Figure 2: Schematic diagram of the protocol used to directly differentiate human iPSCs into functional platelets. (A) Undifferentiated hiPS cells cultured in StemLine hematopoietic stem cell expansion medium plus hBMP4, VEGF, and bFGF for 2 days, and SCF, TPO, and Flt-3L were added for another 6 days to generate hematopoietic progenitors. (B) On day 8 of differentiation, flow cytometric analysis showed the generation of hiPSC-derived CD34+ hematopoietic progenitors. (C) After culturing in platelet differentiation medium for 6 days, a population of CD61+ progenitorswas obtained. Cells were collected and grown on OP9 feeder cells or continuously grown in platelet differentiation medium for an additional 8 days. (D) Cells released to the culture medium on day 22 of differentiation (end of differentiation) were analysed by flow cytometry. A platelet gate was fixed in the forward- and side-scatter profiles of human blood platelets (left panel) to establish proper size gating for platelets derived from iPSCs. Majority of the cells collected from the differentiation medium localized in this gate. This platelet gate was used for the following FACS analysis of culture-derived platelets. (E) Percentage of platelet marker expression (CD41a, CD42b, and CD61) on day14 and day 22 of differentiation derived from feeder-free (FF) culture system and OP9 coculture system.(F) Coexpression of CD41 and CD61 was found on Day22 of differentiation. All data represented here are from more than three independent experiments using two different human iPSC lines.