Cellular Cardiomyoplasty: Methods and Protocols

✍ Scribed by Race L. Kao (editor)

• Publisher: Humana
• Year: 2013
• Tongue: English
• Leaves: 222
• Series: Methods in Molecular Biology; 1036
• Category: Library

✦ Synopsis

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. 

✦ Table of Contents

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 ™ (BioCardia Inc., San Carlos)
3.3 Pressure-Volume Loop
4 Notes
References
Index