Lisa Azzola

To realize the therapeutic potential of human embryonic stem cells (hESCs), it is necessary to regulate their differentiation in a uniform and reproducible manner. We have developed a method in which known numbers of hESCs in serum-free medium were aggregated by centrifugation to foster the formation of embryoid bodies (EBs) of uniform size (spin EBs). These spin EBs differentiated efficiently and synchronously, as evidenced by the sequential expression of molecular markers representing stem cells, primitive streak, and mesoderm. In the presence of hematopoietic growth factors, reproducible differentiation was achieved with blood cells formed in more than 90% of EBs. Using chimeric EBs generated from mixtures of green fluorescence protein-positive (GFP(+)) and GFP(-) hESCs in a clonogenic assay, hematopoietic precursor frequency was estimated to be approximately 1:500 input cells. This method of EB formation provides a generally applicable means for modulating and objectively monitoring the directed differentiation of hESCs.

We have utilized a serum- and stromal cell-free "spin embryoid body (EB)" differentiation system to investigate the roles of four growth factors, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), stem cell factor (SCF), and basic fibroblast growth factor (FGF2), singly and in combination, on the generation of hematopoietic cells from human embryonic stem cells (HESCs). Of the four factors, only BMP4 induced expression of genes that signaled the emergence of the primitive streak-like population required for the subsequent development of hematopoietic mesoderm. In addition, BMP4 initiated the expression of genes marking hematopoietic mesoderm and supported the generation of hematopoietic progenitor cells at a low frequency. However, the appearance of robust numbers of hematopoietic colony forming cells and their mature progeny required the inclusion of VEGF. Finally, the combination of BMP4, VEGF, SCF, and FGF2 further enhanced the total yield of hematopoietic cells. These data demonstrate the utility of the serum-free spin EB system in dissecting the roles of specific growth factors required for the directed differentiation of HESCs toward the hematopoietic lineage.