Blood was collected from donors, and iPSC lines were derived from the blood cells as previously described in detail (Yang et al., 2012). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from blood and cultured in media supplemented with cytokines for expansion of erythroblasts. Erythroblast-enriched cultures were transduced with Sendai viral (SeV) vectors (CytoTune; Thermo Fisher Scientific) expressing the four Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc). Transduced cells were then transferred onto irradiated mouse embryonic fibroblast (MEF) feeder cells (Global Stem) and cultured at 37 °C at 5% oxygen/5% CO2 in hESC medium consisting of DMEM/F12 supplemented with 20% knockout serum replacement (KOSR), 2 mM L-glutamine, MEM nonessential amino acids (NEAA), 1% penicillin/streptomycin (P/S), and 10 ng/mL basic fibroblast growth factor (bFGF). All ingredients for hESC medium were obtained from Thermo Fisher Scientific. iPSC colonies were picked and further cultured in hESC medium to at least cell passage 12 to establish iPSC lines. Two to four iPSC lines were established from each blood sample. Established iPSC lines were transitioned to feeder-free culture conditions for at least 5 passages in mTeSR1 medium and on Geltrex-coated tissue culture plates before they were used for HLC differentiation.
