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Current Protocols in Stem Cell Biology

Current Protocols in Stem Cell Biology

Online ISBN: 9780470151808

DOI: 10.1002/9780470151808

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  1. PREFACE Current Protocols in Stem Cell Biology
    1. PREFACE Preface
    2. Foreword
  2. 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
    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
    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
    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
    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 A Protocol to Quantify Mammary Early Common Progenitors from Long-Term Mammosphere Culture
      9. Unit 1E.8 Amniotic Membrane: Separation of Amniotic Mesoderm from Amniotic Epithelium and Isolation of Their Respective Mesenchymal Stromal and Epithelial Cells
    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
    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 Embryonic Stem Cells into Anterior Definitive Endoderm
    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
  3. 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 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
    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
    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. 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 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
    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
    6. Section F Gut Stem Cells
      1. Unit 2F.1 In Situ Hybridization to Identify Gut Stem Cells
    7. Section G Lung Stem Cells
      1. Unit 2G.1 Isolation and Clonal Assay of Adult Lung Epithelial Stem/Progenitor Cells
  4. 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
  5. 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
    2. Section B Nuclear Transfer
      1. Unit 4B.1 Heterokaryon-Based Reprogramming for Pluripotency
  6. 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 Functional Analysis of 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
    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
    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
  7. Appendix 1 Useful Information
    1. 1A Guidelines for the Conduct of Human Embryonic Stem Cell Research
    2. 1B ISSCR Guidelines for the Clinical Translation of Stem Cells
  8. Appendix 2 Laboratory Equipment
    1. 2 Standard Laboratory Equipment
  9. Appendix Suppliers
    1. Selected Suppliers of Reagents and Equipment

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