Derivation and Characterization of Hematopoietic Cells From Human Embryonic Stem Cells
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In vitro, the aggregation of pluripotent human embryonic stem cells (hESC) into cell clusters termed embryoid bodies (EB) allows for the spontaneous differentiation of hESC into progeny representing endoderm, mesoderm, and ectoderm lineages. During human EB (hEB) differentiation, stochastic emergence of hematopoietic cells can be enhanced by a combination of hematopoietic cytokines and the ventral mesoderm inducer bone morphogenetic protein (BMP)-4. Dependent on the presence of hematopoietic cytokines and BMP-4, vascular endothelial growth factor (VEGF-A165) selectively promotes erythropoietic development toward the primitive lineage. The effects of VEGF-A165 can be augmented by erythropoietin (EPO). Hematopoietic cells are derived from a rare subpopulation of hemogenic precursors during hEB development. These hemogenic precursors lack CD45, but express PECAM-1, Flk-1, and VE-cadherin (hereinafter CD45(neg)PFV) and are solely responsible for hematopoietic cell fate. Human ESC-derived hematopoietic cells have similar colony and cellular morphologies to those derived from committed adult hematopoietic tissues, and also show repopulating capacity in immune deficient mice after intrabone marrow transplantation. In this chapter, we describe methods that have been successfully applied in our laboratory, including (1) generation of hematopoietic cells by EB formation; (2) augmentation of hematopoiesis by use of hematopoietic cytokines and BMP-4; (3) promotion of erythropoietic development by addition of VEGF-A165 and EPO; (4) isolation of CD45(neg)PFV hemogenic precursors and generation of hematopoietic cells from these precursors; and (5) characterization of hESC-derived hematopoietic cells in vitro and in vivo.
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