Reprogramming of human primary somatic cells by OCT4 and chemical compounds.
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ABSTRACT:
SUBMITTER: Zhu S
PROVIDER: S-EPMC3812930 | biostudies-literature | 2010 Dec
REPOSITORIES: biostudies-literature
Publications
Reprogramming of human primary somatic cells by OCT4 and chemical compounds.
Zhu Saiyong S Li Wenlin W Zhou Hongyan H Wei Wanguo W Ambasudhan Rajesh R Lin Tongxiang T Kim Janghwan J Zhang Kang K Ding Sheng S
Cell stem cell 20101201 6
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Project description: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.
2010-12-03 | GSE25218 | GEO
Project description: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. Global gene expression analysis of hiPSCs
2010-12-03 | E-GEOD-25218 | biostudies-arrayexpress
Project description:Multiple methods exist that can reprogram differentiated cells to a pluripotent state similar to that of embryonic stem cells (ESCs). These include somatic cell nuclear transfer (SCNT), fusion-mediated reprogramming (FMR) of somatic cells with ESCs, and the production of induced pluripotent stem cells (iPSCs). All of these methods yield cells in which the endogenous Oct4 gene is reactivated. We were interested in comparing the activity of the Oct4 promoter in three different classes of pluripotent cells, including normal ESCs, FMR cells (FMRCs), and iPSCs. We prepared cells of all three types that harbor a transgene composed of the mouse Oct4 promoter driving green fluorescent protein (Oct4-GFP). All cell derivations started with a characterized transgenic Oct4-GFP mouse, and from this we derived ESCs, FMRCs, and iPSCs with the Oct4-GFP transgene present in an identical genomic integration site in all three cell types. Using flow cytometry we assessed Oct4 promoter expression, cell cycle behavior, and differentiation kinetics. We found similar levels of GFP expression in all three cell types and no significant alterations in pluripotency or differentiation. Our results suggest that the pluripotent condition is a potent "local attractor" state, because it can be achieved through three vastly different avenues.
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