Induction of HSC differentiation from iPSCs using different culture systems. (A) Representative FCM results showing the expression of various surface markers on iPSCs obtained by coculturing WT iPSC clone G1 with OP9 cells for 21 days. (B) The percentages of SSEA-4^–, CD34⁺, CD108⁺, CD184⁺, and CD45⁺ expressed by iPSCs during the differentiation process of iPSCs cultured with OP9 cells. The percentages represent the mean ± standard deviation (SD) of the data from 5 experiments using 3 different WT iPSC clones analyzed at 7, 14, and 21 days of culture. (C) Changes in the SSEA-4 expression of WT clone G1 with time after culture. (D) HSC- and iPSC-related gene expression patterns of iCD34⁺ cells. Total RNA extracted from original iPSC clones (WT and 6pLOH clones), iCD34⁺ cells induced from the same iPSCs, CD34⁺ cord blood cells, and a stem cell-like acute monoblastic leukemia (AML) cell line (AML-MT) was subjected to an RT-PCR analysis for 4 genes: HoxA9, CD45, CD41, and OCT3. (E) Representative images of hematopoietic cells induced from iPSCs in various hematopoietic differentiation media (StemPro-34 serum-free medium [SFM] and OP9, WEHI, and OP9 and WEHI conditioned media). Images were captured by using a light microscope at day 14. (F) The expression of cell surface markers on iPSCs cultured under the 4 different methods. Cells were harvested and analyzed by FCM at the indicated time points (0, 7, 14, 21, and 28 days). The data represent results from 2 independent experiments and are expressed as the mean ± standard error of the mean (SEM) of the percentage of cells expressing the given marker. FSC-W, forward scatter width; SFM, serum-free medium; SSC-H, side scatter height; UCB, umbilical cord blood.