Epiblast Stem Cells: Methods and Protocols (Methods in Molecular Biology, 2490)

✍ Scribed by Pierre Osteil (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 301
• Category: Library

✦ Synopsis

This book aims to deliver the most up-to-date protocols in using epiblast stem cells (EpiSC) to answer critical questions on mammalian development. Beginning with a section on the spectrum of mouse primed pluripotent stem cells, the volume continues with chapters on toolkits to characterize and study primed stem cells, as well as primed stem cells to engineer gastrulation models. Written for the highly successful Method in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Epiblast Stem Cells: Methods and Protocols serves as an ideal guide to overcome the reproducibility problem by gathering detailed state-of-the-art protocols to perform comprehensive analyses, enabling the reader to opt for the appropriate cell type to model the epiblast.

✦ Table of Contents

Preface
Contents
Contributors
Part I: The Spectrum of Mouse Primed Pluripotent Stem Cells
Chapter 1: Establishment of Mouse Epiblast Stem Cells
1 Introduction
2 Materials
2.1 Epiblast Dissection
2.2 EpiSC Culture
3 Methods
3.1 Epiblast Dissection
3.2 EpiSC Derivation and Maintenance
4 Notes
References
Chapter 2: L-Proline Supplementation Drives Self-Renewing Mouse Embryonic Stem Cells to a Partially Primed Pluripotent State: ...
1 Introduction
1.1 The Early Primitive-Ectoderm-Like (EPL) Cell
2 Materials
3 Methods
3.1 Routine Passaging Protocol for mESCs and for the Conversion of mESCs to EPL Cells
4 Notes
References
Chapter 3: Generation of Epiblast-Like Cells
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Tissue Culture Reagents and Equipment
2.3 Tissue Culture Solutions and Media (See Note 2)
2.4 Immunofluorescence
3 Methods
3.1 ECS Culture (See Note 5)
3.2 Maintenance of MEF Cells
3.2.1 Mitomycin C Treatment of MEF Feeder Layers for Feeder-Dependent ESCs
3.3 Generation of Epiblast-Like Cells (EpiLCs) from naïve ESCs
3.3.1 Naïve ESCs
3.3.2 EpiLCs Differentiation
3.4 Molecular Analysis
3.5 Immunofluorescence Analysis
4 Notes
References
Part II: Toolkits to Characterize and Study Primed Stem Cells
Chapter 4: Identification and Visualization of Protein Expression in Whole Mouse Embryos by Immunofluorescence
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 5: Small Interfering RNA (siRNA) Transfection in Epiblast Stem Cells
1 Introduction
2 Materials
2.1 Specialized Equipment Required
2.2 Materials Required
3 Methods
3.1 siRNA Preparation
3.2 Preparing mEpiSCs for Transfection
3.3 mEpiSC Transfection
3.4 Evaluating mEpiSC Transfection Efficiency
4 Notes
References
Chapter 6: Measuring Endocytosis and Endosomal Uptake at Single Cell Resolution
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Probe Preparation and Processing
2.3 Prepare Stock Solutions
2.4 Flow Cytometry
3 Methods
3.1 Prepare Epiblast-Like Cells (EpiLCs)
3.2 Prepare Cells for Addition of Labeled Probes
3.3 Addition of pHrodo-Conjugated Transferrin to EpiLC
3.4 Addition of pHrodo-Conjugated Dextran to EpiLC
3.5 Addition of Both pHrodo-Conjugated Transferrin and pHrodo-Conjugated Dextran to EpiLC for Multiplex Analysis
3.6 Treat Cells with Appropriate Controls/Inhibitors to Test Endocytic Mechanisms
3.7 Measure Fluorescence at Single Cell Resolution Using Flow Cytometry
4 Notes
References
Chapter 7: 3D Immunofluorescent Image Colocalization Quantification in Mouse Epiblast Stem Cells
1 Introduction
2 Materials
3 Methods
3.1 Coverslip Preparation
3.2 Staining Preparation
3.3 Simultaneous Primary Antibody Staining
3.4 Mounting and Slide Preparation
3.5 Immunofluorescent Colocalization Optimization and Analysis
4 Notes
References
Chapter 8: Flow Cytometric Characterization of Pluripotent Cell Protein Markers in Naïve, Formative, and Primed Pluripotent St...
1 Introduction
2 Materials
3 Methods
3.1 Experiment Planning
3.2 Live Pluripotent Stem Cell Preparation
3.3 Fixed Pluripotent Stem Cell Preparation
3.4 Flow Cytometric Analysis
3.5 Statistical Representations
4 Notes
5 Discussion
References
Chapter 9: Exploring Chromatin Accessibility in Mouse Epiblast Stem Cells with ATAC-Seq
1 Introduction
2 Materials
2.1 General Equipment and Materials
2.2 Preparation of Nuclei: Nuclear Lysis Buffer
2.3 Transposition of Nuclei and Library Preparation
3 Methods
3.1 Preparation of Nuclei from EpiSC
3.2 Tagmentation of Accessible Chromatin
3.3 Initial PCR Amplification of Transposed DNA
3.4 Additional Cycle Determination Using qPCR
3.5 Final Amplification of Library
3.6 Magnetic Bead-Assisted Purification of Amplified Library
4 Notes
References
Chapter 10: A Reproducible and Dynamic Workflow for Analysis and Annotation of scRNA-Seq Data
1 Introduction
2 Materials
2.1 Download and Install Cell Ranger
2.2 Download and Unpack Mouse Reference Genome
2.3 Install R Packages
3 Methods
3.1 Mapping 10x Reads
3.2 Setting up General Parameters
3.3 Creating Seurat Object and Quality Control
3.4 Subsetting Data in Seurat Object
3.5 Subsampling Seurat Object
3.6 Normalization and Clustering
3.7 Regress out for Cell Cycle Genes
3.8 Convert Seurat Object into singleCellExperiment (SCE) Object
3.9 Removing Empty Droplets and Doublets from Datasets
3.10 Calculate Pseudotime Using Slingshot
3.11 Cell Type Annotation Using Clustifyr
3.12 DEGs Analysis Using Seurat
3.13 Determine Cell Proportion of Annotated Cells
3.14 Co-expressed Genes
3.15 Clustering Using Hclust
3.16 Session Info
4 Notes
5 Code Availability
References
Chapter 11: Complete Transcriptome Analysis by 5′-End Single-Cell RNA-Seq with Random Priming
1 Introduction
2 Materials
2.1 AutoPrep System and Reagents
2.2 C1 CAGE Oligonucleotides
2.3 Materials and Laboratory Equipment
2.4 Reagents
2.5 Library Preparation
3 Methods
3.1 Prepare C1 CAGE Master Mixes
3.2 Prime the C1 Array
3.3 Prepare Cell Suspension
3.4 Load Cell, Imaging and Run C1 CAGE Script on the C1 System (Cell Lysis, RT, and cDNA Amplification)
3.5 Transfer cDNA Amplified Product into 96-Well Plate and Perform QC
3.6 Tagmentation and Amplify Fragmented DNA
3.7 Purify and Clean Up the Library
3.8 NGS
4 Notes
References
Chapter 12: Global Proteomic Profiling of Embryonic Stem Cells Using iTRAQ Isobaric Tags with LC-MS/MS Quantification
1 Introduction
2 Materials
2.1 Cell Lysates
2.2 Bradford Assay
2.3 iTRAQ Labeling
2.4 High pH Reverse Phase Liquid Chromatography (RPLC)
2.5 Desalting
2.6 Liquid Chromatography-Mass Spectrometry (LC-MS/MS)
2.7 Data Analysis
3 Methods
3.1 Cell Lysates
3.2 Protein Assay
3.3 Sample Digestion and iTRAQ Labeling
3.4 High pH Reverse Phase Liquid Chromatography (RPLC) and Fractionation
3.5 Desalting
3.6 Liquid Chromatography-Mass Spectrometry (LC-MS/MS)
3.7 Data Analysis
4 Notes
References
Chapter 13: Comprehensive and Comparative Structural Glycome Analysis in Mouse Epiblast-like Cells
1 Introduction
2 Materials
2.1 Disruption of Cultured Cells
2.2 Ethanol Precipitation
2.3 N-Glycan Preparation
2.4 GSL and fOS Glycan Preparation
2.5 O-Glycan Preparation and Beta Elimination Reaction
2.6 PMP-Labeled O-Glycan Purification
2.7 GAGs Preparation
2.8 HPLC Analysis
2.9 Glycoblotting Combined with Aminolysis-SALSA
2.10 MALDI-TOF MS Analysis
3 Methods
3.1 Cell Disruption
3.2 Ethanol (EtOH) Precipitation
3.3 N-Glycan Preparation
3.4 GSL Preparation
3.5 FOS Preparation
3.6 O-Glycan Preparation and β-Elimination Reaction
3.7 PMP-Labeled O-Glycan Purification
3.8 GAG Preparation and 2-AB Labeling
3.9 HPLC Analysis
3.10 Glycoblotting Combined with Aminolysis-SALSA
3.11 aoWR-Labeled Glycan Purification
3.12 MALDI-TOF MS Analysis
4 Notes
References
Part III: Primed Stem Cells to Engineer Gastrulation Model
Chapter 14: Generation of Gastruloids from Epiblast-Like Cells
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Tissue Culture Reagents and Equipment
2.3 Tissue Culture Solutions and Media (See Note 2)
2.4 Immunofluorescence Reagents and Equipment
3 Methods
3.1 Culture Conditions Prior to Gastruloids Formation
3.2 Generation of EpiLCs-Derived Gastruloids
3.3 Gastruloids Characterization
3.3.1 Morphometric Analysis
3.3.2 Immunofluorescence Analysis (See Note 12)
4 Notes
References
Chapter 15: Definitive Endoderm from EpiSC Aggregates in Matrigel
1 Introduction
2 Materials
2.1 Media Preparation
2.2 Culture Dishes
2.3 Proteolytic Enzymes
3 Methods
3.1 Adapting EpiSCs to the Serum-Free Culture Medium (See Note 2)
3.2 Plating Dissociated EpiSCs into Nonadherent Culture Dishes
3.3 Embedding Floating EpiSC Aggregates in Matrigel for Further Cell Culture
3.4 Isolating DE Cells from Cell Aggregates
4 Notes
References
Chapter 16: In Vitro Differentiation of Murine Embryonic Stem Cells (ESCs) into Primordial Germ Cell-like Cells (PGCLCs)
1 Introduction
2 Materials
2.1 Feeder-Free and Serum-Free Culture of ESCs and Cryopreservation
2.2 EpiLC Differentiation
2.3 PGCLC Induction
2.4 Detection of PGCLCs by FACS
2.5 Gene Expression Analysis of Spheroids Using qRT-PCR
2.6 Processing Spheroids for Immunofluorescent Staining
3 Methods
3.1 Feeder-Free and Serum-Free Culture of ESCs and Cryopreservation (for One Well of a 12-Well Culture Dish)
3.2 EpiLC Differentiation (for One Well of a 12-Well Culture Dish)
3.3 PGCLC Induction
3.4 Detection of PGCLCs by FACS
3.5 Gene Expression Analysis of Spheroids Using qRT-PCR
3.6 Processing Spheroids for Immunofluorecent Staining
4 Notes
References
Chapter 17: Differentiation of Human-Induced Pluripotent Stem Cells (hiPSCs) into Human Primordial Germ Cell-like Cells (hPGCL...
1 Introduction
2 Materials
2.1 General Reagents, Disposables, and Equipment
2.1.1 Reagents
2.1.2 Disposables
2.1.3 Equipment
2.2 Feeder-Free and Serum-Free Culture of Primed hiPSCs
2.3 Incipient Mesoderm-like Cell (iMeLC) Differentiation
2.4 Human Primordial Germ Cell-like Cells (hPGCLCs) Induction
2.5 Identification of hPGCLCs by Fluorescence-Activated Cell Sorting (FACS)
3 Methods
3.1 Feeder-Free and Serum-Free Culture of Primed hiPSCs
3.2 Incipient Mesoderm-like Cell (iMeLC) Differentiation
3.3 Human Primordial Germ Cell-like Cells (hPGCLCs) Induction
3.4 Identification of hPGCLCs by Fluorescence-Activated Cell Sorting (FACS)
4 Notes
References
Chapter 18: Differentiation of EpiLCs on Micropatterned Substrates Generated by Micro-Contact Printing
1 Introduction
2 Materials
2.1 Fabrication of the SU8 Mold
2.2 Fabrication of PDMS Stamps and PDMS-Coated Coverslips
2.3 Micro-Contact Printing of Fibronectin on Coverslips
2.4 Mouse Embryonic Stem Cells Culture
2.5 EpiLC Conversion and Seeding on Micropatterns
2.6 Initiation of Differentiation and Characterization of Cell Fate by Immunofluorescence
2.7 EpiSC Culture and Differentiation on Micropatterned Substrates
3 Methods
3.1 Fabrication of the SU8 Mold
3.2 Fabrication of PDMS Stamps and PDMS-Coated Coverslips
3.3 Micro-Contact Printing of Fibronectin on Coverslips
3.4 mESC Culture
3.5 Initiation of EpiLC Conversion
3.6 EpiLC Seeding on Micropatterned Substrates
3.7 Initiation of Differentiation and Characterization of Cell Fate of Differentiated EpiLC Colonies by Immunofluorescence
3.8 EpiSC Passaging and Maintenance
3.9 EpiSC Seeding on Micropatterned Substrates
3.10 Initiation of Differentiation and Characterization of Cell Fate of Differentiated EpiSC Colonies by Immunofluorescence
4 Notes
References
Chapter 19: Grafting of Epiblast Stem Cell into the Epiblast and Whole-Embryo Imaging to Unveil Lineage Competence
1 Introduction
2 Materials
2.1 Cell Culture
2.2 PB1 Media (See Note 1)
2.3 Dissection
2.4 Embryo Culture
2.5 Holding and Grafting Pipette Generation
2.6 Embryo Grafting
2.7 Embryo Imaging
2.8 Image Analysis
3 Methods
3.1 Dissection of E7.5 Embryos
3.2 Embryo Culture
3.3 Fabrication of Holding and Microinjection (Grafting) Pipette
3.3.1 Holding Pipette
3.3.2 Microinjection (Grafting) Pipette
3.4 EpiSC Preparation
3.5 Embryo Grafting
3.6 Embryo Imaging
4 Notes
References
Chapter 20: Modeling Epiblast Shape in Implanting Mammalian Embryos
1 Introduction
2 Mathematical Model
2.1 Model Description
2.1.1 Equation of Motion
2.1.2 Internal and Cytoskeleton Forces
2.1.3 External Forces
2.2 Model Discretization
2.3 Example: Simulating Apical Constriction
3 Making of a Rosette-Shaped Epiblast: The Role of Polarized-Apical Constriction
4 Trophectoderm Morphogenesis Regulates Epiblast Shapes in Humans and Mouse
4.1 Lumenogenesis
4.2 Trophectoderm Outgrowth
5 Conclusion
References
Index