In this paper, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin ĝ̂' CD34 + CD43 + CD45 + multipotent progenitors. The protocol comprises three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells; (ii) short-term expansion of multipotent myeloid progenitors with a high dose of granulocyte-macrophage colony-stimulating factor; and (iii) directed differentiation of myeloid progenitors into neutrophils, eosinophils, dendritic cells, Langerhans cells, macrophages and osteoclasts. The generation of multipotent hematopoietic progenitors from hPSCs requires 9 d of culture and an additional 2 d to expand myeloid progenitors. Differentiation of myeloid progenitors into mature myeloid cells requires an additional 5g-19 d of culture with cytokines, depending on the cell type.
Bibliographical noteFunding Information:
acknoWleDGMents This study is supported by funds from the National Heart, Lung, and Blood Institute (R01 HL081962, R21 HL085223) and by National Institutes of Health grant P51RR000167 to the National Primate Research Center, University of Wisconsin, Madison. We thank T. Nakano for providing OP9 cells.