Hematopoietic differentiation of doxycycline inducible human FANCA patient IPSCs. (A) Schema for the differentiation of human IPSCs to HPCs adapted from published protocols.^23,27  The first 8-day period involves morphogen-directed specification of hemogenic endothelium within EBs (see "Methods"). This period of culture incorporates 2 phases: BMP-4 and bFGF-mediated mesoderm induction, followed by HE differentiation driven by vascular endothelial growth factor, IL-11, insulin-like growth factor 1, and hematopoietic cytokines. At day 8, EBs were dissociated, and HE was isolated based on CD34⁺ selection. HE was plated on a 2-dimensional Matrigel substratum for an 8-day EHT culture in the presence of hematopoietic cytokines with or without doxycycline to modulate FANCA expression. At the end of the 8-day EHT culture, round floating HPCs were harvested for analysis in assays of clonogenicity and hematopoietic differentiation. (B) Representative image of day 8 EBs before dissociation (scale bar, 200 μm). (C) The entire cellular composition of dissociated day 8 EBs derived from the indicated human IPSC lines was stained with the indicated antibodies and definitive HE (CD34⁺GLYA⁻KDR⁺CD43⁻) was analyzed by flow cytometry gated on viable singlet cells. (D) Representative images of cultured HE at the indicated time points during EHT culture (scale bar, 50 μm). (E) Representative images of day 8 EHT culture in the presence or absence of doxycycline (scale bar, 100 μm). (F-G) Nonadherent, round cells were harvested at day 8 EHT and analyzed for expression of the indicated surface markers of HPCs by flow cytometry. Percent CD34⁺CD45⁺ HPCs within the nonadherent population was quantified over 6 independent experiments (results presented as mean ± SEM, analyzed by a paired Student t test).