Human Pluripotent Stem Cell Culture: Feeder-Free Cell Culture
3.3.3 Third Step of Human Pluripotent Stem Cell Culture: Embryoid Body Formation
3.4 EB Transplantation
3.4.1 Mouse Embryoid Body Cells Suspension for Transplantation
3.4.2 Human Embryoid Body Cells Suspension for Transplantation
3.5 Mouse and Human Embryoid Body Transplantation and Tissue Processing
4 Notes
References
A Modified SMART-Seq Method for Single-Cell Transcriptomic Analysis of Embryoid Body Differentiation
1 Introduction
2 Materials
2.1 mESCs Maintenance and Embryoid Body Differentiation
2.2 Single-Cell RNA-Seq
3 Methods
3.1 Thawing and Culturing mESCs in Feeder-Free Condition
3.2 Passaging Feeder-Free mESCs
3.3 EB Differentiation
3.4 Prepare Single-Cell Suspension
3.5 Single-Cell Sorting
3.6 Single-Cell cDNA Amplification
3.7 Single-Cell Pooling, cDNA Purification, and Quality Check
3.8 Sequencing Library Preparation and Quality Check
3.9 Data Processing and Quality Control
4 Notes
References
Osteogenic Differentiation from Mouse Embryonic Stem Cells
1 Introduction
2 Materials
3 Methods
4 Notes
References
Murine Embryonic Stem Cell Culture, Self-Renewal, and Differentiation
1 Introduction
2 Materials
2.1 Equipment
2.2 Feeder-Dependent ESCs Culture
2.3 ESCs Passaging
2.4 ESCs Freezing
3 Methods
3.1 Isolation and Expansion of MEF
3.2 Mitotic Inactivation of MEF
3.3 Plates Preparation for ESCs Culture (See Note 7)
3.4 Plating ESCs and Maintenance of Pluripotency
3.5 ESCs Passaging
3.6 ESCs Differentiation
4 Notes
References
Mouse Embryonic Stem Cell Culture in Serum-Containing or 2i Conditions
1 Introduction
2 Materials
2.1 Solution Preparations
2.2 Media Preparations
3 Methods
3.1 mESC Growth in Low-Serum 2i4 Medium
3.1.1 Gelatin Coating of Culture Dishes
3.1.2 Thawing mESCs
3.1.3 Passaging mESCs
3.1.4 Freezing and Storage
3.2 mESC Growth in High-Serum Media with MEFs
3.2.1 MEF Growth and Maintenance
Thawing MEFs
Passaging MEFs
Mitomycin-C Treatment of MEFs (See Note 13)
Freezing MEFs
3.2.2 Thawing mESCs on mitoMEFs
3.2.3 Passaging mESCs on mitoMEFs
3.2.4 Removal of mitoMEFs from mESCs (De-MEFfing)
3.2.5 Freezing and Storage
3.2.6 Adaptation of mESCs to Low-Serum and Serum-Free 2i Media
3.2.7 Adaptation of mESCs to High-Serum Medium with MEFs
4 Notes
References
Directed Differentiation of Mouse Embryonic Stem Cells to Mesoderm, Endoderm, and Neuroectoderm Lineages
1 Introduction
2 Materials
2.1 Solution Preparations
2.2 Media Preparations
3 Methods
3.1 Endoderm and Mesoderm Differentiation of mESCs
3.2 Neuroectoderm Differentiation of mESCs
4 Notes
References
Accessing the Human Pluripotent Stem Cell Translatome by Polysome Profiling
1 Introduction
2 Materials
2.1 Sucrose Gradients
2.2 Cell Procedure
2.3 Fraction Isolation
2.4 RNA Extraction and cDNA Synthesis
2.5 RT-qPCR Analysis
3 Methods
3.1 Preparation of Sucrose Gradients
3.2 Cycloheximide-Treated Cells and Lysis (See Note 11)
3.3 Puromycin-Treated Cells and Lysis (See Note 11)
3.4 Setting Up the Gradient
3.5 Setting Up the Fractionation System
3.6 Polysome Fractionation and Sample Collection (See Note 16)
4 Downstream Analysis of Polysome Profile and Fractions
4.1 Extraction of RNA from Sucrose Gradient Fractions (See Note 22)
4.2 cDNA Synthesis
4.3 RT-qPCR Analysis
5 Notes
References
Genome Engineering Human ESCs or iPSCs with Cytosine and Adenine Base Editors
1 Introduction
2 Materials
2.1 gRNA Cloning and Base Editor Plasmids
2.2 Efficiency Testing and Clonal Screening
3 Methods
3.1 gRNA Design and Cloning
3.2 iPSC Lipofection and Sorting
3.3 Measuring Editing Efficiency
3.4 Subcloning and Genotyping
3.5 Optional: Single Allele Genotyping
4 Notes
References
Proteomic Analysis of Human Neural Stem Cell Differentiation by SWATH-MS
1 Introduction
2 Materials
2.1 Cell Culture and Differentiation
2.2 Consumables for Cell Lysis and Sample Preparation
2.3 Chemicals for Cell Lysis and Sample Preparation
2.4 Hardware and Software for Sample Preparation
2.5 Hardware and Software for Data Acquisition and Analysis of SWATH-MS Measurement in Trap-Elute Mode
2.6 Standards for Nano-LC/MS Analysis
3 Methods
3.1 Cell Culture and Differentiation
3.2 Cell Harvesting and Cell Lysate Preparation
3.3 In-Solution Cell Lysate Digestion and Peptide Desalting
3.4 LC-MS/MS Methods and Measurements
3.5 Data Analysis
3.5.1 Skyline Document Transition Settings
3.5.2 Spectral Library Building from IDA Measurements in Skyline and Peptide Settings
3.5.3 Importing the SWATH-MS Data into Skyline and Peak Picking with mProphet
3.5.4 Sample Annotation, Group Comparison and Data Export
3.6 Statistical Analysis: Relative Quantification Using R
4 Notes
References
Index