Preface
Contents
Contributors
Part I: ES/iPS
Chapter 1: Rapid and Highly Efficient Isolation and Purification of Human Induced Pluripotent Stem Cells
1 Introduction
2 Materials
3 Methods
3.1 Adaption of iPSCs to Single Cell Culture and Passaging
3.2 iPSC Purification by Magnetic-Activated Cell Sorting (MACS)
4 Notes
References
Chapter 2: Artificial Activation of Murine Oocytes Using Strontium to Derive Haploid and Diploid Parthenotes
1 Introduction
2 Materials
2.1 Superovulation of Mice
2.2 Dissection of Mice
2.3 Oocyte Cumulus Complex (OCC) Collection
2.4 Parthenogenetic Activation of Oocytes
2.5 Denuding of OCC
2.6 Embryo Culture
2.7 Pronuclear (PN) Stage Assessment and Embryo Culture
3 Methods
3.1 Superovulation of Mice
3.2 Dissection of Mice
3.3 Oocyte Cumulus Complex (OCC) Collection
3.4 Parthenogenetic Activation of Oocytes
3.5 Denuding of Oocyte Cumulus Complexes
3.6 Embryo Culture
3.7 Pronuclear (PN) Stage Assessment and Embryo Culture
4 Notes
References
Chapter 3: Generation of Human iPSC from Small Volume Peripheral Blood Samples
1 Introduction
2 Materials
2.1 Isolation of Blood Cells
2.2 Isolation of CD34+ Cells
2.3 Freezing of Donor Cells
2.4 Culture of PBMCs or CD34+ Cells
2.5 Transduction of PBMCs or CD34+ Cells
2.6 Cultivation of Transduced Cells
2.7 Transfer to Feeder Cells
2.8 Subcloning and Expansion of iPSCs
3 Methods
3.1 Isolation of PBMCs from Small Blood Volume
3.2 Isolation of CD34+ Cells from PBMCs
3.3 Freezing of Donor PBMCs
3.4 Culturing of PBMCs/CD34+ Cells
3.5 Transduction of PBMCs/CD34+ Cells for Reprograming
3.6 Further Cultivation of Transduced PBMCs/CD34+ Cells
3.7 Transfer of Reprogrammed Cells for iPSC Colony Formation on Feeder Cells
3.8 Subcloning and Expansion of Individual iPSC Colonies
4 Notes
References
Chapter 4: Distinguishing Between Endodermal and Pluripotent Stem Cell Lines During Somatic Cell Reprogramming
1 Introduction
2 Materials
2.1 Media Preparation
2.2 Preparing Mouse Embryonic Fibroblasts (MEFs)
2.3 Preparing Replication-Incompetent Retroviruses for Overexpression of OSKM for Reprogramming
2.4 Viral Titer of Retrovirus
2.5 OSKM Viral Reprogramming
2.6 Picking and Passaging iXEN and iPS Cell Colonies
2.7 Confocal Imaging of Reprogramming Cells
2.8 Cell Sorting of Reprogramming Cells
3 Methods
3.1 Preparing Mouse Embryonic Fibroblasts (MEFs)
3.2 Making Retrovirus for Reprogramming
3.3 Viral Titer of Retrovirus
3.4 OSKM Viral Reprogramming
3.5 Picking and Passaging iXEN and iPS Cell Colonies
3.6 Fluorescently Activated Cell Sorting (FACS) of Reprogramming Cells
3.7 Confocal Imaging of Colonies Undergoing Reprogramming
4 Notes
References
Chapter 5: Measuring Early Germ-Layer Specification Bias in Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 hPSC Culture and Differentiation
2.2 hPSC Differentiation
2.3 Gene-Expression Analysis by qRT-PCR
2.4 Immunocytochemistry
3 Methods
3.1 hPSC Culture
3.2 hPSC Differentiation
3.3 Gene-Expression Analysis by Quantitative Real-Time PCR
3.4 Immunocytochemistry and Quantification
4 Notes
References
Chapter 6: Detection of Soluble and Insoluble Protein Species in Patient-Derived iPSCs
1 Introduction
2 Materials
2.1 Cell Lysis
2.2 Protein Quantification and Centrifugation
2.3 SDS-PAGE
2.4 Transfer
2.5 Fluorescent Western Blot (See Note 2)
3 Methods
3.1 Triton X-100 Cell Lysis
3.2 Total Protein Quantification and Sample Preparation
3.3 Ultracentrifugation
3.4 SDS-PAGE and Transfer
3.5 Western Blot
3.6 Quantification Using Image Studio Lite (See Note 25)
4 Notes
References
Chapter 7: Extracellular Flux Analysis of Mitochondrial Function in Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 Cell Seeding
2.3 Mitochondrial Stress Test
2.4 ETC Complex Specific Assay
3 Methods
3.1 Day 1: Cell Seeding and Sensor Cartridge Hydration
3.2 Day 2: Extracellular Flux Assays
3.2.1 Mitochondrial Stress Assay
3.2.2 ETC Component Specific Assay
4 Notes
References
Chapter 8: Assessment of Endothelial-to-Hematopoietic Transition of Individual Hemogenic Endothelium and Bulk Populations in D...
1 Introduction
2 Materials
2.1 Supplies/Reagents for hPSC Maintenance
2.2 Supplies/Reagents for hPSC Differentiation into PHE
2.3 Supplies/Reagents to Continue hPSC Differentiation into HSPC
2.4 Supplies/Reagents for Bulk Population EHT Cultures
2.5 Supplies/Reagents for Single-Cell EHT Cultures
2.6 Supplies/Reagents for AHE-Specific Cultures
2.7 Supplies/Reagents for Measuring EHT and Hematopoiesis
2.8 General Supplies/Equipment
3 Methods
3.1 Feeder-Free Maintenance of hPSCs on Vitronectin in E8 Media
3.2 Preparation of hPSC Differentiation on TenC- or ColIV-Coated Plates (Day -1)
3.3 Differentiation into Primordial Hemogenic Endothelium (PHE)
3.4 Continue Differentiation in Bulk Culture Without Purification of HE Subsets
3.5 Isolate PHE on D4 and Culture on Collagen IV+IgG-Fc or Collagen IV+DLL1-Fc Coated Plates to Enhance AHE Specification and ...
3.6 Single-Cell Deposition Assay of D4 PHE on OP9-iDLL4
3.7 Isolate AHE on D5 and Culture on OP9/OP9-DLL4 for the Assessment of Hematopoietic Potential
4 Notes
References
Part II: ES/iPS Derived Tissue Stem Cells
Chapter 9: Homogeneous Differentiation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 iPSC Line
2.2 iPSC Culture Medium
2.3 Reagents
2.4 Hepatocyte Differentiation and Maturation Media and Solutions
2.5 Equipment
3 Methods
3.1 Single Cell Culture of iPSCs
3.2 Plating iPSCs for Differentiation
3.3 Induction of Definitive Endoderm (Duration: 4 Days; Fig. 1)
3.4 Hepatic Specification (Duration: 8 Days)
3.5 Hepatocyte Maturation (Duration: 3 Days)
3.6 Further Maturation and Maintenance of HLCs (Duration: 1-3 Weeks)
4 Notes
References
Chapter 10: Differentiation of Human Induced Pluripotent Stem Cells into Cortical Neurons to Advance Precision Medicine
1 Introduction
2 Materials
2.1 Matrigel-Coated Plates
2.2 Induced Pluripotent Stem Cells (iPSC) Medium
2.3 iPSC Thawing, Maintenance, and Passaging
2.4 iPSC Neural Induction
2.5 Neural Rosettes Cryopreservation
2.6 Neural Progenitor Cell (NPC) Medium
2.7 Poly-l-Ornithine and Laminin (POL) Coated Plates
2.8 NPC Maintenance and Passaging
2.9 NPCs Cryopreservation
2.10 Thawing Neural Progenitor Cells
2.11 NPC Differentiation and Neuronal Cultures
3 Methods
3.1 Induced Pluripotent Stem Cells (iPSC) Thawing
3.1.1 Preparation of Matrigel-Coated Plates
3.1.2 Preparation of iPSCs
3.2 Proliferating iPSC Medium Change (``iPSC Feeding´´) (See Note 12)
3.3 iPSC Passage
3.4 Derivation of Neural Rosettes from iPSC
3.4.1 Day -1 and Before: iPS Cells Expansion
3.4.2 Day 0: Plate Preparation and iPSC Neural Induction
3.4.3 Day 1-4: Daily Medium Change
3.4.4 Day 5: Neural Aggregates Transfer to POL Plate
3.4.5 Day 6-11: Neural Aggregates Maintenance and Rosette Formation
3.4.6 Day 12: Selection and Replating Neural Rosettes
3.4.7 Day 14
3.4.8 Day 16, 18
3.4.9 Day 20: Passage and Cryopreservation of NPC at P1
3.5 Neural Rosettes Cryopreservation
3.6 Cryopreservation of NPCs
3.7 Thawing Neural Progenitor Cells (See Note 24)
3.8 NPC Differentiation: Neuronal Cultures in 6-Well Plates
3.9 NPC Differentiation: Neuronal Cultures in 24-Well Plates with Glass Slides
3.10 NPC Differentiation: Neuronal Cultures in 96-Well Plates
4 Notes
References
Chapter 11: Differentiation of iPS-Cells into Peripheral Sensory Neurons
1 Introduction
2 Materials
2.1 Cells
2.2 Reagents and Supplements
2.3 Cell Culture Media
3 Methods
3.1 Differentiation of Neural Crest-like Cells (d0-d10, Fig. 2)
3.2 Coating of Glass Coverslips with Poly-L-Ornithine, Laminin, and Fibronectin
3.3 Differentiation and Maturation of Sensory Neurons (d10-End, Fig. 3)
3.4 Selection of Neurons by Ara-C Treatment
4 Notes
References
Chapter 12: Culture of Human iPSC-Derived Motoneurons in Compartmentalized Microfluidic Devices and Quantitative Assays for St...
1 Introduction
2 Materials
2.1 Transfection Reagents
2.2 Cell Reagents
2.3 Microfluidic Chambers
2.4 MN Seeding in Microfluidic Chambers (MFCs)
2.5 Axon Branching and Branch point Analysis
2.6 Axotomy Assay
3 Methods
3.1 Generation of NIL and NIP Inducible iPSC Lines
3.2 Motoneuron Differentiation (Fig. 1)
3.3 Fabrication of Microfluidic Chambers (MFCs) in PDMS
3.4 MN Seeding in MFCs
3.5 Axon Branching and Branch point Analysis (Fig. 3)
3.6 Axotomy Assay
4 Notes
References
Chapter 13: iPS Cell Differentiation into Brain Microvascular Endothelial Cells
1 Introduction
2 Materials
2.1 Stem Cell Culture
2.2 Plate Coating for Endothelial Cell Seeding
2.3 TEER Measurements
2.4 Sodium Fluorescein Assay
2.5 Poly-D-Lysine Coating for T-75 Flasks
2.6 Fixing and Permeabilization of Cells
2.7 PBTG Blocking Solution
3 Methods
3.1 General Recommendations to Prevent Contamination During the Differentiation Process
3.2 Maintenance of Stem Cell Culture
3.2.1 Preparation of Matrigel-Coated Flasks
3.2.2 Thawing Stem Cell Stocks
3.2.3 Maintenance and Subculture of Stem Cells
3.3 Differentiation Protocol
3.3.1 Differentiation Protocol Setup
3.4 Freezing Down Differentiated Endothelial Cells
3.5 Co-culture of Stem Cell Derived Brain Microvascular Endothelial Cells with Primary Astrocytes
3.6 Immunofluorescence Staining of Brain Microvascular Endothelial Cells Grown on Transwell Membrane
3.7 Measurement of TEER for Barrier Tightness Assessment
3.8 Sodium Fluorescein Assay
4 Notes
References
Chapter 14: Chromatin Immunoprecipitation in Human Pluripotent Stem Cell-Derived 3D Organoids to Analyze DNA-Protein Interacti...
1 Introduction
2 Materials
2.1 Organoid Dissociation and Crosslinking
2.2 Chromatin Extraction and Sonication
2.3 Chromatin Immunoprecipitation
2.4 DNA Purification
2.5 ChIP-qPCR
3 Methods
3.1 Reagent Preparation
3.2 Organoid Dissociation and Crosslinking
3.3 Chromatin Extraction and Sonication
3.4 Chromatin Immunoprecipitation
3.5 DNA Purification
3.6 ChIP-qPCR
3.7 Preparation for ChIP-Seq
4 Notes
References
Chapter 15: Generation of Embryonic Origin-Specific Vascular Smooth Muscle Cells from Human Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Cell Culture Reagents
2.2 Small Molecules
2.3 Growth Factors
2.4 Preparation of Induction and Maintenance Media
3 Methods
3.1 Maintenance of Human iPSCs
3.2 Generation of Lineage-Specific SMC Intermediate Populations
3.2.1 Cardiac Neural Crest (CNC) Cell Differentiation
3.2.2 Ventral Somite (VS) Differentiation
3.2.3 Second Heart Field (SHF) Differentiation
3.2.4 Septum Transversum (ST) Differentiation
3.2.5 Proepicardium (PE)/Epicardium (EPI) Differentiation
3.3 Differentiation and Maturation of Embryonic Origin-Specific VSMC Subtypes
4 Notes
References
Chapter 16: Generation of Salivary Gland Organoids from Mouse Embryonic Stem Cells
1 Introduction
2 Materials
3 Methods
3.1 Maintenance of Mouse ESCs
3.2 Oral Ectoderm Differentiation from Mouse ESCs
3.2.1 Day 0
3.2.2 Day 1
3.2.3 Day 2
3.2.4 Day 5
3.3 Salivary Gland Organoid Differentiation from Mouse ESC-Derived Oral Ectoderm
3.3.1 Day 8
3.3.2 Day 8-30
3.4 Immunostaining of Salivary Gland Organoids
4 Notes
References
Chapter 17: In Vitro Generation of Heart Field Specific Cardiomyocytes
1 Introduction
2 Materials
2.1 Double Reporter hESC Line
2.2 hESC Line Maintenance
2.3 Cardiac Differentiation
2.4 Validation of hESC-Derived CM Populations
3 Methods
3.1 hESC Line Maintenance
3.1.1 Thawing the hESCs
3.1.2 Passaging hESCs Using ReleSR
3.2 Heart Field Specific Cardiac Differentiation
3.2.1 Basal Media Preparation
3.2.2 First Heart Field Specific Cardiomyocyte Differentiation
3.2.3 Second Heart Field Specific Cardiomyocyte Differentiation
3.3 Validation of hESC-Derived CM Populations
3.3.1 Flow Cytometry Analysis
3.3.2 Immunocytochemistry and qPCR
4 Notes
References
Part III: Tissue Stem Cells
Chapter 18: Isolation and Characterization of Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells
1 Introduction
2 Materials
2.1 EV-Free FBS Medium
2.2 Cell Culture Medium
2.3 SDS Polyacrylamide Gel
2.3.1 12% Separating Gel 10 mL
2.3.2 3% Stacking Gel 5 mL
2.4 Running Buffer (1x)
2.5 Transfer Buffer (1x)
2.6 Antibodies
2.7 Blocking Buffer
2.8 2% Uranyl Acetate
3 Methods
3.1 Culture of huc-MSC
3.2 Isolation of huc-MSC-EVs
3.3 Transmission Electron Microscope Analysis
3.4 Nanoparticle Tracking Analysis
3.5 Western Blot
3.6 Gel Electrophoresis
3.7 Transfer Protocol
4 Notes
References
Chapter 19: Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells
1 Introduction
2 Materials
2.1 Expansion and Culture of HSPCs
2.2 HSPC Genome-Editing
2.3 Genomic DNA Extraction and Quantification
2.4 GUIDE-seq
2.5 Off-Target NGS Library Preparation and Sequencing (See Fig. 1)
3 Methods
3.1 COSMID In Silico off-Target Site Prediction (See Fig. 2)
3.2 Experimental Identification of Off-Target Sites Using GUIDE-seq
3.2.1 Genome Editing in CD34+ HSPCs for Integration of dsODN Tag at Cas9 Cut Sites (See Note 3 for GUIDE-seq in Other Cell Typ...
3.2.2 Confirmation of dsODN Integration at the Target Site
3.2.3 Quantification and Shearing of Genomic DNA (See Note 4)
3.2.4 Y-Adapter Preparation
3.2.5 End Repair
3.2.6 Adapter Ligation
3.2.7 PCR1: Amplification of dsTag Integrated Sites
3.2.8 PCR2: Adaptor Labeling of PCR1 Amplicons
3.2.9 Sample Quantification and Normalization
3.2.10 Illumina Sequencing
3.2.11 Bioinformatic Analysis of Sequencing Data
3.2.12 Primer Design for GUIDE-seq Identified Off-Target Sites
3.3 Identification of Off-Target Activity in CD34+ HSPCs
3.3.1 CD34+ Cell Culture
3.3.2 RNP Delivery Using Electroporation
3.3.3 Off-Target Library Sample Preparation (See Fig. 1)
3.3.4 Quantification and Pooling of Off-Target Amplicons
3.3.5 Illumina Sequencing
3.3.6 Bioinformatic Analysis of Sequencing Data
4 Notes
References
Chapter 20: Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.3 Media
2.4 Antibodies
2.5 Animal Requirement
3 Methods
3.1 PBMNC Isolation
3.1.1 Normal Donors
3.1.2 Beta-Hemoglobinopathy Donors
3.2 HSPC Isolation
3.3 Long Term HSC Characterization
3.4 Genome Engineering of HSPCs
3.4.1 Gene Disruption/Deletion
3.4.2 Gene Correction/Addition
ssODN-Based Donor DNA Delivery
rAAV-Based Donor DNA Delivery
3.5 Base Editing Mediated Gene Modification
3.5.1 Lentiviral Approach
3.5.2 Electroporation
3.6 Ex Vivo Clonal Analysis
3.7 Analysis of Genome Engineered HSPCs
3.8 Ex Vivo Differentiation of Genome Engineered HSPCs
3.8.1 In Vitro Erythropoiesis
3.8.2 In Vitro Megakaryopoiesis
3.9 Engraftment of Genome Engineered Cells in NBSGW Mice
3.9.1 Xenotransplantation
Short-Term Engraftment Analysis (Fig. 6a)
Long-Term Bone Marrow Engraftment Analysis (Fig. 6b)
Long-Term Peripheral Blood Engraftment Analysis
Long-Term Spleen Engraftment Analysis
4 Notes
References
Chapter 21: Generation of Rat Neural Stem Cells to Produce Different Astrocyte Phenotypes
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Immunocytochemistry
2.3 Tissue Dissection
2.4 Cell Culture
2.5 Immunocytochemistry
3 Methods
3.1 Animal Preparation
3.2 Dissection
3.3 Tissue Dissociation
3.4 Differentiation of Neurospheres into Astrocytes
3.5 Astrocyte Phenotypes
3.6 Immunocytochemistry
4 Notes
References
Chapter 22: In Situ Quantification and Isolation of MΓΌller Glial Cells by Fluorescence-Activated Cell Sorting from the Regener...
1 Introduction
2 Materials
2.1 High-Intensity Light Lesion
2.2 Eye Dissection
2.3 Tissue Dissociation
2.4 Cryosectioning and Immunohistochemistry
2.5 Zebrafish Lines and Larval Care
2.6 Fluorescence Activated Cell Sorting
2.7 Confocal Microscopy
3 Methods
3.1 Photoreceptor-Specific Lesion Using High-Intensity Light on Zebrafish Larvae
3.2 Eye Dissection of Zebrafish Larvae
3.3 Dissociation of Cells from the Zebrafish Eye
3.4 Isolation of Live MG Cells by Fluorescence Activated Cell Sorting
3.5 Cryosectioning and Immunohistochemistry of Larval Zebrafish
3.6 Confocal Imaging of Larval Zebrafish Retinal Sections
3.7 Quantification of Proliferating MG Cells
4 Notes
References
Chapter 23: Quantification and Clonal Culture of Neural Stem Cells from the Hippocampus of Adult Mouse
1 Introduction
2 Materials
2.1 Mouse Dissection
2.2 Preparation of Single Cell Suspension
2.3 NCFC Assay
2.4 Quantification of NCFC Assay Derived Colonies
2.5 Colony Isolation and Monolayer Culture
3 Methods
3.1 Dissection of Mouse
3.2 Preparation of Single-Cell Suspension
3.3 NCFC Assay
3.4 Quantification of NCFC Assay Derived Colonies
3.5 Colony Isolation and Monolayer Culture
4 Notes
References
Chapter 24: Reprogramming Mouse Oviduct Epithelial Cells Using In Vivo Electroporation and CRISPR/Cas9-Mediated Genetic Manipu...
1 Introduction
2 Materials
2.1 Injection Solution
2.2 Surgical Equipment
2.3 Anesthetic, Antiseptic, and Pain Relief Solutions
2.4 Mouse Lines
2.5 Other Equipment
3 Methods
3.1 Design and Cloning sgRNA Guides
3.2 Preparation of the Microinjection Needle and Injection Solution
3.3 Preparation of Surgical Area
3.4 In Vivo Electroporation
3.5 Confirming Successful Electroporation and Gene Editing
4 Notes
References
Chapter 25: Generation of Human Liver Chimeric Mice and Harvesting of Human Hepatocytes from Mouse Livers
1 Introduction
2 Materials
2.1 Materials for PHHs Preparation Before Transplantation
2.2 Surgical Materials for PHHs Transplantation by Intrasplenic Injection
2.3 Food and Medications for FRG Mice Husbandry
2.4 Materials for Liver Perfusion, Hepatocyte Isolation, and Cryopreservation
3 Methods
3.1 Preparations Before PHH Transplantation
3.1.1 Animal Preparation
3.1.2 PHHs Preparation for Injection
3.2 Transplantation of PHHs Via Intrasplenic Injection
3.3 Food and Medications for Transplanted FRG Mice
3.4 Liver Perfusion and Hepatocyte Isolation
3.4.1 Preparation
3.4.2 Perfusion and Collagenase Digestion
3.4.3 Hepatocyte Processing (Perform in Biosafety Level II Cabinet)
3.4.4 Cryopreservation of Isolated Hepatocytes
4 Notes
References
Chapter 26: Application of 3D Culture Assays to Study Breast Morphogenesis, Epithelial Plasticity, and Cellular Interactions i...
1 Introduction
2 Materials
2.1 General Maintenance of D492 Cell Lines
2.2 Isolation of Breast Endothelial Cells (BRENCs) from Reduction Mammoplasties
2.3 Preparation of D492 3D Assays
2.4 Isolation of 3D Structures
2.5 Immunostaining of 3D Structures
3 Methods
3.1 General Maintenance of D492 Cell Lines
3.1.1 Collagen Coating of Culture Vessels
3.1.2 Retrieving D492 Cell Lines from Liquid Nitrogen
3.1.3 Maintaining D492 Cell Lines
3.1.4 Passaging D492 Cell Lines
3.2 Isolation of Breast Endothelial Cells (BRENCs) from Reduction Mammoplasties (Fig. 2)
3.3 Preparation of D492 3D Assays (Fig. 3)
3.3.1 D492 Monoculture in Matrigel
3.3.2 D492 Co-culture with Endothelial Cells in Matrigel
3.3.3 Isolation of 3D Colonies in Matrigel
3.3.4 D492 Monoculture on-Top of Matrigel (See Note 27)
3.3.5 Immunostaining of 3D Structures on-Top of Matrigel
4 Notes
References
Chapter 27: A Unified Protocol to Streamline Molecular and Cellular Analysis for Three-Dimensional Cell Cultures
1 Introduction
2 Materials
2.1 3D Culture
2.2 Immunofluorescence (IF)
2.3 Protein Isolation
2.4 Total RNA Isolation
3 Methods
3.1 3D Platforms
3.1.1 3D Culture on Gel Bed
3.1.2 3D Culture in Suspension
3.2 Cell Harvest
3.2.1 Cell Harvest from Matrigel Bed
3.2.2 Cell Harvest from Suspension
3.3 Assays
3.3.1 Live Imaging
3.3.2 Immunofluorescence (IF)
3.3.3 Protein Isolation
3.3.4 Total RNA Isolation
4 Notes
References
Chapter 28: Mesenchymal Stem Cell Seeding on 3D Scaffolds
1 Introduction
2 Materials
2.1 MTS Assay
2.1.1 Reagent Preparation
2.2 MTT Assay
2.2.1 MTT Solution Preparation
2.2.2 Solubilization Solution Preparation
2.3 DAPI Staining
2.3.1 Preparing Solutions
2.3.2 Fixing of Cell-Seeded Scaffolds
2.4 Alamar Blue Assay
2.5 ATP Assay
2.5.1 Reagent Preparation
2.6 PicoGreen dsDNA Assay
2.6.1 Assay Buffer Preparation
2.6.2 PicoGreen Preparation
2.7 Cell Lysis Buffer
3 Methods
3.1 MTS Standard Curve
3.2 MTS Assay
3.3 MTT Assay
3.4 DAPI Staining Test
3.5 Alamar Blue Assay
3.6 ATP Assay
3.7 PicoGreen dsDNA Assay
3.7.1 Standard Curve of 2D Cell Culture
3.7.2 DNA Standard Curve (Using a Lambda DNA at the Standard-Quant-iT PicoGreen dsDNA Kit)
3.7.3 Sample Analysis
3.7.4 Cell-Scaffold Construct
4 Notes
References
Chapter 29: Assaying Candidate Human Skin Keratinocyte Stem Cells by Determining Their Long-Term Serial Proliferative Output i...
1 Introduction
2 Materials
3 Methods
3.1 General Culturing and Maintenance of Swiss 3T3-J2 Cells and Establishing Feeder Cells for Long Term Epidermal Cell Culture
3.2 Epidermal Cell Isolation
3.3 Fractionating Epidermal Cells Based on Cell Surface Phenotype
3.4 Expansion of Basal Keratinocyte Fraction to Determine Proliferation Capacity
4 Notes
References
Chapter 30: Protocol for the Detection of Organoid-Initiating Cell Activity in Patient-Derived Single Fallopian Tube Epithelia...
1 Introduction
2 Materials
2.1 Growth Factors
2.2 Chemicals, Media and Reagents
2.3 Other Equipment
3 Methods
3.1 Cell Enrichment for Tissue Sample
3.2 Assay to Detect Organoid-Initiating Cells from Uterine Tubal Derived Single Cell Suspension
3.3 Storage Procedure for Cells
3.4 Procedure to Snap-Freeze Individual or Pooled Tubal Organoids
3.5 Procedure to Cryofreeze and Thaw Tubal Organoids
4 Notes
References
Chapter 31: Quantification of Muscle Stem Cell Differentiation Using Live-Cell Imaging and Eccentricity Measures
1 Introduction
2 Materials
2.1 Muscle Stem Cell Isolation
2.2 Muscle Stem Cell Differentiation
2.3 Myotube Quantification
3 Methods
3.1 Muscle Stem Cell Isolation
3.2 Muscle Stem Cell Differentiation
3.3 Myotube Quantification Using IncuCyte Zoom
4 Notes
References
Part IV: Malignancy
Chapter 32: The Enrichment of Breast Cancer Stem Cells from MCF7 Breast Cancer Cell Line Using Spheroid Culture Technique
1 Introduction
2 Materials
2.1 Cell Line
2.2 Cell Culture Medium
2.3 Spheroid Culture Medium and Plates
3 Methods
3.1 MCF7 Culture
3.2 MCF7 Cells Harvest and Preparation of Single-Cell Suspension
3.3 Preparing Single-Cell Suspension of 500 Cell/mL Concentration and Culture
3.4 Spheroid Incubation Time, Medium Refresh, and Counting
3.5 Spheroid Dissociation, and Passaging
3.6 Spheroid Efficiency Calculation
3.7 Storing Enriched BCSCs
4 Notes
References
Chapter 33: Assessment of Breast Cancer Stem Cell Activity Using a Spheroid Formation Assay
1 Introduction
2 Materials
3 Methods
3.1 Isolation of Epithelial Cells from Murine Mammary Tissue, Primary Breast Tumors and PDX Tumors
3.2 Isolation of Epithelial Cells from Breast Cancer Cell Lines
3.3 Seeding Cells for Sphere Formation Assay
3.3.1 Cell Suspension Sphere Culture
3.3.2 Adherent Sphere Culture
3.4 Counting of Spheres from Cell Suspensions and Matrigel Matrix
3.5 Passaging of Spheres for Assessment of Self-Renewal (Second Generation)
3.6 Coculture Assay
3.7 Propagation of Spheres for Assessment of Different Functional Studies
4 Notes
References
Chapter 34: Enrichment of Cancer Stem Cells in a Tumorsphere Assay
1 Introduction
2 Materials
2.1 Tissue and Cells
2.2 Digestion Medium
2.3 Reagents and Equipment for Separation (See Note 3)
2.4 Cell Culture Reagents (See Note 3)
3 Methods (See Note 7)
3.1 Enrichment from Primary Solid Tumors
3.1.1 Isolation of Primary Tumor Cells from Mouse Solid Tumors
3.1.2 Culture of Primary Mouse Tumor Cells
3.1.3 Culture of Tumorspheres from Primary Mouse Tumor Cells
3.2 Tumorspheres of Cancer Cells
4 Notes
References
Chapter 35: In Vitro Quantification of Cancer Stem Cells Using a Mammosphere Formation Assay
1 Introduction
2 Materials
2.1 Mammosphere Medium
2.2 Complete Mammosphere Medium
2.3 Additional Materials
3 Methods
3.1 Cell Preparation
3.2 Cell Plating
3.3 Media Addition and Replacement
3.4 Mammosphere Quantification
4 Notes
References
Chapter 36: Designing Genetically Engineered Mouse Models (GEMMs) Using CRISPR Mediated Genome Editing
1 Introduction
2 Materials
3 Methods
3.1 Model Strategy
3.2 Design and Choice of gRNAs
3.3 PCR Strategy Design
3.4 Validation of gRNAs in Mouse Zygotes
3.5 DNA Isolation and Genotyping of Blastocysts
3.6 Sequence Analysis and Interpretation
3.7 Designing a Donor Template for HDR (Fig. 1)
3.8 CRISPR/Cas9 Delivery Methods (Fig. 2)
3.9 Embryo Implantation and Monitoring of Pregnancies
3.10 DNA Isolation from Ear Punch or Tail Snips
3.11 Genotyping of Animals (Fig. 3)
3.12 Breeding F0 Founder Animals
3.13 Long Term Genotyping Strategy
3.14 Cryopreservation
4 Notes
References
Chapter 37: Assays for the Spectrum of Circulating Tumor Cells
1 Introduction
2 Materials
2.1 Reagents and Culture Dishes
2.2 Recipes of Solutions
3 Methods
3.1 CTCs Detection and Isolation (See Notes 1 and 2)
3.1.1 CTC Detection in Mouse Blood Samples by Fluorescence Microscope (See Note 3)
3.1.2 CTC Isolation from Mouse or Human Blood Samples by CELLction Biotin Binder Kit and Biotinylated EpCAM Antibody (See Note...
3.1.3 CTC Isolation from Mouse or Human Blood Samples by the Parsortix System
3.2 CTC Culture (See Notes 6 and 7)
3.3 CTC Tumorsphere Formation Assay (See Notes 7 and 8)
3.4 3-Dimensional Invasion Assay (See Note 7)
3.5 Animal Model Experimental CTC Metastatic Variants
3.6 Tumor Cell Self-Seeding
3.7 CTC Clustering Using PDX (See Note 9)
4 Notes
References
Chapter 38: Limiting Dilution Tumor Initiation Assay: An In Vivo Approach for the Study of Cancer Stem Cells
1 Introduction
2 Materials
2.1 Cells for Injection
2.2 Cell Preparation
2.3 Mice
2.4 Cell Injection
2.5 Tumor Monitoring
2.6 Tumor Harvest
2.7 Additional Equipment
2.8 Software
2.9 Input Data
2.10 Parameters
3 Methods
3.1 Cell Preparation
3.2 Serial Dilution of Cells
3.3 Injection of Cells
3.4 Tumor Monitoring
3.5 Tumor Harvest (See Note 10)
3.6 Limiting Dilution Analysis
3.6.1 Visualization
3.6.2 Multiple Group Comparison
3.7 Calculation of Tumor-Initiating Cell Frequency
3.7.1 Calculation of Tumor-Initiating Cell Frequency
3.7.2 Analysis of Multiple Population Group Data
3.7.3 Analysis in R for a Large Number of Datasets
4 Notes
References
Chapter 39: Orthotopic Xenografts of Colorectal Cancer Stem Cells
1 Introduction
2 Materials
2.1 Cell Processing
2.2 Surgery
2.3 Miscellaneous Equipments
3 Methods
3.1 Cell Processing
3.1.1 Cell Culture
3.1.2 Preliminary Assessment of Spheroid Culture Quality
3.1.3 Spheroid Dissociation
3.2 Orthotopic Xenografting
3.2.1 Analgesia and Anesthesia
3.2.2 Cell Grafting
3.2.3 Development and Detection of Primary Tumor and Metastases
4 Notes
References
Index