Cytokine-induced in vitro hematopoietic differentiation of disease model iPSCs. (A) Schematic protocol to induce hematopoietic differentiation in iPSCs, as described previously.¹⁴  Following the liquid culture phase, cells were sorted at day 7 based on positive staining with anti-CD34 and anti-KDR antibodies, as indicated in the FACS profile shown, and cells were seeded into MethoCult media on OP9 feeder cells.¹⁵  (B) Phase-contrast images at day 19 of colonies derived from AP39P-iPSCs, cultured or not with DOX-induced ADH5 expression. (C) Representative images of the hematopoietic progenitor colonies formed in MethoCult media following KDR⁺CD34⁺ sorting. Each colony was observed microscopically and classified as described previously.¹⁴  (D-E) Number of progenitor cell colonies in MethoCult media at around day 19 from the sorted KDR⁺CD34⁺ fraction of the knockout iPSCs with the indicated genotypes (scale bars, 100 μm) (D) and patient-derived iPSCs (E). Mean ± standard deviation (SD) from 3 biological replicates are shown. The ALDH2 activating compound C1 was added on day 0 (30 μM). The experiment in duplicate culture was repeated twice with different sets of iPSC clones. (F) Levels of γH2AX and FANCD2 foci, scored as the percentage of cells with >5 foci per cell. The KDR⁺CD34⁺ cells were sorted at day 7 following the liquid culture phase, with or without DOX-induced ADH5 expression, and stained with anti-γH2AX or anti-FANCD2 antibody. The number of foci was evaluated by an IN Cell Analyzer 2000, with ≥1500 cells for each sample. Mean ± SD in quadruplicate cultures are indicated. The P values were calculated using an unpaired, 2-tailed Student t test. Representative images of cells are shown in supplemental Figure 7.