Current Protocols in Stem Cell Biology

Current Protocols in Stem Cell Biology

Online ISBN: 9780470151808

DOI: 10.1002/9780470151808

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  1. Foreword
  2. Selected Suppliers of Reagents and Equipment
  3. Part PREFACE Current Protocols in Stem Cell Biology
    1. PREFACE Preface
  4. Chapter 1 Embryonic and Extraembryonic Stem Cells
    1. Section A Isolation of Embryonic Stem Cells
      1. Introduction
      2. UNIT 1A.1 Derivation and Characterization of Nonhuman Primate Embryonic Stem Cells
      3. UNIT 1A.2 Derivation of hESC from Intact Blastocysts
      4. UNIT 1A.3 Reprogramming Primordial Germ Cells (PGC) to Embryonic Germ (EG) Cells
      5. UNIT 1A.4 Derivation and Propagation of hESC Under a Therapeutic Environment
      6. UNIT 1A.5 Generation of Human Embryonic Stem Cells
      7. UNIT 1A.6 Generation of Mammalian Offspring by Haploid Embryonic Stem Cells Microinjection
    2. Section B Characterization of Embryonic Stem Cells
      1. UNIT 1B.1 Proteomic Analysis of Pluripotent Stem Cells
      2. UNIT 1B.2 Gene Expression Analysis of RNA Purified from Embryonic Stem Cells and Embryoid Body–Derived Cells Using a High-Throughput Microarray Platform
      3. UNIT 1B.3 Phenotypic Analysis of Human Embryonic Stem Cells
      4. UNIT 1B.4 Isolation of Human Embryonic Stem Cell–Derived Teratomas for the Assessment of Pluripotency
      5. UNIT 1B.5 Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation
      6. UNIT 1B.6 Characterization of X-Chromosome Inactivation Status in Human Pluripotent Stem Cells
      7. UNIT 1B.7 Preparation of Defined Human Embryonic Stem Cell Populations for Transcriptional Profiling
      8. UNIT 1B.8 Identifying Translationally Regulated Genes During Stem Cell Differentiation
      9. UNIT 1B.9 Quantification of Pluripotency Transcription Factor Levels in Embryonic Stem Cells by Flow Cytometry
      10. UNIT 1B.10 The Streptavidin/Biotinylated DNA/Protein Bound Complex Protocol for Determining the Association of c-JUN Protein with NANOG Promoter
    3. Section C Culture and Maintenance of Undifferentiated Embryonic Stem Cells
      1. Introduction
      2. UNIT 1C.1 Expansion of Human Embryonic Stem Cells In Vitro
      3. UNIT 1C.2 Defined, Feeder-Independent Medium for Human Embryonic Stem Cell Culture
      4. UNIT 1C.3 Isolation and Propagation of Mouse Embryonic Fibroblasts and Preparation of Mouse Embryonic Feeder Layer Cells
      5. UNIT 1C.4 Culture of Mouse Embryonic Stem Cells
      6. UNIT 1C.5 Preparation of Autogenic Human Feeder Cells for Growth of Human Embryonic Stem Cells
      7. UNIT 1C.6 Isolation of Human Placental Fibroblasts
      8. UNIT 1C.7 Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells
      9. UNIT 1C.8 Cryopreservation of Dissociated Human Embryonic Stem Cells in the Presence of ROCK Inhibitor
      10. UNIT 1C.9 Authentication and Banking of Human Pluripotent Stem Cells
      11. UNIT 1C.10 Clump Passaging and Expansion of Human Embryonic and Induced Pluripotent Stem Cells on Mouse Embryonic Fibroblast Feeder Cells
      12. UNIT 1C.11 Expansion of Human Embryonic Stem Cells on Cellulose Microcarriers
      13. UNIT 1C.12 Live-Cell Immunofluorescence Staining of Human Pluripotent Stem Cells
      14. UNIT 1C.13 Adaptation of Stem Cells to 96-Well Plate Assays: Use of Human Embryonic and Mouse Neural Stem Cells in the MTT Assay
      15. UNIT 1C.14 Standardized Cryopreservation of Pluripotent Stem Cells
      16. UNIT 1C.15 Expansion of Human Hematopoietic Stem/Progenitor Cells on Decellularized Matrix Scaffolds
      17. UNIT 1C.16 Generation, Expansion, and Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) Derived From the Umbilical Cords of Newborns
      18. UNIT 1C.17 Cryopreservation of Human Pluripotent Stem Cells in Defined Medium
      19. UNIT 1C.18 Efficient and Scalable Culture of Single Dissociated Human Pluripotent Stem Cells Using Recombinant E8 Fragments of Human Laminin Isoforms
      20. UNIT 1C.19 A Method for Sectioning and Immunohistochemical Analysis of Stem Cell–Derived 3-D Organoids
      21. UNIT 1C.20 Isolation of Stem Cells and Progenitors from Mouse Epidermis
    4. Section D Germ Layer Induction/Differentiation of Embryonic Stem Cells
      1. UNIT 1D.1 Germ Layer Induction in ESC—Following the Vertebrate Roadmap
      2. UNIT 1D.2 Formation and Hematopoietic Differentiation of Human Embryoid Bodies by Suspension and Hanging Drop Cultures
      3. UNIT 1D.3 Directed Differentiation of Human Embryonic Stem Cells as Spin Embryoid Bodies and a Description of the Hematopoietic Blast Colony Forming Assay
      4. UNIT 1D.4 Differentiation of Human Embryonic Stem Cells in Adherent and in Chemically Defined Culture Conditions
      5. UNIT 1D.5 Isolation and Differentiation of Xenopus Animal Cap Cells
      6. UNIT 1D.6 High-Throughput Screening Assay for Embryoid Body Differentiation of Human Embryonic Stem Cells
      7. UNIT 1D.7 Differentiation of Human Embryonic Stem Cells to Cardiomyocytes on Microcarrier Cultures
      8. UNIT 1D.8 Using ES Cells Labeled with GFP for Analyzing Cell Behavior During Differentiation
      9. UNIT 1D.9 Purification of Definitive Endoderm Generated from Pluripotent Stem Cells by Magnetic Cell Sorting
    5. Section E Extraembryonic Lineages
      1. Introduction
      2. UNIT 1E.1 Isolation of Human Placenta-Derived Multipotent Cells and In Vitro Differentiation into Hepatocyte-Like Cells
      3. UNIT 1E.2 Isolation of Mesenchymal Stem Cells from Amniotic Fluid and Placenta
      4. UNIT 1E.3 Isolation of Amniotic Epithelial Stem Cells
      5. UNIT 1E.4 Isolation and Manipulation of Mouse Trophoblast Stem Cells
      6. UNIT 1E.5 Isolation of Amniotic Mesenchymal Stem Cells
      7. UNIT 1E.6 Amnion Epithelial Cell Isolation and Characterization for Clinical Use
      8. UNIT 1E.7 Quantifying Epithelial Early Common Progenitors from Long-Term Primary or Cell Line Sphere Culture
      9. UNIT 1E.8 Amniotic Membrane: Separation of Amniotic Mesoderm from Amniotic Epithelium and Isolation of Their Respective Mesenchymal Stromal and Epithelial Cells
      10. UNIT IE.9 Production of 3-D Airway Organoids From Primary Human Airway Basal Cells and Their Use in High-Throughput Screening
      11. UNIT 1E.10 Isolation of Human Amnion Epithelial Cells According to Current Good Manufacturing Procedures
    6. Section F Mesodermal Lineages
      1. UNIT 1F.1 Differentiation of Embryonic Stem Cells into Cartilage Cells
      2. UNIT 1F.2 Differentiation of Human Embryonic Stem Cells to Cardiomyocytes by Coculture with Endoderm in Serum-Free Medium
      3. UNIT 1F.3 Isolation of Hematopoietic Stem Cells from Mouse Embryonic Stem Cells
      4. UNIT 1F.4 Differentiation of Mouse Embryonic Stem Cells into Blood
      5. UNIT 1F.5 Endothelial Differentiation of Embryonic Stem Cells
      6. UNIT 1F.6 Hematopoietic Differentiation of Human Embryonic Stem Cells by Cocultivation with Stromal Layers
      7. UNIT 1F.7 TLX1 (HOX11) Immortalization of Embryonic Stem Cell–Derived and Primary Murine Hematopoietic Progenitors
      8. UNIT 1F.8 Differentiation of Multipotent Mesenchymal Precursors and Skeletal Myoblasts from Human Embryonic Stem Cells
      9. UNIT 1F.9 Derivation of Vasculature from Embryonic Stem Cells
      10. UNIT 1F.10 Isolation and Functional Characterization of Pluripotent Stem Cell–Derived Cardiac Progenitor Cells
      11. UNIT 1F.11 Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes via the Hanging-Drop and Mass Culture Methods
      12. UNIT 1F.12 Lentiviral Transduction and Clonal Selection of hESCs with Endothelial-Specific Transgenic Reporters
      13. UNIT 1F.13 Serum-Free Generation of Multipotent Mesoderm (Kdr+) Progenitor Cells in Mouse Embryonic Stem Cells for Functional Genomics Screening
      14. UNIT 1F.14 Isolation and Expansion of Mesenchymal Stem Cells from Human Conjunctival Tissue
      15. UNIT 1F.15 Fast and Efficient Neural Conversion of Human Hematopoietic Cells
      16. UNIT 1F.16 Efficient Generation of Cardiac Purkinje-like Cells from Embryonic Stem Cells by Activating cAMP Signaling
      17. UNIT 1F.17 Isolation of Ready-to-Use Adipose-Derived Stem Cell (ASC) Pellet for Clinical Applications and a Comparative Overview of Alternate Methods for ASC Isolation
      18. UNIT 1F.18 Human Umbilical Cord Mesenchymal Stromal Cell Isolation, Expansion, Cryopreservation, and Characterization
      19. UNIT 1F.19 Angiogenesis within Stem Cell–Seeded Silk Scaffolds Cultured on the Chorioallantoic Membrane and Visualized by 3D Imaging
    7. Section G Endodermal Lineages
      1. UNIT 1G.1 The Differentiation of Distal Lung Epithelium from Embryonic Stem Cells
      2. UNIT 1G.2 Pancreas Differentiation of Mouse ES Cells
      3. UNIT 1G.3 Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors
      4. UNIT 1G.4 Differentiation of Hepatocytes from Pluripotent Stem Cells
      5. UNIT 1G.5 Deriving Functional Hepatocytes from Pluripotent Stem Cells
      6. UNIT 1G.6 Small-Molecule-Directed Hepatocyte-Like Cell Differentiation of Human Pluripotent Stem Cells
    8. Section H Ectodermal Lineages
      1. UNIT 1H.1 Differentiation of Mouse Embryonic Stem Cells to Spinal Motor Neurons
      2. UNIT 1H.2 Time-Lapse Imaging of Embryonic Neural Stem Cell Division in Drosophila by Two-Photon Microscopy
      3. UNIT 1H.3 Basic Techniques in Human Mesenchymal Stem Cell Cultures: Differentiation into Osteogenic and Adipogenic Lineages, Genetic Perturbations, and Phenotypic Analyses
      4. UNIT 1H.4 Isolation and Culture of Adult Ciliary Epithelial Cells, Previously Identified as Retinal Stem Cells, and Retinal Progenitor Cells
      5. UNIT 1H.5 Derivation of Cerebellar Neurons from Human Pluripotent Stem Cells
      6. UNIT 1H.6 Dopaminergic Differentiation of Human Pluripotent Cells
      7. UNIT 1H.7 Slice Culture Method for Studying Migration of Neuronal Progenitor Cells Derived from Human Embryonic Stem Cells (hESC)
      8. UNIT 1H.8 Generation of Highly Enriched Populations of Optic Vesicle−Like Retinal Cells from Human Pluripotent Stem Cells
      9. UNIT 1H.9 Neural Crest Cells from Dual SMAD Inhibition
      10. UNIT 1H.10 Derivation of Telencephalic Oligodendrocyte Progenitors from Human Pluripotent Stem Cells
  5. Chapter 2 Somatic Stem Cells
    1. Section A Hematopoietic Stem Cells
      1. Introduction
      2. UNIT 2A.1 Isolation of Mononuclear Cells from Human Cord Blood by Ficoll-Paque Density Gradient
      3. UNIT 2A.2 Isolation of Hematopoietic Stem Cells from Human Cord Blood
      4. UNIT 2A.3 Isolation of Mesenchymal Stem Cells from Human Cord Blood
      5. UNIT 2A.4 Isolation and Assessment of Single Long-Term Reconstituting Hematopoietic Stem Cells from Adult Mouse Bone Marrow
      6. UNIT 2A.5 Analysis of the Hematopoietic Stem Cell Niche
      7. UNIT 2A.6 Analysis and Manipulation of Hematopoietic Progenitor and Stem Cells from Murine Embryonic Tissues
      8. UNIT 2A.7 High Level In Vitro Expansion of Murine Hematopoietic Stem Cells
      9. UNIT 2A.8 Isolation and Visualization of Mouse Placental Hematopoietic Stem Cells
      10. UNIT 2A.9 Preparation of Hematopoietic Stem and Progenitor Cells from the Human Placenta
      11. UNIT 2A.10 Isolation of Human Umbilical Cord Blood Aldehyde Dehydrogenase–Expressing Progenitor Cells that Modulate Vascular Regenerative Functions In Vitro and In Vivo
    2. Section B Non-Hematopoietic Bone Marrow-Derived Stem Cells
      1. UNIT 2B.1 Isolation and Characterization of Mesoangioblasts from Mouse, Dog, and Human Tissues
      2. UNIT 2B.2 Purification and Culture of Human Blood Vessel–Associated Progenitor Cells
      3. UNIT 2B.3 Isolation, Culture, and Differentiation Potential of Mouse Marrow Stromal Cells
      4. UNIT 2B.4 Transplantation Models to Characterize the Mechanisms of Stem Cell–Induced Islet Regeneration
      5. UNIT 2B.5 Preparation and Osteogenic Differentiation of Scaffold-Free Mouse Adipose-Derived Stromal Cell Microtissue Spheroids (ASC-MT)
      6. UNIT 2B.6 Preparation of Anti-Inflammatory Mesenchymal Stem/Precursor Cells (MSCs) Through Sphere Formation Using Hanging-Drop Culture Technique
    3. Section C Cardiovascular Stem Cells
      1. UNIT 2C.1 Isolation and Characterization of Endothelial Progenitor Cells from Human Blood
      2. UNIT 2C.2 Derivation of Epicardium-Derived Progenitor Cells (EPDCs) from Adult Epicardium
      3. UNIT 2C.3 Isolation and Expansion of Cardiosphere-Derived Stem Cells
      4. UNIT 2C.4 Expanding Donor Muscle-Derived Cells for Transplantation
      5. UNIT 2C.5 The Derivation of Primary Human Epicardium-Derived Cells
    4. Section D Neural Stem Cells
      1. UNIT 2D.1 Generation of Cultured Oligodendrocyte Progenitor Cells from Rat Neonatal Brains
      2. UNIT 2D.2 Isolating, Expanding, and Infecting Human and Rodent Fetal Neural Progenitor Cells
      3. UNIT 2D.3 Long-Term Multilayer Adherent Network (MAN) Expansion, Maintenance, and Characterization, Chemical and Genetic Manipulation, and Transplantation of Human Fetal Forebrain Neural Stem Cells
      4. UNIT 2D.4 Culture System for Rodent and Human Oligodendrocyte Specification, Lineage Progression, and Maturation
      5. UNIT 2D.5 Isolation and Culture of Ventral Mesencephalic Precursor Cells and Dopaminergic Neurons from Rodent Brains
      6. UNIT 2D.6 Isolation of Neural Stem Cells from Neural Tissues Using the Neurosphere Technique
      7. UNIT 2D.7 Neural Stem Cell or Human Induced Pluripotent Stem Cell–Derived GABA-ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy
      8. UNIT 2D.8 Reproducible Expansion and Characterization of Mouse Neural Stem/Progenitor Cells in Adherent Cultures Derived from the Adult Subventricular Zone
      9. UNIT 2D.9 Neural Differentiation of P19 Carcinoma Cells and Primary Neurospheres: Cell Morphology, Proliferation, Viability, and Functionality
      10. UNIT 2D.10 Single and Dual Birthdating Procedures for Assessing the Response of Adult Neural Stem Cells to the Infusion of a Soluble Factor Using Halogenated Thymidine Analogs
      11. UNIT 2D.11 In Vitro Generation of Three-Dimensional Substrate-Adherent Embryonic Stem Cell–Derived Neural Aggregates for Application in Animal Models of Neurological Disorders
      12. UNIT 2D.12 Scalable Production of Transplantable Dopaminergic Neurons from hESCs and iPSCs in Xeno-Free Defined Conditions
      13. UNIT 2D.13 Functional Stem Cell Integration Assessed by Organotypic Slice Cultures
      14. UNIT 2D.14 Evaluation of Proliferation of Neural Stem Cells In Vitro and In Vivo
      15. UNIT 2D.15 Revealing Neuronal Circuitry Using Stem Cell-Derived Neurons
      16. UNIT 2D.16 Intraspinal Transplantation of Mouse and Human Neural Precursor Cells
      17. UNIT 2D.17 Isolation and Long-Term Expansion of Functional, Myelinating Oligodendrocyte Progenitor Cells from Neonatal Rat Brain
      18. UNIT 2D.18 Efficient Generation of Viral and Integration-Free Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes
      19. UNIT 2D.19 Magnetic Nanoparticle-Mediated Gene Delivery to Two- and Three-Dimensional Neural Stem Cell Cultures: Magnet-Assisted Transfection and Multifection Approaches to Enhance Outcomes
    5. Section E Germline Stem Cells
      1. UNIT 2E.1 Culturing Ovarian Somatic and Germline Stem Cells of Drosophila
      2. UNIT 2E.2 Time-Lapse Live Imaging of Stem Cells in Drosophila Testis
      3. UNIT 2E.3 Isolation of Undifferentiated Female Germline Cells from Adult Drosophila Ovaries
    6. Section F Gut Stem Cells
      1. UNIT 2F.1 In Situ Hybridization to Identify Gut Stem Cells
      2. UNIT 2F.2 Regional Cell Specific RNA Expression Profiling of FACS Isolated Drosophila Intestinal Cell Populations
    7. Section G Lung Stem Cells
      1. UNIT 2G.1 Isolation and Clonal Assay of Adult Lung Epithelial Stem/Progenitor Cells
  6. Chapter 3 Cancer Stem Cells
    1. UNIT 3.1 Colon Cancer Stem Cells
    2. UNIT 3.2 In Vivo Evaluation of Leukemic Stem Cells through the Xenotransplantation Model
    3. UNIT 3.3 Culture and Isolation of Brain Tumor Initiating Cells
    4. UNIT 3.4 Isolation and Expansion of Regionally Defined Human Glioblastoma Cells In Vitro
    5. UNIT 3.5 Organotypic Explant Culture of Glioblastoma Multiforme and Subsequent Single-Cell Suspension
    6. UNIT 3.6 Culture and Isolation of Melanoma-Initiating Cells
    7. UNIT 3.7 In Vitro Enrichment of Tumor-Initiating Cells from Human Established Cell Lines
    8. UNIT 3.8 Isolation of Human Melanoma Stem Cells Using ALDH as a Marker
  7. Chapter 4 Manipulation of Potency
    1. Section A iPS Cells
      1. UNIT 4A.1 Human iPS Cell Derivation/Reprogramming
      2. UNIT 4A.2 Generation and Characterization of Human Induced Pluripotent Stem Cells
      3. UNIT 4A.3 Derivation of Induced Pluripotent Stem Cells from Human Peripheral Circulating T Cells
      4. UNIT 4A.4 Using MicroRNAs to Enhance the Generation of Induced Pluripotent Stem Cells
      5. UNIT 4A.5 Excision of a Viral Reprogramming Cassette by Delivery of Synthetic Cre mRNA
      6. UNIT 4A.6 cGMP Generation of Human Induced Pluripotent Stem Cells with Messenger RNA
      7. UNIT 4A.7 Laser-Assisted Generation of Human Induced Pluripotent Stem Cells
      8. UNIT 4A.8 Teratoma Formation: A Tool for Monitoring Pluripotency in Stem Cell Research
      9. UNIT 4A.9 Derivation of Transgene-Free Induced Pluripotent Stem Cells from a Single Drop of Blood
      10. UNIT 4A.10 Reprogramming of Pancreatic Acinar Cells to Functional Beta Cells by In Vivo Transduction of a Polycistronic Construct Containing Pdx1, Ngn3, MafA in Mice
      11. UNIT 4A.11 Rhesus Macaque iPSC Generation and Maintenance
    2. Section B Nuclear Transfer
      1. UNIT 4B.1 Heterokaryon-Based Reprogramming for Pluripotency
  8. Chapter 5 Genetic Manipulation of Stem Cells
    1. Section A Lineage Tracers in Stem Cells
      1. UNIT 5A.1 Imaging Neural Stem Cell Fate in Mouse Model of Glioma
      2. UNIT 5A.2 Identifying Adult Stem Cells Using Cre-Mediated Lineage Tracing
      3. UNIT 5A.3 Magnetic Resonance Imaging of Human Embryonic Stem Cells
      4. UNIT 5A.4 Lineage Tracing in the Intestinal Epithelium
      5. UNIT 5A.5 Kaede-Centrin1 Labeling of Mother and Daughter Centrosomes in Mammalian Neocortical Neural Progenitors
      6. UNIT 5A.6 Retroviral Gene Expression Control in Primary Organoid Cultures
      7. UNIT 5A.7 Generation of GFP Reporter Human Induced Pluripotent Stem Cells Using AAVS1 Safe Harbor Transcription Activator-Like Effector Nuclease
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        UNIT 5A.8 CRISPR-Mediated Gene Targeting of Human Induced Pluripotent Stem Cells
      9. UNIT 5A.9 Tetracycline-Inducible and Reversible Stable Gene Expression in Human iPSC-Derived Neural Progenitors and in the Postnatal Mouse Brain
    2. Section B Homologous Recombination in Stem Cells
      1. UNIT 5B.1 Generation of Human Embryonic Stem Cell Reporter Knock-In Lines by Homologous Recombination
      2. UNIT 5B.2 Genetic Manipulation of Human Induced Pluripotent Stem Cells
      3. UNIT 5B.3 TALEN- and CRISPR/Cas9-Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid-Based Transfection
      4. UNIT 5B.4 Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells
      5. UNIT 5B.5 Self-Cloning CRISPR
      6. UNIT 5B.6 Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells
    3. Section C RNA Interference in Stem Cells
      1. UNIT 5C.1 Generation of Inducible shRNAi Human Embryonic Stem Cell Lines
      2. UNIT 5C.2 High-Efficiency Transfection and siRNA-Mediated Gene Knockdown in Human Pluripotent Stem Cells
      3. UNIT 5C.3 Array-Based High-Throughput Screening in Mouse Embryonic Stem Cells with shRNAs
  9. Appendix 1 Useful Information
    1. APPENDIX 1A Guidelines for the Conduct of Human Embryonic Stem Cell Research
    2. APPENDIX 1B ISSCR Guidelines for the Clinical Translation of Stem Cells
  10. Appendix 2 Laboratory Equipment
    1. APPENDIX 2 Standard Laboratory Equipment

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