Preface
Contents
Contributors
Heterogeneity of Stem Cells: A Brief Overview
1 Introduction
2 Embryonic Stem Cells
3 Adult Stem Cells
4 Cancer Stem Cells
5 Induced Pluripotent Stem Cells
References
Establishment and Characterization of NaΓ―ve Pluripotency in Human Embryonic Stem Cells
1 Introduction
2 Materials
2.1 Tissue Culture
2.2 Karyotyping
2.3 Alkaline Phosphatase Assay for Pluripotency
2.4 Immunocytochemistry Assay for Pluripotency
2.5 Quantitative Real-Time PCR
3 Methods
3.1 Mouse Embryonic Fibroblast (MEF) Isolation (See Note 21)
3.2 MEF Passaging
3.3 MEF Freezing
3.4 MEF Thawing
3.5 MEF Inactivation and Feeder Layer Seeding
3.5.1 Mitomycin C Treatment
3.5.2 Gelatine Coating
3.5.3 MEF Seeding
3.6 Manual Passage of hESCs
3.7 Enzymatic Passage of hESCs
3.8 NaΓ―ve hESC Conversion of Primed hESCs
3.9 Passaging NaΓ―ve hESCs
3.10 Cryopreservation of NaΓ―ve and Primed hESCs
3.11 Thawing of hESCs
3.12 Karyotyping NaΓ―ve and Primed hESCs
3.12.1 Arresting Cell Cycle at Metaphase
3.12.2 Producing Single Cell Populations
3.12.3 Fixation of the Cells
3.12.4 G-Banding
3.13 Alkaline Phosphatase Staining Assay for Pluripotency
3.14 Immunocytochemistry for Pluripotency
3.14.1 Preparation of MEF Feeder Cells on Glass Coverslips (See Note 79)
3.14.2 Cell Fixation
3.14.3 Staining with Primary Antibodies
3.14.4 Staining with Secondary Antibodies
3.15 NaΓ―ve Pluripotency Gene Expression Analysis
3.15.1 RNA Isolation and Reverse Transcription (See Note 96)
3.15.2 Quantitative Real-Time PCR
4 Notes
References
An Effective and Reliable Xeno-free Cryopreservation Protocol for Single Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Reagents
2.2 Cell Lines
2.3 Disposables
2.4 Equipment
2.5 Reagent Setup
2.6 Preparation of PDL-Coated Dishes
3 Methods
3.1 Culture of hESCs
3.2 Cryopreservation of hESCs
3.3 Thawing of hESCs
3.4 Characterization of hESCs
4 Notes
References
Clonal Analysis of Cells with Cellular Barcoding: When Numbers and Sizes Matter
1 Introduction
2 Barcoded Library Design
2.1 Vectors
2.2 Barcode Design
2.3 Making a Barcode Library
3 Validation of the Library
3.1 Library Sequencing
3.2 Skewing
3.3 Are Big Libraries Better Than Small?
4 Experimental Design
4.1 Classic Cellular Barcoding
4.2 Libraries for Clonal Competition
5 Identification of Barcodes by Sequencing
5.1 Sanger Sequencing
5.2 Deep Sequencing
5.3 Noise Filtering by Frequency
5.4 Noise Removal Based on Sequence Similarity
5.5 Additional Filtering Steps
5.6 Accounting for Distribution Bias in Counting Barcodes
5.7 Resolution of Barcoding
6 Recent Developments and Future Directions
6.1 Alternative Methods for Clonal Analysis
6.2 Mutation-Based Clonal Studies
Appendix: Background and Examples of Data Analysis
Probability of Unique Barcoding
How to Estimate Approximate Number of Barcodes from Reading a Crude PCR Barcode SANGER Chromatogram
Major Properties of Barcode Libraries
Randomness
Skewing
Distances Between Barcodes
Resolution of Barcode Detection
Protocol for Converting Raw Deep Sequencing Data into Sets of Barcodes
References
Analysis of Cell Cycle Status of Murine Hematopoietic Stem Cells
1 Introduction
2 Materials
2.1 Staining Whole Murine Bone Marrow
2.2 Fixation and Permeabilization
2.3 Flow Cytometry
3 Methods
3.1 Labeling the Surface Antigens of Whole Bone Marrow Cells
3.2 Fixation, Permeabilization, and Ki67 Labeling
3.3 DAPI Labeling and Collection of Samples
4 Notes
References
Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Materials
2.2.1 mESC Culture
2.2.2 Cell Fixation
2.2.3 Hybridization and Washing
2.2.4 Cell Mounting
3 Methods
3.1 Cell Culture
3.1.1 Cell Fixation
3.2 Probe Design (Custom Stellaris FISH Probes)
3.3 Preparation of Probe Stocks
3.4 smFISH in Cells in Suspension
3.5 Image Acquisition
3.6 Image Processing
3.7 Data Analysis
3.7.1 Distribution Analysis
3.7.2 Descriptive Analysis
3.7.3 Correlational Analysis
4 Notes
References
Generation of Regionally Specific Neural Progenitor Cells (NPCs) and Neurons from Human Pluripotent Stem Cells (hPSCs)
1 Introduction
2 Materials
2.1 Equipment and Supplies
2.2 Stock Solutions and Reagents
2.3 Medium
3 Methods
3.1 Maintenance of hPSCs in Feeder Free Conditions
3.2 Formation and Growth of EBs
3.3 EB Plating and Formation of Neural Rosettes
3.4 Generation, and Expansion of NPCs
3.5 Characterization of NPCs
3.5.1 Characterization of NPCs by qPCR
3.5.2 Characterization of NPCs by IF Staining
3.6 Differentiation of NPCs to Neurons
3.7 Characterization of Neurons
3.7.1 Characterization of Neurons by qPCR
3.7.2 Characterization of Neurons by IF Staining
4 Notes
References
Isolation and Culture of Embryonic Stem Cells, Mesenchymal Stem Cells, and Dendritic Cells from Humans and Mice
1 Introduction
2 Materials
2.1 Cells
2.2 Culture Medium
2.3 Buffers and Reagents
2.4 Instruments
3 Methods
3.1 Culture and Differentiation of Human Embryonic Stem Cells (hESCs)
3.1.1 Expansion of hESCs
3.1.2 Embryoid Body Formation
3.1.3 Generation of Non-ciliated Pulmonary Epithelial Cells
3.2 Isolation and Culture of Human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs)
3.3 Isolation of Murine Bone Marrow Derived Mesenchymal Stem Cells (mBM-MSCs)
3.4 Isolation of Murine Adipose Tissue Derived MSCs (mAD-MSCs)
3.5 Isolation of Murine Bone Marrow Derived Dendritic Cells (mBMDC)
4 Notes
References
Decoding the Epigenetic Heterogeneity of Human Pluripotent Stem Cells with Seamless Gene Editing
1 Introduction
2 Materials
2.1 Cell Culture Reagents
2.2 Molecular Biology Reagents
3 Methods
3.1 Design of Donor Construct, RFNs, and TALENs
3.2 Passaging, Transfection, and Selection of Targeting Constructs
3.3 Single-Cell Cloning
3.4 Clone Consolidation, Replica Plating, and Freezing
3.5 Targeting Identification and Validation
3.6 Excision of Targeting Construct
3.7 Validation of MLL2 Binding Deficiency and Reduced H3K4me3 by Chromatin Immunoprecipitation-Quantitative PCR (ChIP-qPCR)
4 Notes
References
Stencil Micropatterning for Spatial Control of Human Pluripotent Stem Cell Fate Heterogeneity
1 Introduction
2 Materials
2.1 PDMS Stencils
2.2 Matrigel Coating on Stenciled Substrate
2.3 Cell Harvesting and Seeding Onto Stenciled Substrate
2.4 Passivation of Stenciled Substrate
3 Methods
3.1 Design and Fabrication of PDMS Stencils
3.2 ECM Coating on Stenciled Substrate
3.3 Cell Seeding onto Stenciled Substrate
3.4 Stencil Removal and Passivation of Unpatterned Substrate
4 Notes
References
Visualizing the Functional Heterogeneity of Muscle Stem Cells
1 Introduction
2 Materials
2.1 Myofiber Isolation and Cell Culture
2.2 Immunostaining and PKH26 Staining
3 Methods
3.1 Culture of Satellite Cell Clones
3.1.1 Preparation
3.1.2 Isolation of the EDL Muscle from the Hindlimb
3.1.3 Clonal Culture
3.1.4 Immunostaining
3.2 Identification of Slow-Dividing Cells
4 Notes
References
Isolation and Expansion of Muscle Precursor Cells from Human Skeletal Muscle Biopsies
1 Introduction
2 Materials
2.1 Culture Media and Plastic Ware
2.2 Flow Cytometry Analysis
2.3 Immunofluorescence Analysis
3 Methods
3.1 Cell Isolation
3.2 Sample Freezing
3.3 Sample Thawing
3.4 Cell Expansion
3.5 Cell Characterization
4 Notes
References
Measuring ATP Concentration in a Small Number of Murine Hematopoietic Stem Cells
1 Introduction
2 Materials
2.1 Isolation of Mouse Bone Marrow Cells
2.2 Staining of Bone Marrow Cells
2.3 Depletion by Magnetic-Activated Cell Sorting (MACS)
2.4 Cell Sorting
2.5 Measuring ATP Concentration
3 Methods
3.1 Isolation of Murine Bone Marrow
3.2 Lysis of RBC Cells
3.3 Depletion of Lineage Committed Cells (Notes 10 and 11)
3.4 Cell Sorting (Note 15)
3.5 Preparing Tecan Plate Reader Injection System
3.6 Preparing ATP Standard Serial Dilutions
3.7 Cell Lysis
3.8 Measuring ATP Signal
4 Notes
References
Growth Factor-Free Pre-vascularization of Cell Sheets for Tissue Engineering
1 Introduction
2 Materials
2.1 Labware
2.2 Reagents
2.3 Reagents Setup
3 Methods
3.1 Isolation of SVF Cells from Human Adipose Tissue (See Note 1)
3.2 Cell Counting and Plating
3.3 Hypoxic Conditions (pO2=5%) of Culture
3.4 Analysis
3.4.1 Flow Cytometry (Fig.1)
3.4.2 Immunocytochemistry (Fig.2)
4 Notes
References
In Vitro Culture of Human Hematopoietic Stem Cells in Serum Free Medium and Their Monitoring by Flow Cytometry
1 Introduction
1.1 Overview of the Chapter
2 Materials
2.1 Preparation of Blood Mononuclear Cells and Isolation of CD34+ Cells
2.2 CD34+ Cells Proliferation
2.3 Detection and Quantification of Progenitors
2.4 Flow Cytometry
3 Methods
3.1 Preparation of Mononuclear Cells
3.2 Magnetic Labeling and CD34+ Cell Separation
3.3 Ex Vivo Expansion of CD34+-Enriched Cells
3.4 Culture Analysis
3.4.1 Flow Cytometry
Determination of CD34+ Cells Concentrations and Proportions
Flow Cytometry Analysis of CD34 Phenotypes
SPADE Analysis
3.4.2 Detection and Quantification of Progenitors
4 Notes
References
Aerosol-Based Cell Therapy for Treatment of Lung Diseases
1 Introduction
2 Materials
2.1 For Trachea Collection
2.2 For Isolation of Airway Epithelial Cells (AECs)
2.3 For Skin Collection
2.4 For Skin-Derived Fibroblast Cells (SFCs) Isolation
2.5 For Aerosol Delivery
3 Methods
3.1 Trachea Collection
3.2 AECs Culture Medium and Substrate Preparation
3.3 Isolation of AECs
3.4 Skin Collection
3.5 SFCs Culture Medium and Suspension Preparation
3.6 Isolation of SFCs
3.7 In Vitro Aerosolization
3.8 AECs Aerosolization and Transplantation
4 Notes
References
Induction of Inner Ear Hair Cells from Mouse Embryonic Stem Cells In Vitro
1 Introduction
2 Materials
2.1 ES Cell Culture
2.1.1 ES Cell Line
2.1.2 ES Cell Maintenance Medium
2.1.3 Growth and Maintenance of ES Cells
2.2 Differentiation into HCLs
2.2.1 Differentiation Medium
2.2.2 Conditioned Medium (CM)
2.2.3 Induction of Embryoid Bodies (EBs)
2.2.4 Induction of Hair Cell-Like Cells (HCLs)
2.3 Characterization of HCLs
2.3.1 RT-PCR
2.3.2 Phalloidin Staining
2.3.3 Immunocytochemistry
3 Methods
3.1 Maintenance of ES Cells
3.2 Differentiation into HCLs
3.2.1 Preparation of Various Differentiation Media
3.2.2 Procedure for HCL Induction
Induction of EBs
Induction of HCLs
3.3 Characterization of HCLs
3.3.1 Analysis of Gene Expression in HCLs
3.3.2 Phalloidin Staining of HCLs
3.3.3 Immunocytochemical Analysis of HCLs
4 Notes
References
Maintenance of Dermal Papilla Cells by Wnt-10b In Vitro
1 Introduction
2 Materials
2.1 Isolation of DPCs
2.2 Preparation of Wnt-10b
2.3 DPC Culture
2.4 Characterization of DPCs
2.4.1 ALP staining
2.4.2 Cell Proliferation Assay
2.4.3 RT-PCR
3 Methods
3.1 Isolation of DPCs
3.2 Preparation of Wnt-10b
3.3 DPC Cultures
3.4 Characterization of DPCs
3.4.1 ALP Staining
3.4.2 Cell Proliferation Assay
3.4.3 RT-PCR
4 Notes
References
Maintenance of Skin Epithelial Stem Cells by Wnt-3a In Vitro
1 Introduction
2 Materials
2.1 Isolation of EpSCs
2.2 EpSCs Sequential Culture
2.3 Characterization of EpSCs
2.3.1 Flow Cytometry
2.3.2 Hair Reconstitution Assay
3 Methods
3.1 Isolation of EpSCs
3.2 EpSC Sequential Cultures
3.3 Characterization of EpSCs
3.3.1 Flow Cytometry
3.3.2 Hair Reconstitution Assay
Preparation of Dermal Cells
Preparation of EpSCs as Epithelial Cells
Transplantation
4 Notes
References
Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method
1 Introduction
2 Materials
2.1 Fabrication of Master Molds
2.2 Microfluidic PDMS Device Fabrication
2.3 Cell Culture
2.4 Enzyme-Free Dissociation of Neurosphere by a Microfluidic Chip
3 Methods
3.1 Master Mold Fabrication by Photolithography Technique
3.2 PDMS Device Assembling
3.3 Neurosphere Culture and Dissociation
4 Notes
References
Automated Cell-Based Quantitation of 8-OHdG Damage
1 Introduction
2 Materials
3 Methods
3.1 Immunofluorescence Labeling of 8-OHdG Foci
3.2 Measurement by AKLIDES Nuk
3.3 Electrospray Ionization Mass Spectrometry (Used as the Validation Method)
3.4 Preparation of Samples for Mass Spectrometric Analysis
4 Hints
References
CoCl2 Administration to Vascular MSC Cultures as an In Vitro Hypoxic System to Study Stem Cell Survival
1 Introduction
2 Materials
2.1 Cell Cultures
2.2 Enzymatic Digestion Reagents
2.3 Cobalt (II) Chloride Hexahydrate (CoCl2)
2.4 Cell Viability Assay Components: Cell Fixation and Staining
2.5 Angiogenic Induction Media
2.6 Tubule-Formation Assay
2.7 RNA Extraction, cDNA Synthesis and Real-Time PCR
3 Methods
3.1 MSC Isolation from Healthy and Aneurysm-Affected Aorta
3.1.1 Enzymatic Digestion of Fresh Aortic Tissues
3.1.2 Cell Count and Seeding
3.1.3 Cell Cultures
3.2 Cell Exposure to CoCl2
3.2.1 Cell Seeding for the Viability Assay
3.3 Crystal Violet Staining
3.4 Cell Seeding for the Angiogenic Differentiation Assay
3.5 Evaluation of Angiogenic Response Genes
4 Notes
References
Reporter Systems to Study Cancer Stem Cells
1 Introduction
1.1 Tumor Heterogeneity and Cancer Stem Cells
1.2 Isolation of CSCs by Surface Markers
1.3 Reporter Systems to Identify CSCs
2 Materials
3 Methods
3.1 NANOG Promoter-Driven GFP Reporter
3.1.1 NANOG Is a Pluripotency Gene Involved in Oncogenesis
3.1.2 NANOG-GFP Reporter Enriches CSCs
3.1.3 NANOG-GFP Reporter Is a Dynamic System
3.2 Other Reporter Systems
3.2.1 SOX2
3.2.2 OCT4
3.2.3 SORE6
3.2.4 NOTCH
3.2.5 TERT
3.2.6 s-SHIP
3.2.7 AFP
3.3 Caveats of Using Reporters
3.4 Conclusions
4 Notes
References
Agent-Based Modeling of Cancer Stem Cell Driven Solid Tumor Growth
1 Introduction
2 Materials
2.1 Lattice
2.2 Cell
3 Methods
3.1 Programming Environment
3.2 Simulation Procedure
4 Notes
References
Induction of a Tumor-Metastasis-Receptive Microenvironment as an Unwanted Side Effect After Radio/Chemotherapy and In Vitro a
1 Introduction
2 Materials
2.1 In Vitro Chemotaxis/Chemokinesis Assay for Adherent and Non-adherent Cells
2.2 In Vivo Seeding Efficiency Assay (Detection of Human-Murine Chimerism)
2.3 Preparation of Conditioned Medium for Organs Damaged by Radio/Chemotherapy
3 Methods
3.1 Chemotaxis of Adherent Cells (Fig.2)
3.2 Chemotaxis of Non-adherent Cells
3.3 Chemotaxis vs. Chemokinesis
3.4 In Vivo Seeding Efficiency Assay (Detection of Human-Murine Chimerism)
3.4.1 Preparation of Standard Curve
3.4.2 Detection of Human-Murine Chimerism (Fig.5)
3.5 Harvesting of Conditioned Medium from Organs
3.6 Conclusions
4 Notes
References
Isolation and Propagation of Glioma Stem Cells from Acutely Resected Tumors
1 Introduction
2 Materials
2.1 Plate Preparation for Propagation as Monolayer
2.2 Dissection of Glioma Patient Tissue
2.3 Identification of GSC
2.4 Passaging
2.5 Freezing Cell Stocks in 10% DMSO Plus N2 Medium Without Serum
2.6 Induction of Differentiation Using Serum or CNTF
2.7 Neurosphere Culture and Daily Maintenance with EGF and bFGF
2.8 Preparation of Cells for Establishing Animal Models
3 Methods
3.1 Plate Preparation for Propagation as Monolayer
3.2 Disassociation of Human Glioma Tissue
3.3 Selection of CD133 Expressing GSC
3.4 Passaging
3.5 Freezing Cell Stocks in 10% DMSO Plus N2 Medium Without Serum
3.6 Induction of Differentiation
3.7 Neurosphere Culture and Daily Maintenance with EGF and bFGF
3.8 Preparation of Cells for Establishing Animal Models
4 Notes
References
Isolation and Characterization of Cancer Stem Cells of the Non-Small-Cell Lung Cancer (A549) Cell Line
1 Introduction
2 Materials
2.1 Cell Line
2.2 Cell Culture Media
2.2.1 Cancer Cell Medium
2.2.2 Normal Cell Medium
2.3 Isolating CSCs
2.4 Differentiation Assays
2.4.1 Osteogenic Assay
2.4.2 Adipogenic Assay
2.4.3 Chondrogenic Assay
2.5 Clonogenic Assay
2.6 Spheroid Formation Assay
2.7 Microarray
2.8 In Vivo Tumorigenicity Studies
2.9 Equipment and Plates
3 Methods
3.1 Cell Culture
3.2 Analysis and Sorting of CSCs Using the Cell Sorter
3.3 Differentiation Assay
3.3.1 Osteogenic Differentiation
3.3.2 Osteoblast Detection (Calcium Deposits)
3.3.3 Osteoblast Detection (Alkaline Phosphatase)
3.3.4 Adipogenic Differentiation
3.3.5 Adipocyte Detection
3.3.6 Chondrogenic Differentiation
3.3.7 Chondrocyte Detection
3.4 Clonogenic Assay
3.5 Spheroid Formation Assay
3.6 Microarray Assay
3.6.1 RNA Extraction
3.6.2 Determination of RNA Concentration and RNA Integrity
3.6.3 cDNA Amplification
3.6.4 cDNA Purification
3.6.5 cDNA Fragmentation and Biotin Labeling
3.6.6 Hybridization
3.6.7 Washing and Staining
3.6.8 Scanning
3.6.9 Microarray Statistical Data Analysis
3.7 In Vivo Tumorigenicity Studies
4 Notes
References
Chapter 46: Erratum to: Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method
Index