Generation of Hematopoietic Cells from hiPSCs

The protocol schedule is summarized in Table 1 and a graphical depiction of the process is shown in Figure 1. To induce hematopoietic cells from HiPS-RIKEN-3A-TAL1 and HiPS-RIKEN-4A-TAL1 cells, cells were cultured on OP9 feeder cells in basal differentiation medium composed of IMDM (Sigma) supplemented with 15% fetal bovine serum (FBS; Invitrogen), ITS (10 µg/ml human insulin, 5.5 µg/ml human transferrin, and 5 ng/ml sodium selenite; Sigma), 50 mg/mL ascorbic acid (Sigma), 0.45 mM α-monothioglycerol (Sigma), and PSQ (100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine; Invitrogen) in the presence of VEGF (20 ng/ml) and IGF-II (200 ng/ml). From day 10, the cells were cultured on OP9 cells in the presence of SCF (50 ng/ml), EPO (3 IU/ml) and DEX (10⁻⁶ M). On day 16, the HPV16-E6/E7 expression system was introduced into the cells by lentiviral transduction. Four days later, the cells were cultured on OP9 cells in the presence of DOX (1 µg/ml), SCF (50 ng/ml), EPO (3 IU/ml) and DEX (10⁻⁶ M). The medium was usually changed twice per week, except when cell numbers were low. Around three months after initiation of culture, the cells were able to proliferate without feeder cells. After this time point, we maintained the cells in a serum-free medium, StemSpan SFEM® medium (StemCell Technologies, Vancouver, BC, Canada), in the presence of specific factors.