Induced pluripotent stem cells (iPSCs) are generated by expression of transcription factors OCT4, SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naïve iPSCs, conventional human iPSCs are in a more developmentally advanced state-called primed pluripotency. Here we report that human naïve iPSCs (niPSCs) can be generated directly from less than 1000 primary human somatic cells without stable genetic manipulation by delivery of modified messenger RNAs with microfluidics. Expression of OSKM in combination with NANOG for 12 days generates niPSCs free of transgenes, karyotypically normal, and display transcriptional, epigenetic and metabolic features indicative of the naïve state. Importantly, niPSCs efficiently differentiate into all three germ layers. While niPSCs could also be generated at low frequency under conventional conditions, our microfluidics approach will allow robust and cost-effective production of patient-specific niPSCs for regenerative medicine applications, including disease modelling and drug screening.
Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGFbeta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.
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