Preface
Contents
Contributors
Lineage Tracing by Single-Cell Transcriptomics Decoding Dynamics of Lineage Commitment
1 Introduction
2 Materials
2.1 Reagents and Equipment
2.2 Software
3 Methods
3.1 Sample Collection and Single-Cell Sequencing
3.2 Data Pre-processing
3.3 Dimensionality Reduction, Cell Clustering, and Annotation
3.4 Trajectory Analysis
4 Notes
References
Cleavage Under Targets & Release Using Nuclease (CUT&RUN) of Histone Modifications in Epidermal Stem Cells of Adult Mu
1 Introduction
2 Materials
2.1 Skin Tissue Processing
2.2 Fluorescent Activated Cell Sorting of EpSCs
2.3 CUT&RUN
2.4 Library Preparation and Quality Assessment
3 Methods
3.1 Isolation of Epidermal Cells from Dorsal Skin of Adult Mice
3.2 Fluorescent Activated Cell Sorting of EpSCs
3.3 CUT&RUN of Histone Modifications in Isolated EpSCs
3.3.1 Prepare ConA Beads (Day 1)
3.3.2 Prepare Cells for Binding to Beads (Day1)
3.3.3 Add Antibodies (Overnight)
3.3.4 Cleavage and Release of Target-DNA Complex (Day 2)
3.4 Library Preparation and Quality Assessment
3.5 Sequencing and Data Analysis
4 Notes
References
Targeted Gene Silencing by Using GapmeRs in Differentiating Human-Induced Pluripotent Stem Cells (hiPSC) Toward Pancreatic Pro
1 Introduction
2 Materials
2.1 Equipment
2.2 Disposables
2.3 Reagents
2.4 Reagent Preparation
3 Methods
3.1 Differentiation Toward Pancreatic Endocrine Cell Fate
3.2 Gene Silencing During Differentiation in 2D Conditions
3.3 Evaluation of Cell Viability
3.4 Evaluation of Gene Silencing Efficiency (qPCR)
3.5 Evaluation of GapmeRs Impact on Differentiation Efficiency (Immunofluorescence)
4 Notes
References
Retinoic Acid-Mediated Differentiation of Mouse Embryonic Stem Cells to Neuronal Cells
1 Introduction
2 Materials
2.1 Chemicals
2.2 Cell Line
2.3 Miscellaneous
3 Methods
3.1 Initiation of ESC Culture
3.2 Subculturing ESCs
3.3 Initiation of Embryonic Bodies (EB) Culture
3.4 Differentiation of EBs into Neuronal Progenitor Cells (NPCs).
3.5 Harvesting EBs
3.6 Differentiation of NPCs to Neurons
4 Notes
References
Isolation and Functional Analysis of Myoepithelial Cells from Adult Mouse Submandibular Glands
1 Introduction
2 Materials
2.1 Reagents
2.1.1 Mice
2.1.2 Culture
2.1.3 DNA Cloning
2.1.4 FACS
2.1.5 Immunofluorescence
2.2 Equipment
2.3 Reagent Setup
3 Methods
3.1 Primary Culture of Myoepithelial Cells from Adult Mouse Submandibular Glands
3.1.1 Preparation of Submandibular Glands [Timing: 10 min]
3.1.2 Isolation of Single Cells with Enzymatic Digestion [Timing: 90 min]
3.1.3 Flow Cytometry Analysis and Cell Sorting (FACS) [Timing: 90 min]
3.1.4 Culture of Myoepithelial Cells (Fig. 1c and 1d)
3.2 Establishment of a Myoepithelial Cell Line from Trp53-Null Mice
3.3 Establishment of Dox-Inducible Gene Expression Cell Line
3.3.1 Preparation of Expression Vector (Fig. 1e) (See Note 9)
3.3.2 Transfection
3.3.3 Drug Selection
3.3.4 Isolation by Limiting Dilution and Expansion
3.3.5 Validation of Inducible Expression
3.4 Assays
3.4.1 Sphere Culture (Fig. 1f and 1g)
3.4.2 Immunohistochemistry
3.4.3 Tissue-Specific Markers
4 Notes
References
Chromosomal Analysis in Lineage-Specific Mouse Hematopoietic Stem Cells and Progenitors
1 Introduction
2 Materials
2.1 Preparation of Cell Samples
2.2 Mouse Bone Marrow Cells (MBMCs) Isolation and Culture
2.3 Colony-Forming Unit (CFU) Assay
2.4 Karyotyping Study
3 Methods
3.1 Isolation of Mouse Bone Marrow
3.2 Preparation of Methylcellulose Media
3.3 Enrichment of HSPCs Using Colony-Forming Unit (CFU)
3.4 Analysis of Lineage-Specific HSPCs from CFU Assay
3.5 Slide Preparation for Karyotyping Analysis
3.6 Karyotyping Analysis by Using GenASIs Bandview Software
4 Notes
References
Easy and Rapid Methods for Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Human Umbilical WhartonΒ΄s
1 Introduction
2 Materials
2.1 Biological Materials
2.2 Equipment
2.3 Chemicals and Solutions
3 Methods
3.1 Preparation of DMEM Medium Solution
3.1.1 Human Umbilical Cord Blood Derived-Mesenchymal Stem Cell Procedures
Obtaining of Human Umbilical Cord Blood
Isolation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells
3.1.2 Human Umbilical WhartonΒ΄s Jelly Derived-Mesenchymal Stem Cell Procedures
Preparation of Sterilization Solution for Human Umbilical Cord
Obtaining of Human Umbilical Cord
Isolation of Human Umbilical WhartonΒ΄s Jelly-Derived Mesenchymal Stem Cells
4 Notes
References
Encapsulation of MSCs in PRP-Derived Fibrin Microbeads
1 Introduction
2 Materials
2.1 Equipment and Consumables
2.2 Solutions
3 Methods
3.1 Preparation of PRP
3.2 Culture of BM-MSCs
3.3 Cell Microencapsulation Within PRP-Derived Fibrin
3.4 Scanning Electron Microscopy (SEM)
3.5 Viability Assessment of Encapsulated Cells
4 Notes
References
Assessing Neuronogenic Versus Astrogenic Bias of Neural Stem Cells Via In Vitro Clonal Assay
1 Introduction
2 Materials
2.1 Reagents
2.1.1 Media
Proliferative
Differentiative
2.1.2 Blocking Solution
2.1.3 Antibodies
2.1.4 Other
2.2 Equipment
3 Methods
3.1 Embryonic Dissection
3.2 Setting Up of the Neurosphere Culture
3.3 Clonal Assay
3.4 Clonal Assay Immunocytochemistry
3.5 Clonal Assay Analysis
4 Notes
References
Stem Cell-Based Modeling Protocol for ParkinsonΒ΄s Disease
Abbreviations
1 Introduction
2 Materials
2.1 Induced Pluripotent Stem Cells Culturing
2.2 Embryoid Body (EB) Formation
2.3 In Vitro Differentiation of iPSCs into Midbrain Dopaminergic Neurons (mDMA)
3 Methods
3.1 Cell Culture
3.2 Generation of Embryoid Body (EB)
3.3 Differentiation of iPSCs into Midbrain Dopaminergic Neurons (mDMA)
4 Notes
References
Standard Operating Procedure for Production of Mouse Brown Adipose Tissue-Derived Mesenchymal Stem Cells
Abbreviations
1 Introduction
2 Materials
2.1 Mouse Brown Adipose Tissue Procurement
2.2 Harvesting, Brown Adipose Tissue, Mesenchymal Stem Cells Manufacturing, and Banking
3 Methods
3.1 Brown Adipose Tissue Procurement
3.2 Isolation and Culture of Brown Adipose Tissue-Derived MSCs
3.3 Characterization of Brown Adipose Tissue-Derived MSCs
3.4 Cryopreservation of Brown Adipose Tissue-Derived MSCs
4 Notes
References
Development and Validation of Type 2 Diabetic Zebrafish Model for Cell-Based Treatments
Abbreviations
1 Introduction
2 Materials
2.1 Preparing Animal Model
2.1.1 Fish Maintenance and Induction of Type 2 Diabetes
2.2 Monitoring of Diabetes for Validation of Type 2 Diabetic Zebrafish Model
2.2.1 Measurement of Blood Glucose Level
2.2.2 Gene Expression Analysis by Quantitative Real-Time RT-PCR (RT-qPCR)
3 Methods
3.1 Preparing Animal Model
3.1.1 Fish Maintenance
3.1.2 Induction of Type 2 Diabetes
3.2 Monitoring of Diabetes for Validation of Type 2 Diabetic Zebrafish Model
3.2.1 Measurement of Blood Glucose Level
3.2.2 Gene Expression Analysis by Quantitative Real-Time RT-PCR (RT-qPCR)
4 Notes
References
Neuromuscular Junction-on-a-Chip for Amyotrophic Lateral Sclerosis Modeling
Abbreviations
1 Introduction
2 Materials
2.1 Motor Neuron Spheroids Generation
2.2 Skeletal Muscle Cells Formation and Injection into an NMJ Chip
2.2.1 Coating and Injection of the MN Spheroid into an NMJ Chip
3 Methods
3.1 Motor Neuron Spheroids Generation
3.2 IPSC-Derived Skeletal Myoblasts Preparation and Injection into an NMJ Chip
3.3 Coating and Injection of the Motor Neuron Spheroids into an NMJ Chip
4 Notes
References
Primary Human Leukemia Stem Cell (LSC) Isolation and Characterization
1 Introduction
2 Materials
2.1 Instruments
2.2 Bone Marrow and Blood Samples
2.3 Materials for Separating Mononuclear Cells (MNCs) from Bone Marrow-Peripheral Blood
2.4 Separation Kits for LSC
2.4.1 Recommended Medium
2.5 Markers for LSCs
3 Methods
3.1 Patient Bone Marrow and Blood Samples
3.2 Ficoll Gradient Method for MNC Isolation
3.3 Removal of Erythrocytes with Lysis Solution
3.3.1 Immunophenotyping After Sorting
3.3.2 Separation of LSCs Using Magnetic Selection (MS) Method
3.3.3 Immunophenotyping After MS
4 Notes
References
GMP-Compliant Mesenchymal Stem Cell-Derived Exosomes for Cell-Free Therapy in Cancer
Abbreviations
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Culture Conditioned Media (CCM) Preparation
2.3 Isolation of Mesenchymal Stem Cell-Derived Exosomes (MSC-Exo)
2.4 Identification of Mesenchymal Stem Cell-Derived Exosomes (MSC-Exo)
2.4.1 Scanning Electron Microscopy Photography by Transmission Electron Microscope (TEM)
2.4.2 Western Blot
2.4.3 Enzyme-Linked Immunosorbent Assay (ELISA)
3 Methods
3.1 Cell Culture
3.2 Cell Culture Conditioned Media (CCM) Preparation
3.3 Isolation of Mesenchymal Stem Cell-Derived Exosomes (MSC-Exo)
3.4 Identification of Mesenchymal Stem Cell-Derived Exosomes (MSC-Exo)
3.4.1 Scanning Electron Microscopy Photography by Transmission Electron Microscope (TEM)
3.4.2 Western Blot
3.4.3 Enzyme-Linked Immunosorbent Assay (ELISA)
4 Notes
References
Establishing Brain Tumor Stem Cell Culture from Patient Brain Tumors and Imaging Analysis of Patient-Derived Xenografts
1 Introduction
2 Materials
2.1 Tools and Equipment
2.2 Reagents
3 Methods
3.1 Establishing BTSC Cultures from Human Patients
3.1.1 Making Media
3.1.2 BTSC Isolation Following Surgical Excision of the Tumors
3.2 BTSC Maintenance (Passage, Freezing Down/Thawing)
3.2.1 Passaging Established Cell Lines
3.2.2 Freezing BTSCs
3.2.3 Thawing Frozen BTSCs
3.3 Tumor Induction and Mice Preparation
3.3.1 Preparation of Luciferase-Expressing BTSC Cell Line for Injection
3.3.2 Animal Preparation (Tumor Induction)
3.3.3 D Luciferin Preparation for Injection
3.4 Performing IVIS
3.5 Determining the Kinetic Curve
3.6 Analysis Using the Living Image Software
3.6.1 Create an Account
3.6.2 Initialization of the IVIS Spectrum
3.6.3 Acquire a Luminescent Image
3.7 Magnetic Resonance Imaging
4 Notes
References
An Optimized Protocol for piggyBac-Induced iPSC Generation from hPBMCs by Automatic Electro
1 Introduction
2 Materials
2.1 Peripheral Blood Harvest and Transfer Conditions, PBMC Isolation Preparation, and Culture Conditions
2.2 PBMC Cryopreservation Conditions
2.3 Vector Isolation
2.4 Transfection
2.5 Post-transfection Cell Recovery and iPSC Culture Conditions
2.6 iPSC Cryopreservation Conditions and Passaging
3 Methods
3.1 Peripheral Blood Mononuclear Cell (PBMC) Isolation
3.2 PBMC Cultivation Before Transfection
3.3 PBMC Cryopreservation and Thawing Before Transfection
3.4 Vector Isolation
3.5 Transfection, Post-transfection Cell Recovery, and iPSC Cultivation
3.6 iPSC Passaging, Cryopreservation, and Thawing
4 Notes
References
Signaling Pathways in Trans-differentiation of Mesenchymal Stem Cells: Recent Advances
1 Introduction
2 Genetic Manipulation of MSC
3 Major Signaling Pathways Involved in MSC
3.1 WNT and Ξ²-Catenin Pathways
3.2 Notch Signaling
3.3 TGFΞ²/BMP Superfamily
3.4 FGF Signaling
4 Trans-differentiation Potential of MSC
5 Transcription Factor-Induced Trans-differentiation of MSCs
6 TFs-Induced Direct Transformation of MSCs Lineage Versus Procreation of iPS Cells
7 Mechanisms Behind Plasticity of Adult Mesenchyme
8 Recent Advancements in the Trans-differentiation Capacities of MSCs
9 Conclusion
References
Index