Figure 3.

Timing for directed differentiation of mature myelomonocytic cells from human pluripotent stem cells. To induce hematopoietic differentiation, hES/iPSCs are collected from MEF cocultures and transferred onto overgrown OP9 cells. After 9 days of coculture with OP9, cells should be collected and treated with enzymes to prepare single cell suspensions containing lin⁻CD34⁺CD43⁺CD45⁺ hematopoietic progenitors. To expand these progenitors, single cells should be cultured in non-adherent conditions (pHEMA-coated flasks) in the presence of GM-CSF for 2 days. Expanded lin⁻CD34⁺CD43⁺CD45⁺ progenitors enriched in myeloid CFCs should be isolated by magnetic sorting. Through cell culture with a particular cytokine combination, myeloid progenitors can be differentiated into mature myeloid cells of the desired type. Depending on the cell type, 5 to 19 days of culture are required to obtain a mature cell population.