Pluripotent Stem-Cell Derived Cardiomyocytes (Methods in Molecular Biology, 2320)

✍ Scribed by Yoshinori Yoshida (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 295
• Category: Library

✦ Synopsis

This volume provides methodologies for ES and iPS cell technology on the study of cardiovascular diseases. Chapters guide readers through protocols on cardiomyocyte generation from pluripotent stem cells, physiological measurements, bioinformatic analysis, gene editing technology, and cell transplantation studies. Written in the highly successful Methods in Molecular Biology series format, 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 cutting-edge, Pluripotent Stem-Cell Derived Cardiomyocytes aims to help researchers set up experiments using pluripotent stem cell-derived cardiac cells.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Overview
Chapter 1: Making Cardiomyocytes from Pluripotent Stem Cells
1 Introduction
2 Generating Cardiomyocytes at Large Scale
3 Confirming Function
4 Disease Modeling
5 Conclusions
References
Part II: Generation of Pluripotent Stem Cell-Derived Cardiomyocytes and Cardiac Tissues
Chapter 2: A Method for Cardiac Differentiation, Purification, and Cardiac Spheroid Production of Human Induced Pluripotent St...
1 Introduction
2 Materials
2.1 Cardiac Differentiation
2.2 Reseeding and Purification of Cardiomyocytes
2.3 Production of Cardiac Spheroids
2.4 Immunostaining of hiPSC-Derived Cardiomyocytes
3 Methods
3.1 Passaging hiPSCs and Cardiac Differentiation (Figs. 1 and 2)
3.2 Reseeding and Purification of Cardiomyocytes (Fig. 2)
3.3 Production of Cardiac Spheroids (Fig. 3)
3.4 Immunostaining of hiPSC-Derived Cardiomyocytes (Fig. 4)
4 Notes
References
Chapter 3: Large-Scale Differentiation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Stirring-Type Suspensi...
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 4: Efficient Method to Dissociate Induced Pluripotent Stem Cell-Derived Cardiomyocyte Aggregates into Single Cells
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
4 Notes
References
Chapter 5: Isolation of Cardiomyocytes Derived from Human Pluripotent Stem Cells Using miRNA Switches
1 Introduction
2 Materials
2.1 Synthesis of miRNA Switch
2.2 Preparation of hPSCs and Cardiac Differentiation
2.3 Purification of hPSC-Derived Cardiomyocytes Using miRNA Switch
2.4 Assessment of hPSC-Derived Cardiomyocytes by Immunostaining
3 Methods
3.1 miRNA Switch mRNA Preparation
3.1.1 Preparation of Fragments (ORF, 5′UTR and 3′UTR)
3.1.2 Generating DNA Templates for IVT
3.1.3 IVT
3.2 Preparation of hPSCs and Cardiac Differentiation
3.3 miRNA Switch Transfection (Cell Sorting System)
3.4 miRNA Switch Transfection (Non-sorting System)
3.5 Assessment of hPSC-Derived Cardiomyocytes by Immunostaining
4 Notes
References
Chapter 6: Fabrication of Cardiac Constructs Using Bio-3D Printer
1 Introduction
2 Materials
2.1 Cardiac Spheroid Formation
2.2 Bio-3D Printer
2.3 Fabrication of Cardiac Constructs Using Bio-3D Printer
2.4 Contraction Analysis and Electrical Stimulation
3 Methods
3.1 Cardiac Spheroid Formation
3.1.1 Maintenance of Endothelial Cells and Fibroblasts
3.1.2 Thawing of Human iPSC-CMs
3.1.3 Cardiac Spheroid Formation
3.2 Cardiac Construct Fabrication
3.2.1 Preparation of the Bio-3D Printer
3.2.2 Fabrication of Cardiac Constructs by Bio-3D Printing
3.2.3 Maturation of Cardiac Constructs Using a Bioreactor
3.3 Evaluation of Cardiac Constructs
3.3.1 Contraction Analysis
3.3.2 Electrical Stimulation
4 Notes
References
Chapter 7: Fabrication of Thick and Anisotropic Cardiac Tissue on Nanofibrous Substrate for Repairing Infarcted Myocardium
1 Introduction
2 Materials
2.1 Nanofiber Spinning Materials
2.2 Cell Differentiation and Culture
2.3 Transplantation
3 Methods
3.1 Electrospinning of Nanofiber
3.2 Cardiomyocyte Differentiation
3.3 Cardiomyocyte Culture
3.4 Transplantation
4 Notes
References
Chapter 8: Construction of Three-Dimensional Cardiac Tissues Using Layer-by-Layer Method
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Cell Preparation
3.2 ECM Coating on the Cell Surface
3.3 Construction of 3D Cardiac Tissues
4 Notes
References
Chapter 9: Generation of Cylindrical Engineered Cardiac Tissues from Human iPS Cell-Derived Cardiovascular Cell Lineages
1 Introduction
2 Materials
2.1 Maintenance and Cardiovascular Differentiation of hiPSCs
2.2 Cell Harvest and Lineage Analysis
2.3 ECT Construction
3 Methods
3.1 Maintenance and Cardiovascular Differentiation of hiPSCs
3.2 Cell Harvest and Lineage Analysis
3.3 ECT Construction
4 Notes
References
Part III: Physiological Measurements Using Pluripotent Stem Cell-Derived Cardiomyocytes
Chapter 10: Protocol for Morphological and Functional Phenotype Analysis of hiPS-Derived Cardiomyocytes
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment and Software
3 Methods
3.1 Cell Preparation
3.2 Calcium Transients
3.2.1 Indo-1 Loading
3.2.2 Calcium Transient Measurement
3.2.3 Data Analysis
3.3 Field Membrane Potential
3.3.1 FluoVolt Loading
3.3.2 Field Membrane Potential Recording
3.3.3 Data Analysis
3.4 Optical Mapping
3.4.1 Dye Loading Procedure
3.4.2 Optical Membrane Potential Imaging
3.4.3 Data Processing
3.5 Morphological Analysis
3.5.1 Immunofluorescent Staining
3.5.2 Image Processing
4 Notes
References
Chapter 11: Application of FluoVolt Membrane Potential Dye for Induced Pluripotent Stem Cell-Derived Cardiac Single Cells and ...
1 Introduction
2 Materials
2.1 Embryoid Body (EB) Dissociation
2.2 Single-Cell Seeding onto a Fibronectin-Coated Plate
2.3 Fluorescence-Activated Cell Sorting (FACS) and Cryopreservation
2.4 Seeding iPSC-CM Single Cells on a Glass-Bottom Dish
2.5 Seeding High-Density iPSC-CM Monolayers on Glass-Bottom Dishes
2.6 Loading of FluoVolt (FV)
2.7 Recording of APs from Single Cells and Monolayers of Cardiomyocytes
3 Methods
3.1 Embryoid Body (EB) Dissociation on Day 29 (see Note 2)
3.2 Single-Cell Seeding onto a Fibronectin-Coated Plate
3.3 Fluorescence-Activated Cell Sorting (FACS) and Cryopreservation
3.4 Seeding Single iPSC-CMs on a Glass-Bottom Dish
3.5 Seeding High-Density iPSC-CM Monolayer on a Glass-Bottom Dish
3.6 Loading of FluoVolt (FV)
3.7 Recording APs from Single iPSC-CMs and iPSC-CM Monolayers
4 Notes
References
Chapter 12: Multielectrode Array Assays Using Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes
1 Introduction
2 Materials
2.1 MEA Measurement
2.2 Cell Preparation
3 Methods
3.1 Sterilization of MEA Probes When They Are Used for the First Time
3.2 Coating of MEA Probe
3.3 Plating of iPSC-CMs
3.3.1 Embryoid Body (EB) (See Note 5)
3.3.2 Sheet
3.4 Recording (See Note 7)
3.5 Measurement
3.6 Maintenance of MEA Probes for Reuse
4 Notes
References
Chapter 13: Electrophysiological Analysis of hiPSC-Derived Cardiomyocytes Using a Patch-Clamp Technique
1 Introduction
2 Materials
2.1 Equipment (Figs. 1 and 2)
2.2 Reagents
3 Methods
3.1 Cell Preparation
3.2 Pipette Preparation
3.3 Patch-Clamp Experiment
3.3.1 Current Clamp for Action Potential Recording
3.3.2 Voltage Clamp for Ion Current Recording
3.3.3 Analysis of Each Ion Channel Current
4 Notes
References
Chapter 14: Characterization of Ventricular and Atrial Cardiomyocyte Subtypes from Human-Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 hiPSC Differentiation
2.2 Flow Cytometry
2.3 Quantitative Real-Time PCR
2.4 Whole-Cell Patch-Clamp
3 Methods
3.1 Differentiation of Ventricular- and Atrial-Like Cardiomyocytes
3.2 Flow Cytometry and Cell Sorting
3.3 Quantitative Real-Time PCR
3.4 Whole-Cell Patch-Clamp
4 Notes
References
Chapter 15: Assessment of Contractility in Human iPS Cell-Derived Cardiomyocytes Using Motion Vector Analysis
1 Introduction
2 Materials
3 Methods
3.1 Preparation of iCell Cardiomyocytes
3.2 Application of Compounds
3.3 Video Microscopy for Motion Vector Analysis
3.4 Data Analysis
3.5 Video Microscopy for Motion-Based Propagation Analysis
3.6 Simultaneous Measurement of Motion Vectors and Field Potential
4 Notes
References
Chapter 16: Contractile Force Measurement of Engineered Cardiac Tissues Derived from Human iPS Cells
1 Introduction
2 Materials
2.1 Human iPSC-Derived Cardiomyocyte Culture
2.2 Fibrin Gel Sheet Preparation
2.3 Cardiac Tissue Preparation
2.4 Force Measurement
3 Methods
3.1 Human iPSC-Derived Cardiomyocyte Culture
3.2 Fibrin Gel Sheet Preparation
3.3 Cardiac Tissue Preparation
3.4 Contractile Force Measurement
4 Notes
References
Chapter 17: A Method for Contraction Force Measurement of hiPSC-Derived Engineered Cardiac Tissues
1 Introduction
2 Materials
2.1 ECT Formation
2.1.1 Dissociation of Human iPSC-Derived Cardiomyocytes (iPS-CMs)
2.2 Preparation of Normal Human Dermal Fibroblasts (NHDF)
2.3 ECT Construction
2.4 Contraction Force Measurement
2.5 Analysis of the Acquired Data
3 Methods
3.1 ECT Formation
3.1.1 Dissociation of iPS-CMs
3.1.2 Preparation of NHDF
3.1.3 ECT Construction
3.2 Contraction Force Measurement
3.3 Analysis of the Acquired Data
4 Notes
References
Part IV: Transcriptome and Bioinformatics Analysis
Chapter 18: Single-Cardiomyocyte RNA Sequencing to Dissect the Molecular Pathophysiology of the Heart
1 Introduction
2 Materials
2.1 The Langendorff Perfusion System (Buffers and Reagents)
2.2 The Langendorff Perfusion System (Instruments)
2.3 Single-Cell RNA-Seq
2.4 Single-Cell RNA-Seq Analysis
3 Methods
3.1 Isolation of Adult Mouse Cardiomyocytes Using the Langendorff Perfusion System
3.2 Single-Cell RNA-Seq
3.3 Single-Cell RNA-Seq Analysis (See Note 10)
4 Notes
References
Chapter 19: RNA-Sequencing Analysis of Differentially Expressed Genes in Human iPSC-Derived Cardiomyocytes
1 Introduction
2 Materials
2.1 Induced Cardiomyocytes
2.2 RNA Extraction
2.3 Library Construction
2.4 Sequencing
2.5 Analysis Computer/Server
2.6 Analysis Tools
2.7 Human Genome/Transcriptome Reference Files
3 Methods
3.1 RNA Extraction
3.2 Library Construction
3.3 Sequencing
3.4 Demultiplexing
3.5 Trimming of Adapter Sequences and Removal of Too Short Reads
3.6 Confirmation and Removal of Reads of rRNA Sequences
3.7 Mapping to the Human Genome
3.8 Quality Check by RSeQC
3.9 Counting Reads for Each Transcript
3.10 Normalization
3.11 Further Analysis
4 Notes
References
Chapter 20: Analysis of Transcriptional Profiling of Chamber-Specific Human Cardiac Myocytes Derived from Pluripotent Stem Cel...
1 Introduction
1.1 From Human Induced Pluripotent Stem Cells to Cardiomyocytes
1.2 Sample Preparation and Acquisition of Sequencing Raw Data as a Prerequisite Step in Setting Human iPSC-Derived Cardiomyocy...
1.3 Bioinformatics Workflow Vignettes for Comprehensive Analysis of RNA-Seq Data
1.4 Gene Enrichment Profiling for Cardiac Lineage Specificity
2 Materials
2.1 Computer
2.2 Software
2.3 Data Matrix Files
3 Methods
3.1 Loading the Expression Dataset to Run a Principal Component Analysis (PCA)
4 Notes
References
Part V: Gene Editing and CRISPR Technology for Pluripotent Stem Cells
Chapter 21: Genome Editing in Human Induced Pluripotent Stem Cells (hiPSCs)
1 Introduction
2 Materials
2.1 Plasmid Construction
2.2 Electroporation
2.3 hiPSC Culture and Antibiotics Selection
2.4 Genotype
3 Methods
3.1 Design of gRNA and the HDR Template
3.2 Electroporation into hiPSCs
3.2.1 Electroporation (Day 0)
3.2.2 Puromycin Selection (Day 1~)
3.2.3 Initial Evaluation by Direct Sanger Sequencing (Day 6-7)
3.2.4 Colony Pickup (Day 12-14)
3.2.5 Genotype the Isolated hiPSC Colonies (Day 21~)
4 Notes
References
Chapter 22: Generation of Efficient Knock-in Mouse and Human Pluripotent Stem Cells Using CRISPR-Cas9
1 Introduction
2 Materials
2.1 Mouse Embryonic Stem Cell Culture
2.2 Human Induced Pluripotent Stem Cell Culture
2.3 Electroporation
2.4 Cas9/gRNA-Expressing Plasmid
2.5 Knock-in Construct and Recombinase-Expressing Plasmid
2.6 Antibiotics for Drug Selection
2.7 Software
3 Methods
3.1 Construction of Cas9/gRNA-Expressing Plasmid
3.2 Construction of Knock-In Vectors
3.3 Design of Genotyping Primers
3.4 Knock-in to Mouse ESCs
3.4.1 Gene Targeting with CRISPR-Cas9
3.4.2 Removal of Drug-Resistant Gene
3.4.3 Cloning
3.4.4 Making Stock and Genotyping
3.5 Knock-in to Human iPSCs
3.5.1 Standard Human iPSC Culture
3.5.2 Lipofection
3.5.3 Removal of Drug-Resistant Gene
3.5.4 Cloning
4 Notes
References
Chapter 23: CRISPRi/a Screening with Human iPSCs
1 Introduction
2 Materials
2.1 CRISPRi/a Library
2.2 CRISPRi/a Plasmids
2.3 Lentivirus Production
2.4 iPSC Culture
2.5 Genomic DNA Extraction
2.6 NGS Library Prep and Sequencing
2.7 Data Analysis
3 Methods
3.1 Prepare a CRISPRi iPSC Line
3.1.1 Prepare a CRISPRi iPSC Line and Optimize Culture Condition
3.1.2 Decide the Concentration of Puromycin for Drug Selection
3.2 Test Small-Scale Transduction and Lentiviral Infection
3.2.1 Small-Scale Lentiviral Production
3.2.2 Small-Scale Lentiviral Infection of iPSCs
3.2.3 Evaluate the Knockdown Efficiency in Your Desired Cell Type
3.3 Large-Scale Lentiviral Production for the Pooled gRNA Library
3.3.1 Prepare Lentiviral sgRNA Library
3.3.2 Large-Scale Lentivirus Production
3.3.3 Functional Titration of Lentiviral Library
3.4 Large-Scale Lentiviral Infection of iPSCs
3.4.1 Prepare the Large-Scale Experiments
3.4.2 Lentiviral Library Infection of iPSCs
3.5 Large-Scale CRISPR Screening
3.6 Genomic DNA Extraction
3.7 Next-Generation Sequencing (NGS) to Determine sgRNA Distribution
3.7.1 PCR of Genomic DNA
3.7.2 Purification of Amplified NGS Library by QIAquick Column
3.7.3 Size Selection of NGS Library by AMPure XP Beads
3.7.4 Library QC and Submission for Sequencing
3.7.5 Analyze the Sequencing Results
3.7.6 After Screening
4 Notes
References
Part VI: Transplantation of Pluripotent Stem Cell-Derived Cardiomyocytes
Chapter 24: Transplantation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Mouse Myocardial Infarction Mod...
1 Introduction
2 Materials
3 Methods
3.1 Intratracheal Intubation
3.2 Maintenance of Mechanical Ventilation
3.3 Thoracotomy and Myocardial Infarction Surgery
3.4 Transplantation of iPSC-CMs
3.5 Closure of Incision
4 Notes
References
Chapter 25: Transplantation of Pluripotent Stem Cell-Derived Cardiomyocytes into a Myocardial Infarction Model of Cynomolgus M...
1 Introduction
2 Materials
2.1 Medicines
2.2 Surgery-Related Equipment
3 Methods
3.1 Tubes for Blocking the Coronary Artery
3.2 Preoperative Preparation
3.3 Creation of Myocardial Infarction
3.4 Transplantation of Stem Cells-Derived Cardiomyocytes (On Day 14 After Myocardial Infarction)
4 Notes
References
Index