Figure 1.

Schematic representation of the optimized differentiation protocol used to generate hematopoietic precursors and progenitors from embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) lines. (A): Developmental stages of differentiation of ESCs/iPSCs in culture showing the precursors of the hematopoietic lineage targeted for cellular expansion to putative predefinitive hematopoietic stem cells (HSCs) and hematopoietic progenitor cells. (B): Optimized differentiation protocol showing conditions including media cytokine cocktails (see Materials and Methods for details) and timing of applied factors for more than 3 weeks for the efficient differentiation of ESCs/iPSCs through three distinct stages of development. A representative meso-/hemo-embryoid body (EB) is shown with large cystic growths after 7 days in culture indicates efficient mesoderm differentiation and eventually blood lineage generation. The whole meso-/hemo-EB is then plated onto OP9 feeder layer in the same medium cocktail for 1 week before a hematopoietic cocktail is applied for the final week. Assays used to determine efficiency of generation of hematopoietic progenitors and predefinitive HSCs at the 3 week time point are shown (NSG mouse transplant model). Abbreviations: CFU, colony forming unit; EB, Embryoid body; FACS, flow cytometry; hESC, human embryonic stem cell; HSC, hematopoietic stem cell; iPSC, induced pluripotent stem cell; NSG, NOD/SCID/il2rg^−/−; SFEM HC, serum-free expansion media–hematopoietic cell.