Abstract
SummaryIn contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.
Highlights
Human pluripotent stem cells provide a potent resource for fundamental research into early human development and in addition hold great promise for biomedical applications. human pluripotent stem cells (hPSCs) have been derived by culture of explanted human embryo inner cell masses (ICMs) (O’Leary et al, 2012; Thomson et al, 1998) and by reprogramming of somatic cells (Takahashi et al, 2007; Yu et al, 2007)
We adopted the combination of mRNAs encoding six reprogramming factors, OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28 (OSKMNL), augmented with microRNAs 302 and 367, plus Vaccinia virus immune evasion factors E3, K3, and B18R mRNAs to suppress the interferon response
Using t-distributed stochastic neighbor embedding analysis, we found that methylation profiles of naive and primed PSC cultures clustered apart, with naive cultures adjacent to ICM samples (Figure 5F)
Summary
In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. We investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs
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