Reprogramming of human primary somatic cells by OCT4 and chemical compounds

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE25218 | GEO | 2010/12/03

SECONDARY ACCESSION(S): PRJNA134453

REPOSITORIES: GEO