Generation of human induced pluripotent stem cells

The cDNAs encoding hOct4, hSox2, hKlf4 and c-myc (purchased from Open Biosystems) were subcloned into self-inactivating lentiviral vectors driven by the human phosphoglycerate kinase (PGK) promoter. Lentiviral vector supernatants were produced by triple co-transfection of the plasmid DNA encoding the vector, pCMVΔR8.91 and pUCMD.G into 293T cells. Human fetal lung fibroblasts (MRC-5) purchased from ATCC (CCL-171) were seeded at 1.5×10⁴ cells/cm² in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS). The following day the fibroblasts were transduced with equal amounts of supernatants of the four lentiviral vectors in the presence of 4 ug/ml polybrene for ∼16 hours. Six days after transduction, fibroblasts were harvested by trypsinization and plated at 2×10⁴ cells per 60 mm dish on a feeder layer of mytomycin C-treated mouse embryonic fibroblasts (CF-1). The next day, the medium was switched to hESC medium. The iPS lines were confirmed positive for Tra-1–81, Tra-1–60, SSEA-4 and Nanog by immunofluorescence and flow cytometry. In both hiPSC clones all 4 vector-encoded transgenes were found to be silenced.