Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins.

Cell Culture Human newborn fibroblasts (HNF) were purchased from ATCC (CCL-117) and cultured in Dulbecco’s modified Minimal Essential Medium (DMEM, Invitrogen, Carlsbad, CA), supplemented with 2mM L-glutamine (Invitrogen, Grand island, NY). 1mM β-mercaptoethanol, 1x non-essential amino acids (NEAA; Invitrogen, Carlsbad, CA), 15% fetal bovine serum (FBS, Hyclone, Thermo Scientific, Logan UT), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Grand island, NY). Reprogramming experiments were started when the cultures reached 10-20% confluence. Cultures were maintained at 37°C and 5% CO2, and the media changed every other day. For mouse embryonic fibroblast (MEF) isolation, uteri were isolated from 13.5-day-pregnant CD1 mice and washed with phosphatebuffered saline (PBS). The head and visceral tissues were removed, and the remaining bodies washed with fresh PBS, transferred into a 0.1 mM trypsin/1 mM EDTA solution, and incubated for 20 min. After incubation, MEF culture medium (DMEM containing 15% defined FBS) was added and pipetted up and down to dissociate cells. MEFs were used as feeders at passages one to three.