Cellular Cardiomyoplasty: Methods And Protocols (methods In Molecular Biology)

Embryonic stem cells and adult stem cells are the two major types of stem cells that have been used for experimental and clinical studies. Embryonic stem cells are totipotent cells that have the capability to differentiate into any type of cell in the body. In Cellular Cardiomyoplasty: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study cellular cardiomyoplasty. Methods and techniques described in this volume use only adult stem cells or adult progenitor cells.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 key tips on troubleshooting and avoiding known pitfalls. Authoritative and Practical, Cellular Cardiomyoplasty: Methods and Protocols will benefit the cardiologist, cardiothoracic surgeons, biologist (cell, molecular, or structural), biochemist, and physiologist who are interested in understanding and treating damaged myocardium and failing heart. Preface Contents Contributors Chapter 1: Cellular Cardiomyoplasty: Its Past, Present, and Future 1 Introduction 2 Different Stem Cells for Cellular Cardiomyoplasty 2.1 Skeletal Muscle Stem Cells 2.2 Bone Marrow Stem Cells 2.3 Adipose Tissue Stem Cells 2.4 Cardiac Stem Cells 2.5 Induced Pluripotent Stem Cells 3 Clinical Outcomes Using Different Stem Cells 3.1 Satellite Cells (Myoblasts) 3.2 Mesenchymal Stem Cells 3.3 Adipose Tissue Stem Cells 3.4 Cardiac Stem Cells 3.5 Induced Pluripotent Stem Cells 4 Future Directions in Cellular Cardiomyoplasty References Chapter 2: Skeletal Muscle Stem Cells 1 Introduction 2 Materials 2.1 Skeletal Muscle Sampling 2.2 Satellite Cell Culture 2.3 Labeling of Satellite Cells 2.3.1 Viral Transfection of Satellite Cells 2.3.2 Y Chromosome FISH 3 Methods 3.1 Skeletal Muscle Sampling 3.2 Culture of Satellite Cells 3.3 Labeling of Satellite Cells 3.3.1 Viral Transfection of Satellite Cells 3.3.2 Y Chromosome FISH 4 Notes References Chapter 3: Bone Marrow Stem Cells 1 Introduction: Bone Marrow Stromal Cells for Cellular Cardiomyoplasty 2 Materials 2.1 MSC Isolation and Culture 2.2 MSC Differentiation 2.3 Freezing of MSC 2.4 LacZ Transfection 2.5 Labeling with DAPI 3 Methods 3.1 MSC Isolation and Culture 3.2 MSC Differentiation 3.3 Freezing of MSC 3.4 LacZ Transfection 3.5 Labeling with DAPI 3.6 Prepare BMSC for Injection 3.6.1 Harvesting BMSC 3.6.2 LAD Ligation 3.6.3 Echocardiography 4 Notes References Chapter 4: Adipose Tissue-Derived Mesenchymal Stem Cells: Isolation, Expansion, and Characterization 1 Introduction 2 Materials 2.1 ADSC Isolation and Culture 2.2 Flow Cytometry 2.3 Osteogenic Differentiation 2.4 Adipogenic Differentiation 2.5 Chondrogenic Differentiation 3 Methods 3.1 Ex Vivo Expansion of ADSC 3.1.1 Cell Isolation 3.1.2 Subculture of Cells 3.2 Long-Term Preservation 3.2.1 Cryopreservation 3.2.2 Recovery of Cryopreserved Cells 3.3 Cell Characterization 3.3.1 Flow Cytometry Analysis 3.3.2 ADSC Differentiation Osteogenic Differentiation ( See Note 13) Alizarin Red S Staining Alkaline Phosphatase Test Adipogenic Differentiation ( See Note 14) Oil Red O Staining Chondrogenic Differentiation ( See Note 15) Toluidine Blue Staining 4 Notes References Chapter 5: Cardiac Side Population Cells and Sca-1-Positive Cells 1 Introduction 2 Materials 2.1 Enzymatic Digestion of Adult Murine Hearts for Isolation of Cardiac SP Cells 2.2 Hoechst 33342 Incubation for Isolation of Cardiac SP Cells 2.3 Flow Cytometry and Sorting for Isolation of Cardiac SP Cells 2.4 Enzymatic Digestion of Adult Murine Hearts for Isolation of Cardiac Sca-1 + Cells 2.5 Immunostaining for Isolation of Cardiac Sca-1 + Cells 2.6 Flow Cytometer and Cell Sorting for Isolation of Cardiac Sca-1 + Cells 3 Methods 3.1 Enzymatic Digestion of Adult Murine Hearts for Isolation of Cardiac SP Cells 3.2 Hoechst Incubation for Isolation of Cardiac SP Cells 3.3 Flow Cytometry and Sorting Analysis for Isolation of Cardiac SP Cells 3.4 Enzymatic Digestion of Adult Murine Heart for Isolation of Cardiac Sca-1 + Cells 3.5 Immunostaining for Isolation of Cardiac Sca-1 + Cells 3.6 Cell Sorting for Isolation of Cardiac Sca-1 + Cells 4 Notes References Chapter 6: Two-Step Protocol for Isolation and Culture of Cardiospheres 1 Introduction 2 Materials 2.1 Cell Isolation and Expansion 2.2 MACS Sorting 2.3 Equipment 2.4 Reagent Setup 3 Methods 3.1 Step One: Dissociating and the Primary Tissue Explant 3.2 Step Two: The Cell Enrichment 4 Notes References Chapter 7: Generation of Human iPSCs from Human Peripheral Blood Mononuclear Cells Using Non-integrative Sendai Virus in Chemically Defined Conditions 1 Introduction 2 Materials 2.1 Media 2.2 Reagents 2.3 Equipment 3 Methods 3.1 PBMC Isolation from Blood by Percoll Separation 3.2 Infect PBMCs with Sendai Virus Reprogramming Factors 3.3 Prepare Matrigel Plates 3.4 PBMC Reprogramming 3.5 Colony Purification and Expansion 4 Notes References Chapter 8: Identification of Stem Cells After Transplantation 1 Introduction 2 Materials 2.1 Plasmids for Lentivirus Packaging 3 Methods 3.1 Lentiviral-GFP- 3.2 Stem Cell GFP Labeling 3.3 Myocardial Infarction Model and Cell Transplantation 3.4 Identify Donor Stem Cells in Infarct Heart 4 Notes References Chapter 9: Methods to Study the Proliferation and Differentiation of Cardiac Side Population (CSP) Cells 1 Introduction 2 Materials 2.1 Culture Media 2.2 Kits 2.3 Staining Solutions 2.4 Solutions for Neonatal Rat Ventricular Myocyte (NRVM) Isolation 2.5 FUCCI Reporter System 3 Methods 3.1 Proliferation Assay 3.2 Immunostaining for Proliferation Markers 3.2.1 Ki67 Staining for Flow Cytometric Analysis 3.2.2 Immunocytochemi 3.2.3 Immunocytochemi­cal Staining for 5-Bromo-2′-deoxyuridine (BrdU) 3.3 Cell Cycle Analysis 3.3.1 Propidium Iodide (PI) Staining for Flow Cytometric Analysis 3.3.2 Fluorescence Ubiquitination Cell Cycle Indicators (FUCCI) 3.4 Cardiomyogenic Differentiation: Coculture Assay 4 Notes References Chapter 10: Immune Responses After Mesenchymal Stem Cell Implantation 1 Introduction 2 Mesenchymal Stem Cells (MSCs) as Adult Stem Cells 3 MSCs Characteristics and Subpopulations 4 MSCs as “Universal Donor Cells” 5 Evidence from In Vitro Studies 6 Evidence from In Vivo Studies 7 Clinical Trials Involving Allogeneic Stem Cells Transplantation 8 Conclusion References Chapter 11: Route of Delivery, Cell Retention, and Efficiency of Polymeric Microcapsules in Cellular Cardiomyoplasty 1 Introduction 1.1 Scope of the Disease 1.2 Cellular Cardio- myoplasty: Clinical and Cellular Limitations 1.3 Microcapsules as a Method of Delivery 1.4 Types of Microcapsules 2 Materials 2.1 APA Microcapsules 2.2 GCAC Microcapsules 2.3 Transduction of Cells with Lac Z Gene 2.4 GCAC Microcapsules Encapsulating Stem Cells 2.5 APA Microcapsules Encapsulating Stems Cells 2.6 Monitoring Cell Growth Within GCAC and APA Microcapsules 2.7 Intramyocardial Injections of Microspheres into Rat Myocardium 2.8 Quantitative Analysis of Microspheres 2.9 Histological Analysis of Heart (Lac Z Staining) 2.10 PCR Analysis of Heart When Employing Gender- 3 Methods 3.1 APA Microcapsule Preparation to Encapsulate Microspheres 3.2 GCAC Microcapsule Preparation 3.3 Transduction of Cells with Lac Z Gene Before Encapsulation 3.4 Preparation of GCAC Containing Transduced Stem Cells 3.5 Preparation of APA Microcapsules Containing Stem Cells 3.6 Monitoring Cell Growth Within GCAC and APA Microcapsules 3.7 Intramyocardial Injection of Microencapsulated Stem Cells into Rat Myocardium 3.8 Histological Analysis of Heart (Lac Z Staining) 3.9 PCR Analysis of Heart When Employing Gender- 4 Notes References Chapter 12: Angiogenic Nanodelivery Systems for Myocardial Therapy 1 Introduction 1.1 Myocardial Infarction and Therapeutics 1.2 Nanoparticles: Their Unique Features and Potential in Delivery of Biotherapeutics 1.3 Albumin-Based Nanoparticles: Introduction and Overview of Protocol 1.4 Alginate-Based Nanoparticles: Introduction and Overview of Protocol 2 Materials 2.1 Albumin Nanoparticles 2.2 Alginate Nanoparticles 3 Methods 3.1 Preparation of Albumin NP Co-encapsulating Vegf and Ang1 Angiogenic Proteins 3.2 Procedure to Analyze In Vitro Protein Release Kinetics of NP 3.3 Endothelial Cell Proliferation Assay to Confirm Bioactivity of the Angiogenic NP 3.4 Preparation of Alginate NP Encapsulating PIGF Angiogenic Proteins 4 Notes References Chapter 13: Bio-hybrid Tissue Engineering for Cellular Cardiomyoplasty: Future Directions 1 Background 2 Myocardial Tissue Engineering 3 MAGNUM Clinical Trial (Myocardial Assistance by Grafting a New Bioartificial Upgraded Myocardium) 3.1 Preclinical Study 3.2 MAGNUM Trial 4 RECATABI European Project (REgeneration of CArdiac Tissue Assisted by Bioactive Implants) 5 Ventricular Constraint Therapy 6 Development of Bioartificial Myocardium 6.1 Results 7 Stem Cell Treatment for Chagas Heart Disease 8 Development of Bio-hybrid Ventricular Support Bioprostheses References Chapter 14: Decellularized Whole Heart for Bioartificial Heart 1 Introduction 2 Materials 2.1 Heart Explantation 2.2 Whole-Heart Decellularization Through Coronary Perfusion: Automated Software- 2.3 Whole-Heart Decellularization Through Coronary Perfusion: Decellularization Solutions 2.4 H&E Staining 2.5 Movat’s Pentachrome Staining 2.6 Immunohisto- chemical Staining Using Fast Red/DAB 2.7 DNA Assay 2.8 GAG Assay 3 Methods 3.1 Heart Explantation 3.2 Whole-Heart Decellularization Through Coronary Perfusion 3.3 H&E Staining 3.4 Movat’s Pentachrome Staining 3.5 Immunohisto- chemical Staining for Cytoplasmatic Proteins Using Fast Red 3.6 Immunohisto-chemical Staining for Base Membrane Proteins Using DAB 3.7 DNA Assay 3.8 GAG Assay 4 Notes References Chapter 15: Clinical Trials of Cardiac Repair with Adult Bone Marrow- 1 Introduction 2 Clinical Trials of BMC Therapy for Cardiac Repair 2.1 Bone Marrow Mononuclear Cells 2.2 Mesenchymal Stem Cells 2.3 AC133+ BMCs 2.4 Mobilized BMCs 2.4.1 Circulating Progenitor Cells (CPCs) 2.4.2 CD34+ Cells 2.4.3 Peripheral Blood Stem Cells 3 Meta-analysis of Pooled Data on Effects of BMC Therapy 3.1 LV Ejection Fraction 3.2 Infarct Size 3.3 LV End-Systolic Volume 3.4 LV End-Diastolic Volume 3.5 Other Outcome Parameters 4 Safety of BMC Transplantation 5 Methodological Considerations 5.1 Patient Characteristics 5.1.1 Type of Ischemic Heart Disease 5.1.2 LV Function at Baseline 5.2 BMC Number 5.3 Route of BMC Injection 5.4 Timing of BMC Injection After Acute MI 5.5 Methods of BMC Processing 6 Conclusions References Chapter 16: Clinical Study Using Adipose-Derived Mesenchymal-Like Stem Cells in Acute Myocardial Infarction and Heart Failure 1 Introduction 2 Materials 2.1 Liposuction 2.2 ADRC Isolation 3 Methods 3.1 Intracoronary Infusion 3.2 Intramyocardial Injection by Use of the Helix Catheter TM (BioCardia Inc., San Carlos) 3.3 Pressure-Volume Loop 4 Notes References Index Cellular Cardiomyoplasty : Its Past, Present, And Future / Elizabeth K. Lamb, Grace W. Kao, And Race L. Kao -- Skeletal Muscle Stem Cells / Grace W. Kao, Elizabeth K. Lamb, And Race L. Kao -- Bone Marrow Stem Cells / Minh Ngoc Duong, Yu-ting Ma, And Ray C.j. Chiu -- Adipose Tissue-derived Mesenchymal Stem Cells : Isolation, Expansion And Characterization / Miriam Araña [and 4 Others] -- Cardiac Side Population Cells And Sca-1-positive Cells / Toshio Nagai, Katsuhisa Matsuura, And Issei Komuro -- Two-step Protocol For Isolation And Culture Of Cardiospheres / Lijuan Chen [and 5 Others] -- Generation Of Human Ipscs From Human Peripheral Blood Mononuclear Cells Using Non-integrative Sendai Virus In Chemically Defined Conditions / Jared M. Churko, Paul W. Burridge, And Joseph C. Wu -- Identification Of Stem Cells After Transplantation / Yingjie Wang [and 5 Others] -- Methods To Study The Proliferation And Differentiation Of Cardiac Side Population (csp) Cells / Konstantina-ioanna Sereti [and 3 Others] -- Immune Responses After Mesenchymal Stem Cell Implantation / Rony Atoui And Ray C.j. Chiu -- Route Of Delivery, Cell Retention, And Efficiency Of Polymeric Microcapsules In Cellular Cardiomyoplasty / Alice Le Huu [and 3 Others] -- Angiogenic Nanodelivery Systems For Myocardial Therapy / Arghya Paul, Dominique Shum-tim, And Satya Prakash -- Bio-hybrid Tissue Engineering For Cellular Cardiomyoplasty : Future Directions / Juan Carlos Chachques -- Decellularized Whole Heart For Bioartificial Heart / Hug Aubin [and 4 Others] -- Clinical Trials Of Cardiac Repair With Adult Bone Marrow-derived Cells / Vinodh Jeevanantham [and 3 Others] -- Clinical Study Using Adipose-derived Mesenchymal-like Stem Cells In Acute Myocardial Infarction And Heart Failure / Ilia Alexander Panfilov [and 3 Others]. Edited By Race L. Kao. Includes Bibliographical References And Index. Embryonic stem cells and adult stem cells are the two major types of stem cells that have been used for experimental and clinical studies. Embryonic stem cells are totipotent cells that have the capability to differentiate into any type of cell in the body. In Cellular Cardiomyoplasty: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study cellular cardiomyoplasty. Methods and techniques described in this volume use only adult stem cells or adult progenitor cells. 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 key tips on troubleshooting and avoiding known pitfalls. Authoritative and Practical, Cellular Cardiomyoplasty: Methods and Protocols will benefit the cardiologist, cardiothoracic surgeons, biologist (cell, molecular, or structural), biochemist, and physiologist who are interested in understanding and treating damaged myocardium and failing heart.