Preface
Contents
Contributors
Part I: Introduction
Chapter 1: Consistent Inclusion of Mesenchymal Stem Cells into In Vitro Tumor Models
1 Tumor-Associated Mesenchymal Stem Cells-A New Paradigm in Cancer-Stroma Cooperation
2 Mesenchymal Stem Cells-Origin and Characterization
2.1 Selecting the Source of MSCs-Human Vs Animal-Derived Stem Cells
2.2 Tissue-Specific Phenotype Variations in Human-Derived MSCs
2.3 Distinguishing Distinct Tissue-Specific Subpopulations
2.4 Recognition versus Stimulation of Immunosupportive/Immunosuppressive TA-MSCs Phenotypes
2.5 Characterizing MSCs Phenotype Through Secretome Analysis
3 Standardization of In Vitro Culture Conditions
3.1 Passaging and Variations in Proliferative and Differentiation Potential
3.2 Influence of Culture Substrates on Mesenchymal Stem Cells Phenotype Acquisition
3.3 Defining Co-Culture Ratios and Analyzing Overtime Population Variations
4 Conclusion
References
Chapter 2: Pluripotent Stem Cells for Cell Therapy
1 Introduction
2 Use of PSC in Modeling and Treating Cardiovascular Disease
3 Use of PSC in Modeling and Treating CNS Diseases
4 Use of PSC in Modeling and Treating Retinal Diseases
5 Use of PSC in Modeling and Treating Diabetes Mellitus
6 Conclusion
References
Part II: Disease Modeling
Chapter 3: In Vitro Methods for the Study of Glioblastoma Stem-Like Cell Radiosensitivity
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Clonogenic Survival Assay
2.3 Limiting Dilution Assay
2.4 Ξ³H2AX Staining
2.5 Ionizing Radiation Source
3 Methods
3.1 Clonogenic Survival Assay
3.2 Limiting Dilution Assay (See Note 8)
3.3 Ξ³H2AX Foci
4 Notes
References
Chapter 4: Bioimaging of Mesenchymal Stem Cells Spatial Distribution and Interactions with 3D In Vitro Tumor Spheroids
1 Introduction
2 Materials
2.1 Cell Culture and 3D Spheroids Fabrication
2.2 Cell Membrane Labeling
2.3 3D Cell Tracking in Internal Organization and Migration Assays
3 Methods
3.1 Heterotypic 3D Tumor Spheroids Fabrication
3.2 Cell Membrane Labeling for Labeled 3D Spheroids Generation
3.3 MSCs Internalization Cell Tracking Assays in 3D Spheroids
4 Notes
References
Chapter 5: Investigation of the MSC Paracrine Effects on Alveolar-Capillary Barrier Integrity in the In Vitro Models of ARDS
1 Introduction
2 Materials
2.1 Animals
2.2 Bone Marrow Mesenchymal Stromal Cells (MSCs) Isolation
2.3 Lung Endothelial Cells Isolation
2.4 Lung Epithelial Cells Isolation
2.5 Investigation of the MSC Paracrine Effect
2.6 Assays to Measure Barrier Permeability
2.7 Equipment
3 Methods
3.1 Mesenchymal Stromal Cells Isolation from Bone Marrow
3.2 Lung Endothelial Cells Isolation
3.3 Lung Alveolar Epithelial Cells Isolation
3.4 Investigation of MSC Paracrine Effect
3.4.1 Generation of Conditioned Medium
3.4.2 Indirect Co-Culture
3.4.3 Isolation of Extracellular Vesicles
3.5 Assays to Measure Barrier Permeability
3.5.1 Permeability to FITC-Dextran
3.5.2 XCELLigence Real-Time Cell Analysis (RTCA)
3.5.3 Transepithelial/Endothelial Electrical Resistance (TEER)
3.6 Assays to Investigate Effects of MSC on Reparative Capacities
3.6.1 Scratch Wound Healing Assay
3.6.2 Immunofluorescence Staining of Scratch Wound Assays with Ki67
4 Notes
References
Chapter 6: In Vitro Methods to Evaluate the Effects of Mesenchymal Stem Cells on TGF-Ξ²1-Induced Pulmonary Fibrosis
1 Introduction
2 Materials
2.1 Equipment
2.2 Cell Lines
2.3 Media, Buffers, and Solutions
2.4 Collagen Detection
2.5 Western Blot
2.6 RNA Isolation and Quantitative Real-Time RT-PCR
2.7 Software to Represent Data
3 Methods
3.1 Conditioned Medium from MSCs
3.2 Co-Culture MSCs with Fibroblasts: Transwell Assay
3.2.1 RNA Isolation and Quantitative Real-Time RT-PCR
3.2.2 Western Blot
3.2.3 Determination of Total Collagen Content
3.3 Co-culture MSCs with Fibroblasts: Determine Paracrine Effects
4 Notes
References
Chapter 7: Study of Mesenchymal Stem Cell-Mediated Mitochondrial Transfer in In Vitro Models of Oxidant-Mediated Airway Epithe...
1 Introduction
2 Materials
2.1 Cells
2.2 Cell Culture Reagents, Media, and Materials
2.3 Cell Staining
2.4 Flow Cytometry/Cell Sorting
2.5 Imaging
2.6 Determination of ATP Content
3 Methods
3.1 Culture of Human iPSC-MSCs and BM-MSCs
3.2 Culture of BEAS-2B Cells
3.3 Culture of ASMCs
3.4 Staining and Co-Culture of iPSC-MSCs with BEAS-2B Cells or ASMCs
3.5 Imaging of Mitochondrial Transfer Using Fluorescence Microscopy
3.6 Quantification of Mitochondrial Transfer by Flow Cytometry
3.7 Determination of ATP Content of Cells that Have Received Mitochondria
4 Notes
References
Chapter 8: Co-Culture of Peripheral Blood Mononuclear Cells and Endothelial Colony Forming Cells from Cord Blood of Preterm Bo...
1 Introduction
2 Materials
2.1 Isolation of PBMCs from Peripheral Cord Blood
2.2 Cultivation of PBMCs
2.3 Phenotypic Characterization of PBMCs and ECFCs
2.4 Expression of Molecular Markers for Peripheral Blood Mononuclear Cells and ECFCs
2.5 Functional Characterization of ECFC
2.5.1 In Vitro Tube Formation Assay on Matrigel and the Influence of Varying Oxygen Concentrations
2.5.2 Acetylated Low-Density Lipoprotein (LDL) Uptake and Ulex europaeus Lectin Binding
2.5.3 Single Cell Clonogenic Assay
2.5.4 Wound Healing (or Scratch) Assay
2.6 Co-Culture of Endothelial Colony-Forming Cells With Peripheral Blood Mononuclear Cells
2.7 Microarray Analysis
2.8 Validation by real-time PCR Analysis
3 Methods
3.1 Isolation of PBMCs from Peripheral Cord Blood
3.2 Cultivation of PBMCs into ECFCs
3.3 Phenotypic Characterization of PBMCs and ECFCs
3.4 Analyzing the Expression of Molecular Markers for Peripheral Blood Mononuclear Cells and ECFCs
3.5 Functional Characterization of ECFC
3.5.1 In Vitro Tube Formation Assay on Matrigel and the Influence of Varying Oxygen Concentrations
3.5.2 Acetylated Low-Density Lipoprotein (LDL) Uptake and Ulex europaeus Lectin Binding
3.5.3 Single Cell Clonogenic Assay
3.5.4 Wound Healing (or Scratch) Assay
3.6 Co-Culture of Endothelial Colony Forming Cells with Peripheral Blood Mononuclear Cells
3.7 Microarray Analysis
3.8 Real-Time PCR Analysis
4 Notes
References
Chapter 9: Ex Vivo Model of Spontaneous Neuroretinal Degeneration for Evaluating Stem CellsΒ΄ Paracrine Properties
1 Introduction
2 Materials
2.1 Ex Vivo Neuroretina and Stem Cells Co-Culture
2.2 Neuroretinal Explants Processing
2.2.1 Paraffin Wax Embedding
2.2.2 Cryopreservation
2.2.3 Resin Embedding
3 Methods
3.1 Ex Vivo Neuroretina and Stem Cells Co-Culture
3.1.1 Stem Cell Preparation
3.1.2 Neuroretina Explants Preparation (See Note 2)
3.1.3 Neuroretinal Explants and Stem Cells Co-Culture
3.2 Neuroretinal Explants Processing
3.2.1 Paraffin Wax Embedding (for Light or Epifluorescence Microscopy)
3.2.2 Cryopreservation (for Epifluorescence or Confocal Microscopy)
3.2.3 Epoxy Resin Embedding (for Transmission Electron Microscopy)
4 Notes
References
Chapter 10: Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model: An Experimental Setting for Organ Recondition and Pharmaco...
1 Introduction
2 Materials
2.1 Animals
2.2 Surgery
2.3 Normothermic Machine Perfusion System
2.4 Perfusion Solution
2.5 Analysis
3 Methods
3.1 Normothermic Machine Perfusion System (See Fig. 1)
3.1.1 Setting of Open Perfusion Circuit
3.1.2 Setting of Warm Water Circuit
3.2 Surgery
3.2.1 Blood Collection for Perfusion Solution
3.2.2 Hepatectomy for Perfusion Procedure (See Fig. 2a, b)
3.3 Perfusion Procedure
3.4 Analysis and Results
3.4.1 Preliminary Analysis and Operating Parameters
3.4.2 Perfusate Analysis
3.4.3 Biochemistry Analysis
3.4.4 Bile Analysis
3.4.5 Histological Analysis (See Note 8)
4 Notes
References
Chapter 11: Co-Culture of Human Mesenchymal Stromal Cells and Primary Mouse Hepatocytes
1 Introduction
2 Materials
2.1 Cell Culture Media
2.2 Isolation of Human Bone Marrow-Derived Mesenchymal Stromal Cells (MSC)
2.3 Culture Plate Coating
2.4 Culture of MSC
2.5 Cryopreservation of MSC
2.6 Thawing of MSC
2.7 Hepatogenic Differentiation of MSC
2.8 Isolation of HC
2.9 Co-Culture
2.10 Oil Red O (ORO) Staining of Lipids
2.11 Staining of Co-Culture for F-Actin
2.12 Equipment and Non-Disposable Materials
2.13 Disposable Materials
3 Methods
3.1 Isolation of MSC
3.2 Cryopreservation of MSC
3.3 Coating Well Plates or Coverslips
3.4 Thawing and Growing of Frozen MSC
3.5 Hepatogenic Differentiation of MSC
3.6 Isolation of HC
3.7 Co-Culture of HC and Hepatogenic Differentiated MSC
3.8 Staining Lipids with ORO
3.9 Staining of Co-Culture with Phallodin
4 Notes
References
Chapter 12: A 3D Dynamic In Vitro Model of Inflammatory Tendon Disease
1 Introduction
2 Materials
2.1 MSC Cultivation
2.2 Scaffold Preparation
2.3 Seeding of Tendon Scaffolds
2.4 Dynamic Culture of Tendon Constructs
2.5 Inflammatory Culture Conditions-Leukocyte Recovery and co-Culture
2.6 Inflammatory Culture Conditions-IL-1Ξ² and TNF-Ξ± Supplementation
2.7 Sample Harvesting
3 Methods
3.1 MSC Cultivation (See Note 1)
3.2 Scaffold Preparation (See Note 2)
3.3 Seeding of Tendon Scaffolds (See Note 3)
3.4 Dynamic Culture of Tendon Constructs (See Note 4)
3.5 Inflammatory Culture Conditions-Leukocyte Recovery and Co-Culture (See Note 5)
3.6 Inflammatory Culture Conditions-IL-1Ξ² and TNF-Ξ± Supplementation (See Note 6)
3.7 Sample Harvesting (See Note 7)
4 Notes
References
Chapter 13: Generation of Epidermal Equivalents from Hair Follicle Melanocytes, Keratinocytes, and Dermal Fibroblasts
1 Introduction
2 Materials
2.1 Equipment
2.2 Media and Buffers
2.3 Cells, Reagents, and Antibodies
3 Methods
3.1 Protocol for the Feeder Layer
3.2 Primary Culture of Human Keratinocytes from the Outer Root Sheath (HUKORS)
3.2.1 Preparations 2 Days Ahead of Sampling Follicles d(-2)
3.2.2 Hair Plucking (See Fig. 1)
3.2.3 Follicle Preparation, HUKORS Cultivation and Harvest (See Fig. 2)
3.2.4 Cryopreservation of HUKORS
3.3 Secondary 3D Culture: Non-Pigmented Epidermal Model Consisted of HUKORS (NP-EPI)
3.3.1 Preparation of Inserts with Feeder Layer d(-1)
3.3.2 NP-EPI Culture
3.4 Primary Culture of Human Melanocytes from the Outer Root Sheath (HUMORS)
3.4.1 Procedure of Generating HUMORS
3.4.2 Characterizing the HUMORS in the ORS Cell Culture
3.5 Secondary 3D Culture Pigmented Epidermal Model Consisted of HUKORS and HUMORS (P-EPI)
3.5.1 Preparation of Inserts with Feeder Layer
3.5.2 P-EPI Culture
3.6 Characterization of Epidermal Equivalents
3.6.1 Immunocytochemistry in 2D Cell Cultures (See Fig. 5)
3.6.2 Melanin Content of Epidermal Equivalents
3.6.3 Histological Characterization of Equivalents
3.6.4 Immunohistochemistry of Equivalents
3.7 Dermatological Scoring of Histological Sections (See Fig. 6)
4 Notes
References
Part III: Characterization and Pre-Conditioning
Chapter 14: Using Gene Expression Music Algorithms (GEMusicA) for the Characterization of Human Stem Cells
1 Introduction
2 Materials
2.1 Software and Hardware Requirements
2.2 Gene Expression Data
3 Methods
3.1 Preparing the Environment
3.2 Step A: Selection and Pre-Processing of Gene Expression Data
3.3 Step B.1: Calculation, Filtering, and Sorting of Frequencies and Tone Length
3.4 Step B.2: (Optional) Adjust Melodies to a Reference Model
3.5 Step C: Generate Audio Files
3.6 Step D: (Optional) Visualization and Quantitative Analysis of Frequencies
4 Notes
References
Chapter 15: Evaluation of Extracellular Vesicles from Adipose Tissue-Derived Mesenchymal Stem Cells in Primary Human Chondrocy...
1 Introduction
2 Materials
2.1 AD-MSC Isolation, Culture, and Analysis
2.2 EVs Isolation and Analysis
2.3 OA Chondrocyte Isolation and Culture
3 Methods
3.1 Isolation of AD-MSCs
3.2 AD-MSC Culture
3.3 Phenotypic Characterization of AD-MSCs
3.4 Isolation of EVs from AD-MSC CM
3.5 Analysis of EVs
3.6 Isolation and Culture of OA Chondrocytes
3.7 Stimulation and Treatment of OA Chondrocytes
4 Notes
References
Chapter 16: Generation of Neural Stem Cells from Pluripotent Stem Cells for Characterization of Early Neuronal Development
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.3 Buffers and Media
3 Methods
3.1 Coating Culture Plates
3.1.1 Matrigel-Coated Nunc Cell Culture 6-Well Plates
3.1.2 Poly-L-Ornithine-Coated Dishes
3.1.3 Poly-L-Ornithine-/Laminin-Coated Dishes
3.2 Generation of NSC Lines from Human iPS Cells
3.2.1 Generation of 3D EB Cultures
3.2.2 Generation of Neural Tube-Like Structures
3.2.3 Generation of Neurospheres in Suspension Cultures
3.2.4 Generation of NSC Cultures
3.3 Permanent Cell Culture of NSCs Representing a Stable NSC Line
3.4 Characterization and Cryopreservation of NSC Lines
4 Notes
References
Chapter 17: Engineered Tissues Made from Human iPSC-Derived Schwann Cells for Investigating Peripheral Nerve Regeneration In V...
1 Introduction
2 Materials
2.1 Differentiation of hiPSCs to Schwann Cells
2.2 Tethered Mini Collagen Hydrogels
2.3 Immunocytochemistry and mRNA Extraction
3 Methods
3.1 Differentiation of hiPSCs to Schwann Cells Via an Expandable Schwann Cell Precursor Stage
3.2 Tethered Collagen Hydrogels
3.3 Immunocytochemistry and RNA Extraction
3.4 Neuronal Co-Culture for Peripheral Nerve Regeneration
4 Notes
References
Chapter 18: Modulation of a Stem Cell Gene: LGR4 Knockout in a Human Cell Line by CRISPR/Cas Method
1 Introduction
2 Material
2.1 Generation of a Vector
2.1.1 Linearization of the Vector
2.1.2 Phosphorylation of the Vector
2.1.3 Ligation
2.1.4 Amplification of the Vector
2.2 Transfection of the Cells
2.3 Sorting of the Cell
2.4 Analysis of Cell Clones
2.4.1 Sequencing
2.4.2 Molecular Biological Tests Like the TOPFlash Assay
3 Methods
3.1 Generation of a Vector and Linearization of the Vector
3.2 Phosphorylation of the DNA Oligos
3.3 Ligation
3.4 Amplification of the Vector
3.5 Transfection of the Cell System
3.6 Sorting of the Cells
3.7 Analysis of Cell Clones
3.7.1 Sequencing
3.7.2 Molecular Biological Tests-TOP/FOPFlash Assay
4 Notes
References
Chapter 19: In Vitro Tool: 3D Cell Culture of Human Adipose Tissue-Derived Mesenchymal Stromal Cells on Low Stiffness Silicone...
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 Isolation and Cell Culture of Human MSC from Adipose Tissue
2.1.2 2D and 3D Cell Culture
2.1.3 Differentiation
2.1.4 Characterization of Differentiated Cells by Histological Staining
2.2 Equipment
3 Methods
3.1 Preparation of Human Adipose Tissue-Derived MSC
3.1.1 Isolation of Human Adipose Tissue-Derived MSC
3.1.2 Cryopreservation of Human Adipose Tissue-Derived MSC
3.2 3D Culture and Differentiation of Human Adipose Tissue-Derived MSC
3.2.1 Coating
3.2.2 Thawing of Human Adipose Tissue-Derived MSC
3.2.3 Seeding and 3D Culture of Human Adipose Tissue-Derived MSC
3.2.4 Differentiation of Human Adipose Tissue-Derived MSC
3.3 Histological Characterization
3.3.1 Hepatogenic Differentiation by PAS Reaction
3.3.2 Osteogenic differentiation by BCIP/NBT Staining
3.3.3 Adipogenic Differentiation
4 Notes
References
Index