Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology, 1416)

✍ Scribed by Massimiliano Gnecchi (editor)

• Publisher: Springer
• Year: 2016
• Tongue: English
• Leaves: 572
• Category: Library

✦ Synopsis

This volume aims to outline the current status of the Mesenchymal Stem Cells(MSC) field in regenerative medicine and to propose clear and reproducible protocols to better define the identity, function and use of these cells that are today, more than ever, “under the spotlight”. Mesenchymal Stem Cells: Methods and Protocols, Second Edition is organized into four sections. The first guides the reader through a series of state-of-the-art reviews summarizing the use of MSC for the treatment of various diseases. The other three sections are a collection of methodological chapters covering several aspects: isolation and characterization of MSC; expansion of MSC for clinical use; production and characterization of the MSC secretome. Written in the highly successful Methods in Molecular Biology series format, the method chapters include introductions to their respective topics, complete lists of the necessary materials and reagents, step-by-step, readily reproduciblelaboratory protocols, and tips on troubleshooting which will help the researcher to avoid known pitfalls.

Authoritative and cutting-edge, Mesenchymal Stem Cells: Methods and Protocols, Second Edition, aims to ensure successful results in the further study of this vital field.

✦ Table of Contents

Preface
Acknowledgments
Contents
Contributors
Part I: Overview of Mesenchymal Stem Cells For Cell Therapy
Chapter 1: Mesenchymal Stromal Cells in Hematopoietic Stem Cell Transplantation
1 Introduction
2 Mesenchymal Stem Cell Characterization and Immunomodulatory Properties
2.1 Characterization of Ex-Vivo Expanded Mesenchymal Stem Cells
2.2 Immuno-modulatory Properties of Mesenchymal Stem Cells
3 Mesenchymal Stem Cell Therapy in Hematopoietic Stem Cell Transplantation
3.1 Mesenchymal Stem Cell Therapy to Promote Hematopoietic Engraftment
3.2 Mesenchymal Stem Cell Therapy to Treat Graft-Versus-Host Disease
4 Conclusions and Future Directions
References
Chapter 2: Bone Tissue Engineering: Past–Present–Future
1 Past Cell Therapy
1.1 Cells
1.2 Biomaterials
1.3 Obstacles
2 Present Challenges
2.1 Informative Substrates
2.2 Scaffold Physical Properties
2.3 Topographical Surface Modifications of Scaffolds
2.4 Micro-environment
3 Will Tissue Engineering and Cell Therapy Still Be Valuable?
4 Conclusions
References
Chapter 3: Mesenchymal Stem Cells for Osteochondral Tissue Engineering
1 Introduction
2 Mesenchymal Stem Cells for Engineering Bone
3 Highlight: “Engineering Anatomically Shaped Human Bone Grafts” Grayson et al. [48]
4 Engineering Cartilage Using Mesenchymal Stem Cells
5 Highlight: Self-Assembly of Human Mesenchymal Stem Cells into Functional and Stratified Cartilage [62]
6 Engineering Osteochondral Composites Using Mesenchymal Stem Cells
7 Highlight: Osteochondral Composites and the Calcified Cartilage
8 Future Directions
References
Chapter 4: Mesenchymal Stem Cells in Cardiology
1 Introduction
2 Cardiovascular Disease
3 Mesenchymal Stem Cells
4 Cardiac Immunobiology
5 Cardiomyogenesis and Neoangiogenesis
6 Mesenchymal Stem Cell Modification
7 Mesenchymal Stem Cell Preconditioning
8 Clinical Trials with Mesenchymal Stem Cells for Heart Disease
9 Conclusions
References
Chapter 5: Mesenchymal Stem Cells in Kidney Repair
1 Introduction
2 Mesenchymal Stem Cells
3 Mesenchymal Stem Cell-Based Therapy in Acute Kidney Injury
3.1 Acute Kidney Injury
3.2 Role of Mesenchymal Stem Cells in Acute Kidney Injury
3.3 Mechanism of Action of Mesenchymal Stem Cells
4 Mesenchymal Stem Cell-Based Therapy in Chronic Kidney Disease
5 Conclusions
References
Chapter 6: Mesenchymal Stem Cells in Lipogems, a Reverse Story: from Clinical Practice to Basic Science
1 Introduction
2 The Method and the Device
2.1 The Lipogems Kit
2.2 Features of the Lipogems Product
3 The Expansion of Lipogems-Derived hASCs
4 The Lipogems Product Is Efficiently Cryopreserved and Can Be Obtained from Cadaveric Donors
5 Lipogems-Derived hASCs Vigorously Respond to Both Chemical and Physical Stimuli
6 Future Directions
References
Chapter 7: Paracrine Mechanisms of Mesenchymal Stem Cells in Tissue Repair
1 Introduction
2 Paracrine Effects
2.1 Cytoprotective Effect
2.2 Provasculogenic Effects
2.3 Anti-inflammatory Effect
2.4 Effects on Endogenous Regeneration
2.5 Antifibrotic Effect
2.6 Effects on Metabolism
3 Characterization of the Stem Cell Secretome
4 Exosomes
5 Protein and Molecular Therapies
References
Part II: Isolation and Characterization of Mesenchymal Stem Cells
Chapter 8: Protocols for in vitro Differentiation of Human Mesenchymal Stem Cells into Osteogenic, Chondrogenic and Adipogenic Lineages
1 Introduction
2 Materials
2.1 Equipment
2.2 Cell Culture of MSC
2.3 Osteogenic Differentiation
2.3.1 Osteogenic Medium (See Note 1)
2.3.2 Alkaline Phosphate Staining (Early Differentiation)
2.3.3 Von Kossa Staining (Late Differentiation) (See Note 5)
2.4 Chondrogenic Differentiation
2.4.1 Chondrogenic Medium
2.4.2 Alcian Blue Staining
2.5 Adipogenic Differentiation
2.5.1 Adipogenic Medium (See Note 9)
2.5.2 Oil Red O Staining
2.6 RNA Isolation and Gene Expression Analysis
3 Methods
3.1 Osteogenic Differentiation Protocol
3.1.1 Alkaline Phosphatase Staining (Early Differentiation)
3.1.2 Von Kossa Staining (Late Differentiation)
3.2 Chondrogenic Differentiation Protocol
3.2.1 Alcian Blue Staining
3.3 Adipogenic Differentiation Protocol
3.3.1 Oil Red O Staining
3.4 Expression Analysis of Osteo-­Chondro-­Adipo Specific Genes
3.4.1 RNA Isolation
3.4.2 RT-PCR Analysis
4 Notes
References
Chapter 9: Colony Forming Unit Assays
1 Introduction
2 Materials
2.1 Isolation and Culture of Human Bone Marrow- Derived MSCs
2.2 Colony Forming Unit Assay
3 Methods
3.1 Isolation and Culture of Human Bone Marrow-Derived MSCs
3.2 Colony Forming Unit Assays
3.2.1 Colony Forming Unit: Fibroblast Assay(CFU-F)
3.2.2 Colony Forming Unit: Single Cell Assay(sc-CFU)
4 Notes
References
Chapter 10: Methods and Strategies for Lineage Tracing of Mesenchymal Progenitor Cells
1 Introduction
1.1 Are Mesen­chymal Stem Cells Real Stem Cells?
1.2 Mesenchymal Stem Cells and Stem Cell Niches
1.3 Mesenchymal Progenitors and Mesenchymal Stem Cells in Tissue Regeneration
1.4 Nestin and BM-MSCs
1.5 LepR and BM-MPs
1.6 Pdgfra and Sca1: MP and “MSC” Lineage Markers
1.7 Gremlin 1 and BM-MSC/ SSCs
1.8 Gli1 and Tissue-­Resident MPs
1.9 Mesenchymal Lineage Markers
2 Materials
2.1 Tamoxifen Preparation and Administration
2.2 Tissue Fixation, Collection, Processing, and Cryosectioning
2.3 Detection of Native FP and Immunofluorescence
2.4 In Situ and Whole-­Mount LacZ Staining
3 Methods
3.1 Reporter Gene Induction with Tamoxifen
3.2 Tissue Processing for Detection of Native FP Fluorescence
3.3 Combined FP Reporter Visualization with Immuno-­fluorescence
3.4 Whole-Mount LacZ Reporter Staining
3.5 LacZ In Situ Reporter Staining
3.6 Combination IF and LacZ In Situ Staining
4 Notes
References
Chapter 11: Isolation of Mouse Bone Marrow Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 Isolation and Culture of Murine Bone Marrow
2.2 Harvesting Plastic-Adherent Marrow Cells
2.3 Preparation of Antibody-­Conjugated Dynabeads®
2.4 Immuno-depletion
2.5 Phenotypic Characterization of Immunodepleted Murine MSCs (IDmMSCs)
2.6 Differentiation
2.6.1 Adipogenic Differentiation
2.6.2 Chondrogenic Differentiation
2.6.3 Osteogenic Differentiation
2.6.4 Radiation-­Induced Growth Arrest
3 Methods
3.1 Isolation and Culture of Murine Bone Marrow
3.2 Harvesting Plastic-Adherent Marrow Cells
3.3 Preparation of Antibody-­Conjugated Dynabeads®
3.4 Immuno-depletion
3.5 Phenotypic Characterization of IDmMSCs
3.6 Differentiation of IDmMSCs into Connective Tissue Lineages In Vitro
3.6.1 Adipogenic Differentiation of IDmMSCs
3.6.2 Chondrogenic Differentiation of IDmMSCs
3.6.3 Osteogenic Differentiation of IDmMSCs
3.7 Radiation-
4 Notes
References
Chapter 12: Isolation of Pig Bone Marrow-Derived Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 List of Reagents and Materials
2.2 Preparation of Materials
3 Methods
3.1 Bone Marrow Collection
3.2 Isolation of Porcine Mononuclear Cells (pMNCs)
3.3 Seeding and Culturing Porcine Mesenchymal Stem Cells (pMSCs)
3.4 Freezing/Storing Mesenchymal Stem Cells
4 Notes
References
Chapter 13: Isolation, Culture, and Phenotypic Characterization of Mesenchymal Stromal Cells from the Amniotic Membrane of the Human Term Placenta
1 Introduction
2 Materials
2.1 Isolation of Mesenchymal Stromal Cells
2.2 Cell Expansion
2.3 Characterization: Immuno-phenotyping
3 Methods
3.1 Isolation of Amniotic Mesenchymal Stromal Cells
3.2 Cell Expansion
3.3 Characterization: Immuno-phenotyping
4 Notes
References
Chapter 14: Isolation, Culture, and Characterization of Human Umbilical Cord Blood-Derived Mesenchymal Stromal Cells
1 Introduction
2 Materials
2.1 Isolation
2.2 Culture
2.3 Cryopreservation of Expanded CB-MSC
2.4 Characterization
2.4.1 CFU-f
2.4.2 Flow Cytometry
2.4.3 Differentiation
3 Methods
3.1 Isolation
3.2 Culture
3.3 Cryopreservation (See Note 4)
3.4 Characterization
3.4.1 Morphology and Phenotype (See Note 2)
3.4.2 Expansion Potential
3.4.3 CFU-F Assay
3.4.4 Flow Cytometry (See Note 5)
3.4.5 Differentiation Assays (Osteogenic, Adipogenic, Chondrogenic) (See Note 6)
4 Notes
References
Chapter 15: Isolation, Expansion, and Immortalization of Human Adipose-Derived Mesenchymal Stromal Cells from Biopsies and Liposuction Specimens
1 Introduction
2 Materials
3 Methods
3.1 Informed Consent
3.2 Sample Delivery and Storage
3.3 Nomenclature and Processing
3.4 Enzymatic Digestion
3.5 Neutralization
3.6 Centrifugation and Resuspension
3.7 Plating
3.8 Culture and Expansion
3.9 Freezing and Thawing
3.10 Cell Transduction and Immortalization
4 Notes
References
Chapter 16: Optimization of Mesenchymal Stem Cells to Increase Their Therapeutic Potential
1 Introduction
1.1 Limitation of Cell Therapy
1.2 Mesenchymal Stem Cells
2 Materials
2.1 MSCs Isolated from
2.2 Media
2.3 Other Materials
3 Methods
3.1 Isolation and Culture of Rat MSCs (Same Principle for Mice)
3.2 Isolation and Culture of Human MSCs
3.3 Conditioning
3.4 Testing the Efficacy of the Conditioning Agents
3.4.1 Oxidative Challenge (H2O2)
3.4.2 Hypoxic Challenge
3.5 Viability Assay
3.6 Proliferation Assay
3.7 Characterization
3.7.1 Differentiation Protocol
Adipocyte Differentiation
Osteogenic Differentiation
3.7.2 FACS
3.8 In Vivo Tests and Future Perspectives
4 Notes
References
Chapter 17: Directed Differentiation of Human-Induced Pluripotent Stem Cells to Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment and Supplies
2.3 Media
2.4 Staining Solutions
3 Methods
3.1 Feeder-Free Maintenance and Expansion of Human iPSC Lines
3.2 Derivation of Single Cell-Derived MSC Culture from Human iPSCs
3.3 Surface Antigen Analysis of iPSC-MSCs
3.4 Differentiation of Human iPSC-MSCs: Adipogenesis, Osteogenesis, and Chondrogenesis
3.4.1 Adipogenesis
3.4.2 Osteogenesis
3.4.3 Chondrogenesis
4 Notes
References
Part III: Mesenchymal Stem Cells for Clinical Use
Chapter 18: Isolation and Manufacture of Clinical-Grade Bone Marrow-­Derived Human Mesenchymal Stromal Cells
1 Introduction
2 Materials
2.1 D5 Medium
2.2 Isolation of Bone Marrow Mononuclear Cells
2.3 Plating of Bone Marrow Mononuclear Cells, Mesenchymal Stem Cell Culture, and Cryopreservation
2.4 Release Testing
3 Methods
3.1 Preparing Expired Platelet Lysate
3.2 Release Testing of Platelet Lysate
3.3 Preparation of D5 Medium
3.4 Isolating Bone Marrow Mononuclear Cells from Whole Bone Marrow
3.5 Plating Bone Marrow Mononuclear Cells
3.6 Passaging Mesenchymal Stem Cells
3.7 Cryopreservation
4 Notes
References
Chapter 19: Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Methods for ATMP Release
1 Introduction
2 Materials
2.1 Microbiological Control for Cellular Products
2.2 Endotoxin Assay
2.3 Mycoplasma Assay
2.4 Cell Count and Viability
2.5 Identity Assay (Immunophenotype)
2.6 Clonogenic Potential (CFU-F Assay)
3 Methods
3.1 Microbiological Control for Cellular Products
3.1.1 Procedure
3.1.2 Specification
3.1.3 Procedure for Subculture in the Case of Positive Results
3.1.4 Final Results
3.2 Endotoxin Assay
3.2.1 Procedure
3.2.2 Acceptance Criteria and Specification
3.3 Mycoplasma Assay
3.3.1 Procedure
Sample Preparation
3.3.2 PCR Mix Preparation
3.3.3 PCR Reaction
3.3.4 Gel Electrophoresis
Acceptance Criteria and Specification
3.4 Cell Count and Viability
3.4.1 Procedure
3.4.2 Acceptance Criteria and Specifications
3.5 Identity Assay (Immunophenotype)
3.5.1 Procedure
3.5.2 Acceptance Criteria and Specifications
3.6 Clonogenic Potential (CFU-F Assay)
3.6.1 Procedure
3.6.2 Preparation of StemMACS MSC Supplements
3.6.3 Preparation of Complete Medium
3.6.4 Cell Seeding
3.6.5 Cell Staining
3.6.6 CFU-F Colonies Enumeration
3.6.7 Acceptance Criteria and Specification
4 Notes
References
Chapter 20: Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Validation Strategy
1 Introduction
2 Materials
3 Methods
3.1 Microbiological Control for Cellular Products
3.1.1 Validation Samples
3.1.2 Validation Materials
3.1.3 Validation Strategy
3.1.4 Acceptance Criteria for Validation
Specificity
Detection Limit
Repeatability
Intermediate Precision
3.2 Endotoxin
3.2.1 Validation Samples
3.2.2 Validation Strategy
Specificity
Repeatability
Intermediate Precision
3.3 Mycoplasma Assay (NAT Technique)
3.3.1 Validation Samples
3.3.2 Validation Materials
3.3.3 Validation Strategy
3.3.4 Acceptance Criteria for Validation
Specificity
Detection Limit
Repeatability
Intermediate Precision
Robustness
3.4 Cell Count and Viability
3.4.1 Validation Samples
3.4.2 Validation Strategy
3.4.3 Acceptance Criteria for Validation (See Note 9)
Specificity
Repeatability
Intermediate Precision
Accuracy
Linearity and Range
3.5 Identity Assay (Immunophenotype)
3.5.1 Validation Samples
3.5.2 Validation Strategy
3.5.3 Acceptance Criteria for Validation
Specificity
Repeatability
Intermediate Precision
Accuracy
3.6 Clonogenic Potential (CFU-F Assay)
3.6.1 Validation Samples
3.6.2 Validation Strategy
3.6.3 Acceptance Criteria for Validation
Specificity
Repeatability
Intermediate Precision
4 Notes
References
Chapter 21: Cryopreservation and Revival of Human Mesenchymal Stromal Cells
1 Introduction
2 Materials
2.1 Cell Culture and Harvest
2.2 Cryopreservation of Human Mesenchymal Stem Cells for Research Applications
2.3 Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications
2.4 Recovery of Cryopreserved Mesenchymal Stem Cells
2.5 Viability of Thawed Mesenchymal Stem Cells
3 Methods
3.1 Human Mesenchymal Stem Cell Culture and Harvest for Research Applications
3.2 Human Mesenchymal Stem Cell Culture and Harvest for Clinical Applications
3.3 Cryopreservation of Mesenchymal Stem Cells for Research Applications
3.4 Cryopreservation of Mesenchymal Stem Cells for Clinical Applications
3.5 Thawing of Cryopreserved Mesenchymal Stem Cells in Cryovials
3.5.1 Thawing of Cryopreserved MSCs in Screw Cap Cryovials
3.5.2 Thawing of Cryopreserved MSCs in CellSeal
3.6 Viability of Thawed Mesenchymal Stem Cells
3.6.1 Trypan Blue Staining
3.6.2 NucleoCounter Analysis
4 Notes
References
Chapter 22: Clinical-Grade Manufacturing of Therapeutic Human Mesenchymal Stem/Stromal Cells in Microcarrier-Based Culture Systems
1 Introduction
2 Materials
2.1 Cells
2.2 Solutions
2.3 Equipment and Supplies
3 Methods
3.1 ASC and BM MSC Thawing
3.2 Expansion of ASC and BM MSC Under Static Conditions
3.3 Expansion of ASC and BM MSC Under Dynamic Conditions
3.3.1 Preparation of Microcarriers
3.3.1.1 Ready-to-Use Enhanced Attachment® and Synthemax® Microcarriers
3.3.1.2 SoloHill Plastic Microcarriers
3.3.2 Spinner Flask Cultures
3.3.3 Stirred-Tank Bioreactor Cultures
3.4 Monitoring the Cell Culture in the Spinner Flask and Stirred-Tank Bioreactor
3.4.1 Cell Count and Viability
3.4.2 Metabolite Analysis
3.4.3 Cell Distribution in Microcarriers
3.5 MSC Characterization After Expansion Under Stirred Conditions
3.5.1 Immunophenotypic Analysis
3.5.2 Telomere Length
3.5.3 Quantitative RT-PCR Analysis of Differentiation Markers
4 Notes
References
Chapter 23: GMP-Compliant Expansion of Clinical-Grade Human Mesenchymal Stromal/Stem Cells Using a Closed Hollow Fiber Bioreactor
1 Introduction
1.1 Quantum ® Cell Expansion System
1.2 Quantum Cell Expansion System Hydraulics
1.3 Operation of the Quantum System
2 Materials
2.1 Equipment
2.2 Reagents
2.3 Disposables
2.4 MSC Culture Media
3 Methods
3.1 Overview on the Expansion Process
3.2 Tasks
3.3 Loading the Cell Expansion Set (CES)
3.4 Prime Cell Expansion Set
3.5 Filling Media Bags
3.6 Filling Cell Inlet Bags (CIBs)
3.7 Loading Media Bags
3.8 Loading Cell Inlet Bags (CIBs)
3.9 Coating of the Bioreactor
3.10 Inlet Line Washout
3.11 IC EC Washout
3.12 Condition Media
3.13 Expansion of MSC from Bone Marrow (P0): Loading of BM, Removal of Non-­adherent Cells, and Feeding
3.14 Expansion of MSC from Pre-­cultured MSC (P1): Loading of MSC, Removal of Non-­adherent Cells, and Feeding
3.15 Taking Samples from the Sample Coil
3.16 Taking Samples from the Sample Port
3.17 Measuring Glucose and Lactate Concentrations
3.18 Harvest of MSC
3.19 Unload the Cell Expansion Set
3.20 Post-
3.21 Taking hMSC Samples for Quality Controls
3.22 Cryopreser-vation of P0 and P1 hMSC
3.23 Preparation of P1 hMSC for Direct Administration to the Patient
3.24 Quality Controls
3.25 Release Criteria
4 Notes
References
Chapter 24: Engineering Small-Scale and Scaffold-Based Bone Organs via Endochondral Ossification Using Adult Progenitor Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 Nude Mice Implantation Reagents
2.2 Reagent Setup
2.3 Equipment
2.3.1 Cell Culture Equipment
2.3.2 Nude Mice Implantation Equipment
3 Methods
3.1 Preparation of Human Cell Suspension
3.2 Small Scale: Transwell-Based Scaffold-Free Cell Aggregates
3.2.1 Small Scale: Transwell-Based Scaffold-Free Cell Aggregates Preparation and Culture
3.2.2 Small-Scale Transwell-Based Scaffold-Free Cell Aggregate Nude Mice Implantation
3.2.3 Small Scale: Transwell-Based Scaffold-Free Cell Aggregate Characterization
3.3 Large-Scale Scaffold-Based Constructs
3.3.1 Large-Scale Scaffold-Based Construct Preparation and Culture
3.3.2 Large-Scale Scaffold-Based Construct Nude Mice Implantation
3.3.3 Large-Scale Scaffold-Based Construct Characterization
3.4 Procedure for Nude Mice Implantation
4 Notes
References
Chapter 25: Fabrication of Elasticity-Tunable Gelatinous Gel for Mesenchymal Stem Cell Culture
1 Introduction
2 Materials
2.1 Preparation of Vinylated Glass Substrates for Chemical Immobilization of StG Gel
2.2 Preparation of Styrenated Gelatin (StG)
2.3 Preparation of Gelatin Sol Solution and Fabrication of the Gel Film
2.4 Measurement of Young’s Modulus of Elasticity-­Tunable Gelatin Gel by Microindentation Test with an Atomic Force Microscope
2.5 Characterization of MSCs on Elasticity-Tunable Gelatin Gel
2.5.1 MSC Culture
2.5.2 RNA Collection
2.5.3 Protein Collection for Western Blotting
3 Methods
3.1 Preparation of Vinylated Glass Substrates for Chemical Immobilization of StG Gel
3.1.1 Pre-cleaning of Glass Substrate for Silane Coupling
3.1.2 Hydroxylation of Glass Surface with Hot Piranha Treatment (in the Fume Hood)
3.1.3 Vinyl Silane Coupling on Glass Substrate (in the Fume Hood)
3.2 Preparation of Styrenated Gelatin (StG)
3.2.1 StG Synthesis
3.2.2 Measurement of the Degree of Derivatization of the StG
3.3 Fabrication of the Photo-­Crosslinked Gelatin Gel
3.3.1 Preparation of Gelatin Sol
3.3.2 Fabrication of Photo-­Crosslinked Gelatin Gel (Under Ambient Atmosphere)
3.3.3 Fabrication of Photo-­Crosslinked Gelatin Gel (Under Nitrogen Atmosphere; Highly Recommended)
3.3.4 Fabrication of Thermally Crosslinked Gelatin Gel
3.4 Measurement of Young’s Modulus of Elasticity-­Tunable Gelatin Gel by Microindentation Test with an Atomic Force Microscope
3.5 Characterization of Mesenchymal Stem Cells on Elasticity-Tunable Gelatin Gel
3.5.1 MSC Culture on the Gelatin Gel
3.5.2 RNA Collection
3.5.3 Protein Collection for Western Blotting
4 Notes
References
Part IV: Mesenchymal Stem Cell Secretome
Chapter 26: Testing the Paracrine Properties of Human Mesenchymal Stem Cells Using Conditioned Medium
1 Introduction
2 Materials
2.1 General Supplies
2.2 Production of Conditioned Medium
2.3 Cytoprotection Assays
2.4 Matrigel Assay with Human Endothelial Progenitor Cells
2.5 Migration Assay with Human Endothelial Progenitor Cells
2.6 Proliferation Assay of Cardiac Progenitor Cells
2.7 Cardiac Progenitor Cell Migration Assay
3 Methods
3.1 Conditioned Medium
3.2 Concentrated Conditioned Medium
3.3 Cytoprotection Assay
3.4 Angiogenesis Assays
3.4.1 Matrigel Assay
3.4.2 Endothelial Progenitor Cell Migration Assay
3.5 Regeneration Assays
3.5.1 Cardiac Progenitor Cell Proliferation Assay
3.5.2 Cardiac Progenitor Cell Migration Assay
4 Notes
References
Chapter 27: Tips on How to Collect and Administer the Mesenchymal Stem Cell Secretome for Central Nervous System Applications
1 Introduction
1.1 Mesenchymal Stem Cells
1.2 Mesenchymal Stem Cell Secretome
2 Materials
2.1 Mesenchymal Stem Cell Secretome Collection for Proteomic Analysis
2.2 Mesenchymal Stem Cell Secretome Transplantation in the Dentate Gyrus (DG) of the Hippocampus
3 Methods
3.1 Mesenchymal Stem Cell Secretome Collection for Proteomic Analysis
3.2 Mesenchymal Stem Cell Secretome Transplantation in the Dentate Gyrus (DG) of the Hippocampus
4 Notes
References
Chapter 28: Soluble Factors from Human Fetal Bone Marrow-Derived Mesenchymal Stem Cells: Preparation of Conditioned Medium and Its Effect on Tumor Cells
1 Introduction
2 Materials
2.1 Isolation of Human Fetal MSC (hfMSC)
2.2 Culture and Expansion of hfMSC
2.3 Characterization of hfMSC
2.4 Production and Concentration of Conditioned Medium from hfMSC
3 Isolation of Fetal Mesenchymal Stem Cells
3.1 Expansion of hfMSC
3.2 Characterization of hfMSC
3.3 Production of Conditioned Medium from hfMSC
3.3.1 Concentrated Conditioned Medium from hfMSC
3.4 Assay Conditioned Medium Effect on Tumor Cell Viability
4 Notes
References
Chapter 29: Isolation and Characterization of Exosome from Human Embryonic Stem Cell-Derived C-Myc-Immortalized Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 Generation of Lentivirus Particles Carrying MYC
2.2 MYC Immortalization of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells
2.3 Mesenchymal Stem Cell Expansion
2.4 HPLC Isolation of Mesenchymal Stem Cell Exosome
2.5 Exosome Protein Quantification
2.6 Sucrose Density Gradient Assay
2.7 NanoSight
3 Methods
3.1 Generation of Lentivirus Particles Carrying MYC
3.1.1 Lentivirus Production
3.1.2 MYC Immortalization of hES-MSC
3.1.3 Clonal Selection
3.2 Passaging hES-MSCs
3.3 Expansion of E1MYC Using a Cell Stack
3.4 Conditioning of E1MYC
3.5 HPLC Purification of Mesenchymal Stem Cell Exosomes
3.5.1 Tangential Flow Filtration
3.5.2 Size Exclusion Chromatography and Concentration
3.5.3 Analysis of Purified Exosomes
3.6 Exosome Protein Quantification
3.6.1 BSA Standard Preparation
3.6.2 Sample Preparation
3.6.3 Absorbance Measurement
3.6.4 Protein Concentration Calculation
3.7 Sucrose Density Gradient Assay
3.8 Nanosight
4 Notes
5 Appendix: Purification Method
References
Chapter 30: Transcriptomic Analysis of Adult Renal Derived Mesenchymal Stem-Like Cells
1 Introduction
2 Materials
2.1 Mouse Kidney Harvest
2.2 Isolation, Expansion, and Cryopreservation
2.3 Characterization: Immunophenotyping
3 Methods
3.1 Mouse Kidney Collection
3.2 Mouse Kidney CD44+ Mesenchymal Stem Cell FACSorting (See Note 5)
3.3 Mouse Kidney CD44+ Mesenchymal Stem Cell Culture, Expansion, and Stocks
3.4 Determination of Mesenchymal Stem Cell Multipotency
3.5 Mouse Kidney CD44+ Mesenchymal Stem Cell Differentiation to Renin Expressing Cells with cAMP
3.6 Mouse Kidney Mesenchymal Stem Cell Flow Cytometry Characterization
3.6.1 RNA Isolation (RNeasy Plus Micro)
3.6.2 RNA Isolation (PicoPure RNA Isolation) (See Notes 17 and 18)
RNA Extraction from Cell Pellets
RNA Isolation
Gene Expression Profiling
4 Notes
References
Chapter 31: Proteomic Analysis of Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 Cell Isolation and Culture Components
2.2 Components for Sample Preparation and Proteomic Analysis
3 Methods
3.1 Isolation and Culture of Mesenchymal Stem Cells from Umbilical Cord Veins (See Note 4)
3.2 Isolation and Culture of Mesenchymal Stem Cells from Bone Marrow (See Note 4)
3.3 Immunopheno­typical and Functional Characterization
3.4 Proteomic Analysis
4 Notes
References
Chapter 32: Unraveling Mesenchymal Stem Cells’ Dynamic Secretome Through Nontargeted Proteomics Profiling
1 Introduction
2 Materials
2.1 Secretome Concentration
2.2 TCA/Acetone Precipitation
2.3 Protein Solubilization
2.3.1 Protein Solubilization for 1D-SDS-PAGE
2.3.2 Protein Solubilization for 2D-IEF-­SDS- PAGE
2.3.3 Protein Solubilization for Liquid Digestion
2.4 Protein Resolving by 1D-SDS-­PAGE
2.5 Protein Resolving by 2D-IEF-­SDS-PAGE
2.6 Gel Staining
2.6.1 Flamingo Staining
2.6.2 Colloidal Coomassie Staining
2.7 In Gel Digestion and Peptide Extraction
2.8 Liquid Digestion
2.9 iTRAQ
2.9.1 iTRAQ Sample Preparation
2.9.2 iTRAQ 1D-LC or 2D-LC-MS/MS Analysis
2.10 Peptide Cleanup by C18 Solid Phase Extraction
2.11 LC-MS/MS Data Acquisition in IDA and DIA
2.12 Data Processing
3 Methods
3.1 Secretome Concentration
3.2 TCA/Acetone Precipitation
3.3 Protein Solubilization
3.3.1 1D-SDS-PAGE Protein Solubilization
3.3.2 2D-IEF-SDS-
3.3.3 Solubilization for Liquid Digestion
3.4 Protein Resolution by 1D-SDS-­PAGE
3.5 Protein Resolution by 2D-IEF-­SDS-PAGE
3.6 Gel Staining
3.6.1 Flamingo Staining
3.6.2 Colloidal Coomassie Staining
3.7 In Gel Digestion and Peptide Extraction
3.8 Liquid Digestion
3.8.1 Standard Procedure
3.8.2 Fast Procedure
3.9 iTRAQ
3.9.1 iTRAQ Sample Preparation
3.9.2 iTRAQ 1D-LC Peptide Fractionation
3.10 Peptide Cleanup by C18 Solid Phase Extraction
3.11 LC-MS/MS Data Acquisition in IDA and DIA Modes
3.11.1 LC Method
3.11.2 Information-­Dependent Acquisition (IDA) Method for Identification/Label-Free Quantification and for iTRAQ
3.11.3 Data-­Independent Acquisition: SWATH (Sequential Windowed Data-­Independent Acquisition of the Total High-­Resolution Mass Spectra) Acquisition Method
3.12 Data Processing
3.12.1 2D Image Analysis
3.12.2 Protein Identification Using ProteinPilot™ Software
3.12.3 Protein Quantification
Relative Peptide Query (rPQ)
Area Under the Curve (AUC) of Precursor Ions
SWATH Data
3.12.4 iTRAQ Labeled Peptide Relative Quantification
4 Notes
References
Chapter 33: Identification of Factors Produced and Secreted by Mesenchymal Stromal Cells with the SILAC Method
1 Introduction
2 Materials
2.1 Cell Culture and SILAC Labeling
2.2 Collection of Conditioned Media and Preparation of Protein Extracts
2.3 Separation and Visualization of Proteins by SDS-PAGE and Gel Staining
2.4 In-Gel Protein Digestion
2.5 NanoLC-MS/MS and Data Analysis
3 Methods
3.1 Cell Culture and SILAC Labeling
3.2 Collection of Conditioned Media
3.3 Preparation of Secretome Extracts and SDS-PAGE
3.4 In-Gel Protein Digestion
3.5 NanoHPLC- MS/ MS
3.6 Data Analysis
4 Notes
References
Index