Note: Intro -- Contents -- 1 Generation of Hemangioblasts from Human Pluripotent Stem Cells -- Abstract -- 1.1 Introduction -- 1.2 Hemangioblast Generation from hESCs Cultured on MEF [17, 18] -- 1.2.1 Materials -- 1.2.2 Methods -- 1.2.2.1 Prepare Primary Mouse Embryo Fibroblasts (PMEFs) from 12.5 dpc CD-1 Mouse Embryos [21] -- 1.2.2.2 Culture of Undifferentiated hESCs [22] -- 1.2.2.3 Generation of Hemangioblasts (Blast Cells) from hESCs [17, 18] -- 1.3 Hemangioblast Generation from hESCs and iPSCs Cultured Under 3D Condition [19] -- 1.3.1 Materials -- 1.3.2 Methods -- 1.3.2.1 Two-Dimensional Growth and Expansion of hESCs and iPSCs -- 1.3.2.2 Culturing hESCs and iPSCs on 3D Microcarriers -- 1.3.2.3 Passaging Cells Grown on Microcarriers -- 1.3.2.4 Embryoid Body Formation and Hemangioblast Generation -- References -- 2 Derivation of Mature Erythrocytes from Human Pluripotent Stem Cells by Coculture with Murine Fetal Stromal Cells -- Abstract -- 2.1 Introduction -- 2.2 Materials -- 2.2.1 hPSCs Lines -- 2.2.2 mFLSCs (Adapted from Refs. [27, 34]) -- 2.2.3 mAGMS-3 Cell Line Culture (Adapted from Ref. [32]) -- 2.2.4 Induction of Multipotential Hematopoietic Progenitors -- 2.2.5 Suspension Culture in Liquid Medium -- 2.2.6 Hematopoietic Colony Culture -- 2.2.7 Flow Cytometric Analysis of hPSC-Derived Erythrocytes -- 2.2.8 Immuno-Staining Assay of hPSC-Derived Erythrocytes -- 2.3 Methods -- 2.3.1 Maintenance of hPSCs Lines -- 2.3.2 Establishment of mFLSCs -- 2.3.3 Maintenance of mAGMS-3 Cell Line -- 2.3.4 Coculture of Undifferentiated hPSCs with mFLSCs/mAGMS-3 -- 2.3.5 Colony Formation Analysis of Hematopoietic Progenitors Derived from hPSCs Coculture with mFLSCs/mAGMS-3 -- 2.3.6 Erythrocytes Derived from hPSCs Coculture with mFLSCs/mAGMS-3 -- 2.3.6.1 Expansion of Erythroid Progenitors -- 2.3.6.2 Differentiation and Maturation of Erythrocytes. , 2.3.7 Characterization of hPSC-Derived Erythrocytes by Flow cytometry Assay -- 2.3.8 Hb Components of hPSC-Derived Erythrocytes Detected by Immuno-Staining Assay -- 2.4 Future Perspects -- References -- 3 Derivation of Megakaryocytes and Platelets from Human Pluripotent Stem Cells -- Abstract -- 3.1 Introduction -- 3.2 Materials -- 3.2.1 Cell Lines -- 3.2.2 Hematopoietic Progenitor Cells Generation from Human iPS Cells -- 3.2.3 Megakaryocytes and Platelets Generation from the HPS -- 3.3 Methods -- 3.3.1 Maintenance and Expansion of hiPSC Lines -- 3.3.1.1 Preparation of VTN-N-Coated Plates (6-Well Plate) -- 3.3.1.2 EDTA Passaging (6-Well Plate) -- 3.3.2 Generation of MKs from hiPSCs -- 3.3.3 Plateletlike Particles Generation from the MKs and Characterization -- 3.3.4 Functional Analysis of Plateletlike Particles -- 3.4 Notes -- Acknowledgments -- References -- 4 Derivation of Functionally Mature Eosinophils from Human Pluripotent Stem Cells -- Abstract -- 4.1 Introduction -- 4.2 Materials -- 4.2.1 hPSC Lines and Culture Medium -- 4.2.2 Murine AGMS-3 Cell Line and Fetal Liver Stromal Cells (mFLSCs) -- 4.2.3 Induction of Multipotential Hematopoietic Progenitor Cells -- 4.2.4 Induction of Eosinophils -- 4.2.5 Flow Cytometric Analysis of hPSC-Derived Eosinophils -- 4.2.6 Transmission Electron Microscope (TEM) Analysis of hPSC-Derived Eosinophils -- 4.2.7 May-Gr03CBnwald-Giemsa Staining -- 4.2.8 Immunochemical Staining -- 4.2.9 RT-PCR -- 4.3 Methods -- 4.3.1 Maintenance of hPSC Lines -- 4.3.2 Coculture of Undifferentiated hPSC with AGMS-3 (or mFLSCs) -- 4.3.3 Induction of Eosinophils -- 4.3.4 Characterization of hPSC-Derived Eosinophils by Flow Cytometric Analysis -- 4.3.5 Characterization of hPSC-Derived Eosinophils by Transmission Electron Microscope (TEM) Analysis -- 4.3.6 May-Gr03CBnwald-Giemsa Staining -- 4.3.7 RT-PCR -- 4.3.8 Immunostaining. , 4.3.9 The Functional Assay of hPSC-Eosinophils -- 4.4 Discussion and Future Prospectives -- References -- 5 Human Pluripotent Stem Cells as a Renewable Source of Natural Killer Cells -- Abstract -- 5.1 Introduction -- 5.2 Materials -- 5.2.1 Cell Lines -- 5.2.2 Hematopoietic Differentiation of hES/iPS Cells by Spin EBs -- 5.2.3 Natural Killer Cell Differentiation from Differentiated Spin EBs -- 5.2.4 Natural Killer Cell Expansion -- 5.3 Methods -- 5.3.1 TrypLE Adaptation of hESC/iPSCs -- 5.3.2 Generation of Hematopoietic Progenitor Cells from hES/iPS Cells by Spin EB Formation -- 5.3.3 Natural Killer Cell Differentiation with or without Stromal Cells -- 5.3.4 Clinical Scale Expansion of hESC-/iPSC-Derived NK Cells for Immunotherapy -- 5.4 Notes -- Acknowledgments -- References -- 6 Generation of T-Lineage Cells from iPS Cells and Its Application -- Abstract -- 6.1 Introduction -- 6.1.1 Repeated DLI Treatment Prolongs Mouse Survival -- 6.2 Materials -- 6.2.1 Cells and Cell Lines -- 6.2.2 Reagent and Culture Ware for General Use -- 6.2.3 Culture Medium and Cell Culture -- 6.2.4 Flow Cytometry -- 6.3 Methods -- 6.3.1 Differentiation of T-Lineage Cells from Murine Pluripotent Stem Cells -- 6.3.2 Functional Analysis of IPS-T Cells -- 6.3.3 Preparation of Bone Marrow Cells for Bone Marrow Transplantation in DLI Model -- 6.3.4 Treatment of DLI Mouse Model Using T-Lineage Cells from IPS Cells -- 6.4 Notes -- References -- 7 Reprogramming of Human Cord Blood CD34+ Cells into Induced MSCs -- Abstract -- 7.1 Introduction -- 7.2 Materials -- 7.2.1 Cord Blood CD34+ Cells -- 7.2.2 Cell Culture -- 7.2.3 Episomal Vector and Nucleofection Kits -- 7.2.4 Lentiviral Vector and Transduction -- 7.2.5 Plastics and Equipment -- 7.2.6 Immunohistochemistry and Flow Cytometry -- 7.2.7 Trilineage Differentiation of iMSCs -- 7.3 Methods -- 7.3.1 CD34 Enrichment. , 7.3.2 CD34 Cell Culture -- 7.3.3 Nucleofection -- 7.3.4 Generation of Integration-Free iMSCs -- 7.3.5 Immortalization of iMSCs -- 7.3.6 Long-Term Culture of iMSCs -- 7.3.7 Freezing Down iMSCs -- 7.3.8 Phenotyping of iMSCs by Flow Cytometry -- 7.3.9 Trilineage Differentiation of iMSCs -- 7.4 Notes -- References -- 8 CRISPR/Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells -- Abstract -- 8.1 Introduction -- 8.2 Materials -- 8.2.1 Cell Culture -- 8.2.2 Transfection Reagents and Nucleofection Kits -- 8.2.3 DNA Extraction, PCR, and Cloning -- 8.2.4 Plastics and Equipment -- 8.3 Methods -- 8.3.1 sgRNA Design and Vector Cloning -- 8.3.1.1 sgRNA Design -- 8.3.1.2 Cas9-sgRNA Vector Cloning -- 8.3.2 Identification of the Best sgRNA by T7E1 Assay -- 8.3.2.1 Preparation of 293T Cells for Transfection -- 8.3.2.2 Transfection of 293T Cells Using Lipofectamine 2000 -- 8.3.2.3 Harvesting Cells for DNA Extraction -- 8.3.2.4 Amplifying Targeted Locus by PCR -- 8.3.2.5 Digestion of the DNA Heteroduplex with T7E1 -- 8.3.2.6 Calculation of the Cleavage Efficiency -- 8.3.3 Design and Clone Donor Plasmid Based on the Best sgRNA -- 8.3.4 Creating the OCT4-GFP Reporter iPSC Line -- 8.3.4.1 Preparation of iPSCs -- 8.3.4.2 Nucleofection of iPSCs -- 8.3.4.3 Single-Cell Sorting of GFP-Positive iPSCs by FACS -- 8.3.4.4 Confirmation of Gene Knock-in by Junction PCR -- 8.4 Notes -- References -- 9 Humanized Mouse Models with Functional Human Lymphoid and Hematopoietic Systems Through Human Hematopoietic Stem Cell and Human Fetal Thymic Tissue Transplantation -- Abstract -- 9.1 Introduction -- 9.2 Materials -- 9.2.1 Mouse -- 9.2.2 Fetus -- 9.2.3 Human Hematopoietic Stem Cell Purification -- 9.2.4 Materials for Human Fetal Thymic Graft Transplantation -- 9.2.4.1 Anesthetic Agent -- 9.2.4.2 Surgical Instruments for Human Fetal Thymic Graft Transplantation. , 9.2.5 Flow Cytometric Analysis -- 9.2.5.1 Fluorescent-Conjugated mAbs and Isotype-Matched Control Abs -- 9.2.5.2 FACS Medium -- 9.2.6 Macrophage Depletion Regimen -- 9.3 Methods -- 9.3.1 Human CD34 + Fetal Liver Cell (FLC) Purification and Cryopreservation -- 9.3.2 Human Fetal Thymic Tissues Preparation -- 9.3.3 Humanization of NOD/SCID Mice and Their Derivatives -- 9.3.4 Follow the Human Lymphoid Cell Reconstitution of Humanized Mice Every 2--3 Weeks -- 9.3.5 Generation of Humanized Mice with Human RBC and Platelet Reconstitution in Periphery Blood -- 9.3.5.1 Measure Human RBC Reconstitution -- 9.3.5.2 Measure Human Platelet Reconstitution -- References.