Adult Stem Cells: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Safe and Noninvasive Method for Generating Induced Mesenchymal Stem Cells from Urinary Epithelial Cells
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Medium and Growth Factors
	3 Methods
		3.1 Urinary Epithelial (UE) Cell Culture Protocol (Fig. 1a)
		3.2 Reprogramming of Human UE into UE-iPSCs
			3.2.1 Thawing Human  UEs
			3.2.2 Reprogramming of UECs (Fig. 1c)
		3.3 Alkaline Phosphatase Staining (Fig. 2c)
		3.4 Characterization of iPSCs
			3.4.1 Immunostaining Analysis (Fig. 3)
			3.4.2 Western Blot Analysis
			3.4.3 qRT-PCR Analysis
	4 Trilineage Differentiation (Fig. 4): To Confirm the Pluripotency by Performing In Vitro Trilineage Differentiation
		4.1 Hepatocyte Differentiation (Endoderm)
		4.2 Neuronal Differentiation (Ectoderm)
		4.3 Endothelial Differentiation (Mesoderm)
	5 Differentiation of Human Urinary Epithelial Cells Derived Induced Pluripotent Cells (UE-iPSCs) into Mesenchymal Stem Cells (...
		5.1 Maintenance of the UE-iMSCs
		5.2 Characterization of iMSCs (See Note 5)
			5.2.1 Immunostaining Analysis (Fig. 6a)
			5.2.2 Western Blot Analysis
			5.2.3 qRT-PCR Analysis (Fig. 5b)
			5.2.4 Flow Cytometry Analysis (Fig. 6b)
			5.2.5 Functional Assays
	6 Evaluating the In Vitro Trilineage Differentiation Potential of UE-iMSCs (Fig. 7)
		6.1 Differentiation of iMSCs into Osteocytes
		6.2 Differentiation of iMSCs into Chondrocytes
		6.3 Differentiation into Adipocytes
	7 Notes
	References
Chapter 2: A Nonenzymatic Procedure to Obtain Human Mesenchymal Stromal Cells from the Dermis
	1 Introduction
	2 Material
		2.1 Tissue (Cells)
		2.2 Supplies and Equipment
		2.3 Reagents and Buffers
		2.4 Solutions
	3 Methods
		3.1 Donors
		3.2 Skin Biopsy Transportation
		3.3 Processing Human Fetal Skin
		3.4 Processing Human Adult Skin
		3.5 Harvesting Fetal Dermal MSCs
		3.6 Harvesting Adult Dermal MSCs
		3.7 Culturing Fetal Dermal MSCs
		3.8 Culturing Adult Dermal MSCs
		3.9 Cell Immunophenotyping
		3.10 Secretome Collection
		3.11 Cryopreservation of Dermal MSCs
	4 Notes
	References
Chapter 3: Adult Mesenchymal Stem Cells in Presence of Glycosaminoglycans
	1 Introduction
	2 Methods
		2.1 Cell Seeding
		2.2 MSC Growth Within the 3D Matrix: Time Course Analyses
		2.3 Biological Analyses
			2.3.1 Proliferation/Viability Assay
				Dimethylthiazol-Diphenyltetrazolium Bromide (MTT) Assay
				Alamar Blue and Neutral Red Assay
				Neutral Red Assay
				Proliferation Assay
				DNA Content Quantification
			2.3.2 Senescence
			2.3.3 Differentiation Toward a Specific Phenotype
			2.3.4 Analyses of Specific Biomarkers Expression
				Chondrogenic Biomarkers
				Adipogenic Biomarkers
	References
Chapter 4: Mitochondrial Transfer Between Mesenchymal Stem Cells and Cancer Cells
	1 Introduction
	2 Materials
		2.1 Reagents
			2.1.1 Cell Co-culture Assay Reagents and Materials
			2.1.2 Reagents and Software for Microscopy
			2.1.3 Reagents and Software for the FACS Analysis
	3 Methods
		3.1 Microscopy Experiment Design
		3.2 Flow Cytometer Experiment Design
	4 Notes
	References
Chapter 5: Isolation and Characterization of Human Dental Pulp Stem Cells Derived from Dental Pulp of Permanent Teeth
	1 Introduction
	2 Materials
		2.1 Human Dental Pulp Tissue
		2.2 Supplies and Equipment
		2.3 Reagents and Buffers
		2.4 Media Solutions
	3 Methods
		3.1 Preparing of Human Dental Pulp Tissue
		3.2 hDPSCs Isolation
		3.3 hDPSCs Maintenance and Enriching
		3.4 Immunophenotyping of hDPSCs
		3.5 Cell Proliferation Assay
		3.6 Immunostaining of hDPSCs
	References
Chapter 6: A Three-Step Protocol to Differentiate iPSC into Colon Organoids
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cell Culture and Preparation
		3.2 Definitive Endoderm Differentiation (Fig. 2)
		3.3 Hindgut Differentiation (Fig. 3)
		3.4 Colon Differentiation and Organoid Formation (Fig. 4)
		3.5 Characterization of Colon Organoids (Fig. 5)
		3.6 Freezing and Thawing of Colon Organoids
			3.6.1 Freezing
			3.6.2 Thawing
	References
Chapter 7: Comparison of Two Differentiation Protocols of Human-Induced Pluripotent Stem Cells into Cardiomyocytes
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Supplies and Equipment
		2.3 Reagents and Buffers
	3 Methods
		3.1 Culture of Human iPSCs
		3.2 Differentiation of hiPSCs to Cardiomyocytes According to the GiWi Protocol (see Fig. 1)
		3.3 Differentiation of hiPSCs to Cardiomyocytes Using the PSC Cardiomyocyte Differentiation Kit (see Fig. 2)
		3.4 Analysis of hiPSC Differentiation Efficiency into Cardiomyocytes
		3.5 Analysis of Gene Expression (see Note 7)
		3.6 Immunofluorescent Stainings
	4 Notes
	References
Chapter 8: Enriching Cardiomyocytes Derived from hiPSCs by Magnetic-Activated Cell Sorting (MACS)
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Supplies and Equipment
		2.3 Reagents and Buffers
	3 Methods
		3.1 Cultivation and Preparation
		3.2 Magnetic Labeling and Separation of hiPSC-Derived Non-cardiomyocytes (See Notes 2-6) of hiPSC-CMs
			3.2.1 Magnetic Labeling of hiPSC-Derived Non-cardiomyocytes
			3.2.2 Magnetic Separation: Depletion of hiPSC-Derived Non-cardiomyocytes
		3.3 Magnetic Labeling and Separation of hiPSC-Derived Cardiomyocytes (See Notes 2-5)
			3.3.1 Magnetic Labeling of hiPSC-Derived Cardiomyocytes-Enrichment
			3.3.2 Magnetic Separation: Positive Selection of hiPSC-Derived Cardiomyocytes
		3.4 Characterization of hiPSC-CMs
			3.4.1 Characterization of hiPSC-CMs by IF Staining
			3.4.2 Characterization of hiPSC-CMs by FACS
			3.4.3 Characterization of hiPSC-CMs by qRT-PCR (See Notes 7 and 8)
	4 Notes
	References
Chapter 9: Generation of Induced Pluripotent Stem Cells from Erythroid Progenitor Cells
	1 Introduction
	2 Materials
	3 Methods
		3.1 Peripheral Mononuclear Cell Isolation
		3.2 Erythroid Progenitor Cell Expansion
		3.3 The Characterization of Erythroid Progenitor Cells by Flow Cytometry
		3.4 The Transduction of Erythroid Progenitor Cells with Sendai Viral Vectors and The Generation of iPSCs
		3.5 iPSCs Characterization
			3.5.1 qRT-PCR analysis for Pluripotency genes
			3.5.2 Immunocytochemistry of Pluripotency Genes
				PSC 4-Marker Immunocytochemistry
				Alkaline Phosphatase Staining
			3.5.3 Formation and Spontaneous Differentiation of Embryoid Bodies
				Characterization of Embryoid Bodies
		3.6 Cell Expansion and Culture Conditions
		3.7 Cryopreservation and Thawing
	References
Chapter 10: iPSC-Based Disease Modeling and Functional Assessment of Neurons in Patients with Metabolic Disorder
	1 Introduction
	2 Materials
		2.1 PBMC Isolation
		2.2 Reprogramming of PBMCs to iPSCs
		2.3 iPSC Characterization
		2.4 Neuronal Differentiation
		2.5 Multielectrode Array
	3 Methods
		3.1 PBMC Isolation
		3.2 iPSC Reprogramming
		3.3 Neuronal Differentiation
		3.4 Neuronal Activity Using MEA and Data Analysis
	4 Notes
	References
Chapter 11: A Method for Producing Induced Pluripotent Stem Cell-Derived Cardiomyocytes from Leigh Syndrome Patients for Its A...
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Supplies and Equipment
		2.3 Chemicals and Reagents
	3 Methods
		3.1 Reprogramming of Induced Pluripotent Stem Cells from Leigh Patient-Derived Peripheral Blood Mononuclear Cells
		3.2 Differentiation of Leigh Patient-Derived Induced Pluripotent Stem Cells to Cardiomyocytes
		3.3 Characterization of Leigh Patient-Derived Induced Pluripotent Stem Cells and Cardiomyocytes Using Immunofluorescence
		3.4 Systematic Procedure to Coat, Culture, and Then Seed Leigh Patient-Derived iPSC-CMs onto a Multi-electrode Array (MEA) Pla...
		3.5 Assessment of Field Potential in Leigh Patient-Derived iPSC-CMs Using a Multi-electrode Array
	4 Notes
	References
Chapter 12: Generation of Human-Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells of C9ORF72-Associated A...
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Supplies and Equipment
		2.3 Reagents and Buffers
	3 Methods
		3.1 Isolation of PBMC
		3.2 Reprogramming of PBMC Using Sendai Vectors
		3.3 Picking hiPSC colonies
		3.4 Culture of hiPSC
		3.5 Spontaneous Differentiation of hiPSC via Embryoid Bodies (EBs)
		3.6 Immunofluorescent Stainings-Analysis of Pluripotency Markers in hiPSC (See Fig. 2) and the Markers of Three Germ Layers in...
		3.7 Quantitative Analysis of Pluripotency Marker Expression Using Flow Cytometry
	4 Notes
	References
Chapter 13: Cardiac Progenitor Cell Metabolism
	1 Introduction
	2 Materials
		2.1 CPC Isolation and Culture
		2.2 Materials for CPC Characterization
		2.3 Materials for CPC Metabolic Assays
	3 CPC Isolation and Culture
		3.1 CPC Cell Culture
		3.2 Sub-culturing
	4 CPC Characterization
		4.1 Immunoblot
		4.2 Flow Cytometry
		4.3 Proliferation Assays
		4.4 Immunostaining
	5 CPC Metabolic Assays
		5.1 Measuring Glycolysis and Oxidative Phosphorylation
		5.2 Mitochondrial Morphology
		5.3 ATP Determination
		5.4 Measurement of CPC Metabolites
	6 Applications for Stem Cell Biology and Cardiac Repair
	References
Chapter 14: Generation of Directly Reprogrammed Human Endothelial Cells
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Plasmid
		2.3 Supplies and Equipment
		2.4 Reagents and Buffer (If Required Filter Through 0.2 μm Filter)
		2.5 Media and Solutions
	3 Procedure
		3.1 Generation of Tetracycline-Inducible Lentiviral Vectors Expressing ETV2 and Production of Lentiviral Particles
		3.2 Early rEC Reprogramming (See Fig. 1)
		3.3 Late rEC Reprogramming (See Fig. 1)
	4 Notes
	References
Chapter 15: Isolation and Characterization of Extracellular Vesicles from Cell Culture Media
	1 Introduction
	2 Methods
		2.1 Supplies and Equipment
		2.2 Reagents and Buffers
		2.3 Preparation of EV-Depleted Cell-Culture Medium
		2.4 EV Isolation
			2.4.1 Isolation of EV-Containing Fraction
			2.4.2 Isolation of  sEVs
			2.4.3 Isolation of  mEVs
		2.5 EV Quantification and Particle Size Analysis
	3 Notes
	References
Chapter 16: Exosomes Isolation, Purification, and Characterization
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Supplies
		2.3 Equipment
		2.4 Software and Data Analysis
	3 Methods
		3.1 Cell Isolation and Maintenance
		3.2 Exosome Purification Methods
		3.3 Ultracentrifugation Method for Exosome Isolation
	4 Characterization
		4.1 Nanosight
		4.2 Transmission Electron Microscopy
		4.3 Western Blot Analysis
	5 Notes
	References
Chapter 17: Exosomes: Methods for Isolation and Characterization in Biological Samples
	Abbreviations
	1 Introduction
	2 Exosome Biogenesis and Secretion
		2.1 Exosome Biogenesis
			2.1.1 ESCRT-Dependent Pathways
			2.1.2 ESCRT-Independent Pathways
		2.2 Exosome Secretion
	3 Exosome Cargo
		3.1 Protein
		3.2 Lipids
		3.3 Nucleic Acids
	4 Methods of Exosome Isolation
		4.1 Ultracentrifugation
			4.1.1 Differential Ultracentrifugation (dUC)
			4.1.2 Density Gradient Ultracentrifugation
		4.2 Ultrafiltration
		4.3 Size-Exclusion Chromatography (SEC)
		4.4 Polymer-Based Precipitation
		4.5 Immunoaffinity Capture
		4.6 Microfluidics
		4.7 Commercial Kits for Exosome Isolation
	5 Exosome Characterization
		5.1 Electron Microscopy
		5.2 Atomic Force Microscopy (AFM)
		5.3 Nanoparticle Tracking Analysis (NTA)
		5.4 Dynamic Light Scattering (DLS)
		5.5 Tunable Resistive Pulse Sensing (TRPS)
		5.6 Flow Cytometry
		5.7 Western Blotting and Enzyme-Linked Immunosorbent Assay (ELISA)
	6 Potential Challenges Using Exosomes as Therapeutics
	7 Conclusion and Future Perspectives
	References
Chapter 18: Cancer Stem Cells: Detection and Characterization from Solid Tumors
	1 Introduction
	2 Materials
		2.1 Reagents
			2.1.1 Cell Culture Reagents and Materials
			2.1.2 Antibodies
			2.1.3 Side Population Reagents
			2.1.4 ALDEFLUOR Assay Reagents
			2.1.5 Equipment and Plates
	3 Methods
		3.1 Spheres
			3.1.1 Mammospheres
			3.1.2 Lung Tumorspheres
			3.1.3 Sarcospheres
		3.2 Detection and Isolation of CSCs by Flow Cytometry
			3.2.1 Stemness Marker Expression
			3.2.2 Side Population Detection
			3.2.3 ALDEFLUOR Assay
		3.3 Flow Cytometer Setup and Data Acquisition
			3.3.1 Cytometric Detection of Stemness Markers
			3.3.2 Cytometric Analyses of Side Population
			3.3.3 Cytometric Analyses of ALDEFLUOR Assay
			3.3.4 Cell Sorting
	4 Notes
	References
Chapter 19: Generation of Bidimensional and Three-Dimensional Muscle Culture Systems
	1 Introduction
	2 Materials
		2.1 Solutions and Media
		2.2 Preparation of Chick Embryo Extract (CEE)
		2.3 Culture Media
	3 Methods
		3.1 Development of Bidimensional Skeletal Muscle Cell Culture by Enzymatic and Mechanical Dissociation
		3.2 Procedure for Isolation of Satellite Cells by Enzymatic Dissociation of Skeletal Muscles
			3.2.1 Classical Protocol
			3.2.2 Automated Dissociation Protocol
		3.3 Isolation of Satellite Cells from Muscle Fibers
			3.3.1 Detailed Protocol
	4 Storage of Satellite Cells
		4.1 Procedure of Cryopreservation
		4.2 Thawing Procedure
	5 Generation of a Three-Dimensional Skeletal Muscle Culture
	6 Notes
	References
Chapter 20: An Enzyme-Free Method for Isolation and Expansion of Muscle Stem Cells for Cultivated Meat Applications
	1 Introduction
	2 Materials
	3 Methods
		3.1 Tissue Preparation
		3.2 Explant Culture
		3.3 Cell Monitoring and Maintenance
		3.4 Cell Type Characterization
			3.4.1 Flow Cytometry
			3.4.2 Immunofluorescence
			3.4.3 Quantitative Real-Time PCR (qRT-PCR)
	References
Chapter 21: Synthesis of Hydrofluoric (HF) Acid Free, Two-Dimensional (2D) Tantalum Carbide MXene Nanosheets and Quantum Dots
	1 Introduction
	2 Materials
		2.1 Supplies and Equipment
		2.2 Reagents
	3 Methods
		3.1 Exfoliation of Aluminum from Ta4AlC3
		3.2 Delamination of Etched MXenes (See Note 1)
		3.3 Synthesis of MXene Nanosheets (MNSs) (See Fig. 2)
		3.4 Synthesis of MXene Quantum Dots (MQDs) (See Fig. 3 and Note 2)
		3.5 Morphological Characterization of MNSs (See Note 3)
		3.6 Morphological Characterization of MQDs (See Note 4)
	4 Notes
	References
Chapter 22: Three-Dimensional Electrically Conductive Scaffolds to Culture Cardiac Progenitor Cells
	1 Introduction
	2 Methods
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 Printing Solution Preparation
		3.2 Scaffold Design and Stereolithography Setup
		3.3 Electroconductive Scaffold Fabrication
		3.4 Culturing of the Cardiac Progenitor Cells on Conductive Scaffolds
		3.5 Cell Viability
		3.6 Cell Phenotype Characterization
	References
Chapter 23: Omic Evaluation of Nanomaterial-Based Photodynamic Therapy of Cancer
	1 Introduction
	2 Materials
		2.1 Cell Culture
			2.1.1 Cell Lines
			2.1.2 Reagents and Buffers
			2.1.3 Supplies and Equipment
		2.2 Transcriptomic and Proteomic Analysis
			2.2.1 Reagent and Buffers for Protein and RNA Samples
			2.2.2 Equipment for Transcriptome and Proteome Analysis
			2.2.3 Requirements for Bioinformatic Analysis
	3 Methods
		3.1 Evaluation of Cytotoxicity
		3.2 In Vitro Photodynamic Therapy
		3.3 Visualization of ROS Production
		3.4 Proteomic Analysis
			3.4.1 Extraction and Digestion of Proteins
			3.4.2 Measurement of Protein Concentration with BCA Assay
			3.4.3 Profiling of Proteins
		3.5 Transcriptomic Analysis
			3.5.1 Extraction and Preparation of RNA
			3.5.2 Sequencing and Data Processing
		3.6 Bioinformatic Analysis
			3.6.1 Proteomic Analysis
			3.6.2 Transcriptomic Analysis
	4 Notes
	References
Chapter 24: Electrospun Fibers for Use in Implantable Materials to Support Cell Therapy
	1 Introduction
	2 Materials
		2.1 Polymer Formulation
		2.2 Electrospinning Process
		2.3 Filtering Process
	3 Methods
		3.1 Polymer Formulation (See Fig. 1)
		3.2 Electrospinning Process (See Fig. 2)
		3.3 Filtering Process (See Fig. 4)
	4 Notes
	References
Chapter 25: Caco-2 Monolayer as a Model of the Intestinal Barrier: Permeability of Magnesium Salts
	1 Introduction
	2 Materials
		2.1 Cells
		2.2 Supplies and Equipment
		2.3 Reagents and Buffers
		2.4 Media Solutions
	3 Methods
		3.1 Cultivation of Caco-2 Cells
		3.2 Preparing of Caco-2 Monolayers
		3.3 Magnesium Transport Through Caco-2 Monolayers
		3.4 Colorimetric Estimation of Magnesium Ions in Basolateral Samples
	4 Notes
	References
Chapter 26: Antimicrobial Surface for Devices Used in Stem Culture Manipulation and In Vitro Biofabrication of Tissues
	1 Introduction
	2 Methods
		2.1 Reagents
			2.1.1 Antimicrobial Agents
			2.1.2 Polymeric Matrices
			2.1.3 Solvents
			2.1.4 Substrates
		2.2 Preparation and Deposition of Silver-Based Antimicrobial Solutions
			2.2.1 Preparation of Antimicrobial Mixture Using Ag+ and UV Curable Resin Epoxy Cycloaliphatic  ECC
			2.2.2 Preparation of Antimicrobial Mixture Using Ag+ and Commercial Paint
		2.3 Preparation and Deposition of Curcumin-Based Antimicrobial Solutions
	3 Conclusions
	4 Notes
	References
Chapter 27: Manufacturing Process of Prostheses Using Semirigid Molds by Additive Technologies
	1 Introduction
	2 Methods
		2.1 Optimization of Printing Parameters
		2.2 Computational Processing
	3 Conclusions
	References
Chapter 28: Preparation of Monodispersed Nanofibrous Gelatin Microspheres Using Homebuilt Microfluidics
	1 Introduction
	2 Material
		2.1 Supplies and Equipment
		2.2 Homemade Microfluidic Device
		2.3 Homebuilt Microfluidics
		2.4 Reagents
	3 Methods
		3.1 Generate Monodispersed Droplets of Gelatin/Water/Ethanol (GWE) in Mineral  Oil
		3.2 Solvent Exchange
		3.3 Crosslinking
		3.4 Blocking
		3.5 Lyophilization
	4 Notes
	References
Index