Part F: Directed Differentiation of Human Embryonic Stem Cells into Myeloid Cells

The in vitro aggregation of human embryonic stem (ES) cells into three dimensional clusters, termed embryoid bodies (EB), allows for the spontaneous differentiation of cells representing endoderm, mesoderm and ectoderm lineages. During EB differentiation, the stochastic generation of hematopoietic cells is enhanced by a combination of hematopoietic cytokines and the mesoderm inducer bone morphogenetic protein‐4 (BMP‐4). Under this treatment, up to 90% of CD45+ cells with colony forming unit (CFU) activity can be generated. These cells solely derive from a rare subpopulation of hemogenic precursors that lack CD45, but express PECAM‐1, Flk‐1 and VE‐Cadherin (CD45negPFV), and can repopulate the bone marrow of immune deficient mice after intra bone marrow injection, albeit at very low levels. This chapter describes protocols that have been successfully applied in our laboratory for the in vitro and in vivo generation and characterization of hematopoietic cells from EBs and purified CD45negPFV hemogenic precursors.