Cardiac Regeneration: Methods and Protocols (Methods in Molecular Biology (2158))

Preface
Contents
Contributors
Part I: Cardiac Injury Models
	Chapter 1: Myocardial Infarction Techniques in Adult Mice
		1 Introduction
			1.1 Cardiac Injuries
			1.2 Assessing Cardiac Function in a High Variation Background
			1.3 Cell Cycle Markers as Proxies for Proliferation
			1.4 Discrepancies Between Different Attributes
		2 Materials
			2.1 Left Anterior Descending Artery Occlusion
			2.2 Cardiac Function Assessment by Echocardiography
			2.3 Histological Scar Quantification
			2.4 Immunofluorescence Analysis of Cardiomyocyte Proliferation
		3 Methods
			3.1 Left Anterior Descending Artery Occlusion, See Also
				3.1.1 One Day Prior to Surgery
				3.1.2 Preparation for Surgery
				3.1.3 Anesthesia and Intubation
				3.1.4 Surgery
			3.2 Cardiac Function Assessment by Echocardiography, See Also
				3.2.1 Preparation for Echocardiography
				3.2.2 Parasternal Long-Axis Acquisition
				3.2.3 Short-Axis Acquisition
				3.2.4 Analysis of the Long-Axis Echocardiographic Measurements B-Mode
				3.2.5 Analysis of the Short-Axis Echocardiographic Measurements M-Mode
			3.3 Histological Scar Quantification
				3.3.1 Specimen Preparation and Image Acquisition
				3.3.2 Maximum Damage Area Analysis
				3.3.3 Scar Volume Analysis
				3.3.4 Scar Degree Analysis
			3.4 Immunofluorescence Analysis of Cardiomyocyte Proliferation
				3.4.1 Staining
				3.4.2 Imaging and Analysis
		4 Notes
		References
	Chapter 2: Apical Resection and Cryoinjury of Neonatal Mouse Heart
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Mice
			2.3 Reagents
				2.3.1 Picro-Sirius Red Staining
				2.3.2 AFOG Staining
				2.3.3 Cardiomyocyte Cell Cycle Assay by Immunostaining
		3 Methods (Procedure Outlined in Fig. 2, See Note 1)
			3.1 Prepare the Pups for Surgery and Anesthesia by Hypothermia
			3.2 Ventricular Resection
			3.3 Cryoinjury
			3.4 Suture and Post-operative  Care
			3.5 Histological and Proliferation Analysis
				3.5.1 Histological Analysis
				3.5.2 Proliferation Analysis
		4 Notes
		References
	Chapter 3: Left Ventricular Pressure Volume Loop Measurements Using Conductance Catheters to Assess Myocardial Function in Mice
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 4: Myocardial Infarction in Pigs
		1 Introduction
		2 Materials
			2.1 Percutaneous Transluminal Coronary Angioplasty to Induce Coronary Ischemia
			2.2 Surgical Equipment
			2.3 Medications
		3 Methods
			3.1 Anesthetizing the Pig
			3.2 Obtaining Baseline Functional Measurements
			3.3 Inducing Myocardial Ischemia
			3.4 Removing the Arterial and Venous Sheaths
			3.5 Options for Administration of Compounds During the Procedure
			3.6 Post-procedure (24 h-2 Months of Reperfusion, or Longer)
		References
	Chapter 5: Ventricular Cryoinjury as a Model to Study Heart Regeneration in Zebrafish
		1 Introduction
		2 Materials
			2.1 Reagents and Equipment
			2.2 Solutions
			2.3 Material Preparation
		3 Methods
			3.1 Surgical Procedure
			3.2 Heart Dissection and Fixation
		4 Notes
		References
	Chapter 6: Cardiac Resection Injury in Zebrafish
		1 Introduction
		2 Materials
			2.1 Zebrafish Handling
			2.2 Anesthesia
			2.3 Resection Injury
		3 Methods
			3.1 Preparation for Resection Injury
			3.2 Anesthesia
			3.3 Resection Injury
			3.4 Post-injury Procedure
		4 Notes
		References
	Chapter 7: A Genetic Cardiomyocyte Ablation Model for the Study of Heart Regeneration in Zebrafish
		1 Introduction
		2 Materials
		3 Methods
			3.1 Zebrafish Cardiomyocyte Ablation
			3.2 Assay for Cardiomyocyte Proliferation During Regeneration
			3.3 Histological Analysis of Cardiomyocyte Ablation and Regeneration
		4 Notes
		References
	Chapter 8: Cardiac MRI Assessment of Mouse Myocardial Infarction and Regeneration
		1 Introduction
		2 Materials
			2.1 T1-Weighted Gd-Based Contrast Agents (See Note 1)
			2.2 T2-Weighted Iron Oxide Nanoparticles
		3 Methods
			3.1 Animal Preparation for In Vivo CMR
			3.2 CMR Preparation
			3.3 Cine CMR
			3.4 Tagging MRI
			3.5 Dynamic Contrast Enhancement (DCE)
			3.6 Late Gadolinium Enhancement (LGE)
			3.7 Extracellular Volume (ECV)
			3.8 Cellular MRI for Intramyocardial Inflammation
		4 Notes
		References
Part II: Ex Vivo and In Vivo Approaches
	Chapter 9: Isolation, Culture, and Live-Cell Imaging of Primary Rat Cardiomyocytes
		1 Introduction
		2 Materials
			2.1 SADO  Mix
			2.2 Pre-plating and Plating Neonatal and Embryonic Cardiomyocytes
			2.3 MACS Miltenyi Biotec Protocol
			2.4 Adult Cardiomyocyte Isolation
		3 Methods
			3.1 SADO Mix Protocol
				3.1.1 Neonatal Cardiomyocyte Isolation
				3.1.2 Embryonic Cardiomyocyte Isolation
			3.2 MACS Miltenyi Biotec Protocol
			3.3 Adult Cardiomyocyte Isolation
			3.4 Live-Cell Imaging of Cardiomyocytes to Detect and Analyze Mitosis
		4 Notes
		References
	Chapter 10: Generation of Human Induced Pluripotent Stem Cells and Differentiation into Cardiomyocytes
		1 Introduction
		2 Materials
			2.1 Fibroblast Transduction
			2.2 Blood Cell Transduction
			2.3 Checking Transgene by RT-PCR
				2.3.1 Maintenance of Human iPSCs in a Feeder-Free System
				2.3.2 Cardiomyocyte Differentiation Media and Reagents
		3 Methods
			3.1 Fibroblast Transduction
				3.1.1 Fibroblast Preparation
				3.1.2 Transduction
				3.1.3 Clone Expansion
				3.1.4 Confirmation of Vector-Free iPS Clones
			3.2 Blood Cell Transduction
				3.2.1 PBMC Isolation and Expansion
				3.2.2 PBMC Expansion
				3.2.3 Viral Transduction, Clone Expansion, and Confirmation of Vector-Free iPS Clones
			3.3 Maintenance of Human iPSCs
				3.3.1 Preparing Matrigel Plates
				3.3.2 Passaging of iPSCs (See Note 9)
			3.4 Human Cardiomyocyte Differentiation
				3.4.1 iPS Passaging
				3.4.2 Differentiation
				3.4.3 CM Dissociation
		4 Notes
		References
	Chapter 11: Differentiation of Human Induced Pluripotent Stem Cells into Epicardial-Like Cells
		1 Introduction
		2 Materials
			2.1 Differentiation of iPSCs into  iECs
			2.2 Enrichment of CDH1+  iECs
			2.3 Long-Term Maintenance of iPSC-Derived  iECs
			2.4 Differentiation of iECs into Cardiac Fibroblasts and Smooth Muscle Cells
			2.5 Immunostaining of iECs and iEC Derivatives
			2.6 Titration of CHIR99021 to Increase Efficiency of Mesoderm Differentiation
		3 Methods
			3.1 Epicardial Differentiation of iPSCs
			3.2 Enrichment of CDH1+  iECs
			3.3 Long-Term Maintenance of  iECs
			3.4 Differentiation of iECs into Cardiac Fibroblasts and Smooth Muscle Cells
			3.5 Immunostaining Analysis
			3.6 Titration of CHIR99021 to Increase Efficiency of Mesoderm Differentiation
		4 Notes
		References
	Chapter 12: In Vitro Conversion of Murine Fibroblasts into Cardiomyocyte-Like Cells
		1 Introduction
		2 Materials
			2.1 Generation of Retroviruses
			2.2 Generation of Mouse Embryonic Fibroblasts (MEFs)
			2.3 Generation of Neonatal Mouse Fresh Cardiac Fibroblasts
			2.4 Cardiac Reprogramming In Vitro and Evaluation of Reprogramming Efficiency
		3 Methods
			3.1 Generation of Retroviruses (See Note 1)
			3.2 Generation of Mouse Embryonic Fibroblasts (MEFs)
			3.3 Generation of Neonatal Mouse Fresh Cardiac Fibroblasts (See Note 6)
				3.3.1 Generate Cardiac Fibroblasts from Explant Culture Method
				3.3.2 Generate Cardiac Fibroblasts from Enzyme Digestion Method
				3.3.3 Isolation of Thy1.2+ Fibroblasts by Magnetic Activated Cell Sorting (MACS)
			3.4 Cardiac Reprogramming In Vitro
			3.5 Evaluation of Reprogramming Efficiency
				3.5.1 FACS Analysis of Reprogramming Efficiency
				3.5.2 RT-qPCR Analysis to Test Gene Expression
				3.5.3 Immunocytochemical (ICC) Analysis of Reprogramming Efficiency
				3.5.4 Evaluation of Calcium Activity of iCMs at Week 4
		4 Notes
		References
	Chapter 13: Frame-Hydrogel Methodology for Engineering Highly Functional Cardiac Tissue Constructs
		1 Introduction
		2 Materials
			2.1 Manufacturing of Tissue Molds
			2.2 3D Engineered Tissue Culture Media and Cell Sources
			2.3 3D Tissue Generation
		3 Methods
			3.1 Manufacturing of PDMS Tissue Molds
			3.2 Frame Manufacturing
			3.3 Initial Mold Preparation for Tissue Culture
			3.4 Cell Preparation
			3.5 Final Mold Preparation
			3.6 Hydrogel Preparation
			3.7 Casting of Engineered Tissues in PDMS Molds
			3.8 Removing Engineered Tissues from Molds
			3.9 Tissue Culture
		4 Notes
		References
	Chapter 14: Efficient Protocols for Fabricating a Large Human Cardiac Muscle Patch from Human Induced Pluripotent Stem Cells
		1 Introduction
		2 Materials
			2.1 Maintaining hiPSCs
			2.2 Supplies/Reagents for Differentiating hiPSCs into  CMs
			2.3 Supplies/Reagents for Differentiating hiPSCs into  ECs
			2.4 Supplies/Reagents for Differentiating hiPSCs into  SMCs
			2.5 Supplies and Reagents for hCMP Manufacture
			2.6 Supplies and Reagents Needed for Patch Transplantation in Swine MI Model
			2.7 Equipment
		3 Methods
			3.1 HiPSC Culture and Maintenance
			3.2 Differentiation of hiPSCs into  CMs
			3.3 Differentiation of hiPSCs into  ECs
			3.4 Differentiation of hiPSCs into  SMCs
			3.5 Construction of a Large  hCMP
			3.6 hCMP Transplantation
			3.7 Analysis of hCMP Engraftment
		4 Notes
		References
	Chapter 15: Isolation and Characterization of Intact Cardiomyocytes from Frozen and Fresh Human Myocardium and Mouse Hearts
		1 Introduction
		2 Materials
			2.1 Tools and Instruments
			2.2 Reagents
		3 Methods
			3.1 Sample Fixation and Enzyme Digestion
			3.2 Count the Total Number of Cardiomyocytes in the Mouse Heart
			3.3 Immunofluorescence Microscopy
			3.4 Image Acquisition and Cardiomyocyte Characterization
			3.5 Other Species and Organs
		4 Notes
		References
	Chapter 16: Ex Vivo Techniques to Study Heart Regeneration in Zebrafish
		1 Introduction
		2 Materials
			2.1 Zebrafish
			2.2 Reagents and Key Equipment
		3 Methods
			3.1 Preparation
			3.2 Heart Extraction and Cleaning
			3.3 Standard Explant Culture
			3.4 Epicardial Cell Ablation
			3.5 Proliferation Assay
			3.6 Partial Ventricular Explant Culture
			3.7 Chemical Treatments and Screens
		4 Notes
		References
	Chapter 17: Purification of Pluripotent Stem Cell-Derived Cardiomyocytes Using CRISPR/Cas9-Mediated Integration of Fluorescent...
		1 Introduction
		2 Materials
			2.1 iPSC Culture Maintenance, Passaging, and Freezing
			2.2 SgRNA Cloning into PX458 Plasmid
			2.3 Integration of Reporter Constructs into iPSCs
			2.4 iPSC Cardiac Differentiation
			2.5 Fluorescence-Activated Cell Sorting of Fluorescently Labeled Cardiomyocytes
		3 Methods
			3.1 Maintenance of Undifferentiated iPSCs
			3.2 Passaging Undifferentiated iPSCs
			3.3 Freezing Undifferentiated iPSCs
			3.4 SgRNA Design
			3.5 Cloning and Validation of sgRNAs
			3.6 Design of Reporter Constructs for Isolation of PSC-Derived Cardiomyocytes
			3.7 Integration of Reporter Constructs into iPSCs
			3.8 Colony Picking and Clone Validation
			3.9 hiPSC Cardiac-Directed Differentiation
			3.10 Fluorescence-Activated Cell Sorting of Fluorescently Labeled Cardiomyocytes
		4 Notes
		References
Part III: Visualizing and Manipulating Heart Regeneration
	Chapter 18: In Vivo Clonal Analysis of Cardiomyocytes
		1 Introduction
		2 Materials
			2.1 Mouse Models
			2.2 Genotyping Primers
			2.3 Hydroxytamoxifen Preparation and Administration
			2.4 2-Dimensional Quantification of CM Clone Number and  Size
			2.5 3-Dimensional Quantification of CM Clone Volume
				2.5.1 Clarity
				2.5.2 Light-Sheet Imaging
				2.5.3 Imaris Processing
		3 Methods
			3.1 Generation of aMHCCreER;Rainbow
			3.2 Hydroxytamoxifen Preparation and Administration
			3.3 2-Dimensional Quantification of Cardiomyocyte Clones
				3.3.1 Tissue Harvest and Processing for Histological Analysis
				3.3.2 Fluorescent Imaging of Tissue Sections
				3.3.3 Quantification of Clone Number and  Size
			3.4 3-Dimensional Quantification of Clone Volumes
				3.4.1 Clearing of Postnatal Day 2 (P2) Mouse Hearts Using CLARITY
				3.4.2 Light-Sheet Calibration
				3.4.3 Light-Sheet Imaging
				3.4.4 Image Reconstruction and 3D Clonal Volume Measurement Using Imaris
		4 Notes
		References
	Chapter 19: High-Fidelity Quantification of Cell Cycle Activity with Multi-Isotope Imaging Mass Spectrometry
		1 Introduction
		2 Materials
			2.1 In Vivo Labeling
			2.2 Tissue Fixation
			2.3 Tissue Embedding
			2.4 Preparing Silicon Wafers
			2.5 Sectioning and Preparing the Mounted Sample
			2.6 NanoSIMS Analysis
			2.7 Image Visualization and Data Analysis
		3 Methods
			3.1 In Vivo Labeling
			3.2 Tissue Fixation
			3.3 Tissue Embedding
			3.4 Preparing Silicon Wafers
			3.5 Sectioning and Preparing of the Mounted Sample
			3.6 NanoSIMS Analysis
			3.7 Image Visualization and Data Analysis
		4 Notes
		References
	Chapter 20: AAV Gene Transfer to the Heart
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of DNA for AAV Production
			3.2 Transfection of HEK293 Cells
			3.3 Harvesting of Crude AAV  Prep
			3.4 Purification of AAV
			3.5 QPCR-Based Quantification of AAV Titer
			3.6 Delivery of AAV to Mice
		4 Notes
		References
	Chapter 21: In Vitro Synthesis of Modified RNA for Cardiac Gene Therapy
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Supplies
			2.3 In Vitro Synthesis of modRNA
				2.3.1 DNA Plasmid Order
				2.3.2 Plasmid Transformation
				2.3.3 Plasmid Miniprep
				2.3.4 Synthesizing Tailed DNA Template for IVT Reaction
				2.3.5 Purifying PCR Product
				2.3.6 Checking Quality of Tailed  DNA
				2.3.7 In Vitro Transcription Reaction
				2.3.8 Purify modRNA Using MegaClear
				2.3.9 RNA Phosphatase Treatment
				2.3.10 Purify modRNA Using MegaClear
				2.3.11 Concentrate modRNA for In Vivo Use by Amicon Ultra-4 Centrifugal Filters
				2.3.12 Measure the Quality of modRNA by TapeStation
		3 Methods
			3.1 In Vitro Synthesis of modRNA
				3.1.1 Plasmid Order
				3.1.2 Plasmid Transformation
				3.1.3 Plasmid Miniprep
				3.1.4 Synthesizing Tailed DNA Template for IVT Reaction
				3.1.5 Purify PCR Product Using the QIAquick PCR Purification  Kit
				3.1.6 Checking Quality of Tailed  DNA
				3.1.7 In Vitro Transcription (IVT) Reaction
				3.1.8 Purify modRNA Using MEGAclear
				3.1.9 RNA Phosphatase Treatment
				3.1.10 Purify modRNA Using MEGAclear
				3.1.11 Concentrate modRNA for In Vivo Use by Amicon Ultra-4 Centrifugal Filters
				3.1.12 Measure modRNA Quality by Agilent High-Sensitivity RNA Assay
			3.2 modRNA Preparation for In Vitro or In Vivo Delivery, Detection, and Analysis of modRNA Translation
				3.2.1 In Vitro Transfection
				3.2.2 In Vivo Delivery
				3.2.3 Detecting Luciferase Expression Using the IVIS System
		4 Notes
		References
	Chapter 22: Generation and Manipulation of Exosomes
		1 Introduction
		2 Materials
			2.1 Exosome Generation
			2.2 Exosome Manipulation: Cloaking Ischemic Myocardium Homing Peptide
			2.3 Exosome Manipulation: Cloaking Platelet Membrane
		3 Methods
			3.1 Exosome Generation
			3.2 Exosome Manipulation: Cloaking Ischemic Myocardium Homing Peptide
			3.3 Exosome Manipulation: Cloaking Platelet Membrane
		4 Notes
		References
	Chapter 23: Epigenetic Assays in Purified Cardiomyocyte Nuclei
		1 Introduction
		2 Materials
			2.1 Cardiomyocyte Nuclei Isolation
			2.2 ATAC-Seq
			2.3 CUT&RUN and High-Throughput Sequencing
		3 Methods
			3.1 Density Gradient Centrifugation
			3.2 Magnetic Immunoprecipitation of Cardiomyocyte Nuclei
			3.3 CM-Specific ATAC-Seq
			3.4 ATAC-Seq Library Preparation and Quality Control
			3.5 CUT&RUN
			3.6 CUT&RUN DNA Extraction
			3.7 CUT&RUN Library Preparation
		4 Notes
		References
	Chapter 24: Genetic Lineage Tracing of Non-cardiomyocytes in Mice
		1 Introduction
		2 Materials
		3 Methods
			3.1 Generation of c-Kit-Based Lineage-Tracing Mice
			3.2 Tamoxifen Treatment
			3.3 Harvest Tissues for Frozen Sections
			3.4 Immunofluorescence to Identify Cells Derived from c-Kit+ Cells
			3.5 Quantification of c-Kit-Derived Lineages, Such as Cardiomyocytes
		4 Notes
		References
	Chapter 25: Experimental Hypoxia as a Model for Cardiac Regeneration in Mice
		1 Introduction
		2 Materials
			2.1 Hypoxia Chamber (Coy Laboratory Products) (See Fig. 1)
				2.1.1 Principle
				2.1.2 Standard Equipment
			2.2 Live-Animal Waste Filtration System
				2.2.1 Standard Equipment
				2.2.2 Principle
		3 Methods
			3.1 Mild Hypoxia in Neonatal Mice (See Note 2)
			3.2 Severe Hypoxia in Adult Mice (See Schematic)
			3.3 Controlling the Atmosphere by Nitrogen Gas
				3.3.1 Principle
				3.3.2 Calibration Setup
			3.4 O2 Calibration Procedure
				3.4.1 Zero Calibration
				3.4.2 Span Calibration
			3.5 Daily Maintenance of the Hypoxia Chamber
		4 Notes
		References
Index