Reprogramming of human primary somatic cells by OCT4 and chemical compounds

Human induced pluripotent stem cells (hiPSCs) can be practically derived from neonatal human epidermal keratinocytes (NHEKs) transduced with OCT4, by a novel cocktail of small molecules including 5 μM PS48, 0.25 mM NaB, 0.5 μM A-83-01 and 0.5 μM PD0325901. These iPSCs are morphologically, molecularly and functionally similar to pluripotent hESCs. NHEKs were cultured in a 100 mm tissue culture dish and transduced 3 times (3-4 hours each transduction) with freshly produced lentivirus supernatants. 1,000,000 transduced NHEKs were seeded on the irradiated x-ray inactivated CF1 MEF feeder cells in a 100-mm dish and cultured in KCM and treated with 5 μM PS48, 0.25 mM NaB (Stemgent) and 0.5 μM A-83-01 (Stemgent) for 2 weeks, followed by changing half volume of media to hESCM and supplementing with 5 μM PS48, 0.25 mM NaB and 0.5 μM A-83-01 for another 2 weeks. Then cell culture media were changed to hESCM and supplemented with 5 μM PS48, 0.25 mM NaB, 0.5 μM A-83-01 and 0.5 μM PD0325901 (Stemgent) for additional four weeks.The culture was split by Accutase (Millipore) and treated with 1 μM Thiazovivin (Stemgent) in the first day after splitting. The iPSC colonies stained positive by Alexa Fluor 555 Mouse anti-Human TRA-1-81 antibody (BD Pharmingen) were picked up for expansion on feeder cells in hESCM and cultured routinely.