Induced pluripotent stem cells (iPSCs) are generated via the expression of the transcription factors OCT4 (also known as POU5F1), SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naive iPSCs, conventional human iPSCs are in a more developmentally advanced state called primed pluripotency. Here, we report that human naive iPSCs (niPSCs) can be generated directly from fewer than 1,000 primary human somatic cells, without requiring stable genetic manipulation, via the delivery of modified messenger RNAs using microfluidics. Expression of the OSKM factors in combination with NANOG for 12 days generates niPSCs that are free of transgenes, karyotypically normal and display transcriptional, epigenetic and metabolic features indicative of the naive state. Importantly, niPSCs efficiently differentiate into all three germ layers. While niPSCs can be generated at low frequency under conventional conditions, our microfluidics approach enables the robust and cost-effective production of patient-specific niPSCs for regenerative medicine applications, including disease modelling and drug screening.
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RNA-seq and RRBS data of this study have been deposited in the Sequence Read Archive (SRA) under BioProject number PRJNA381757 and GEO under accession code GSE110377. Accession numbers of other published datasets are reported in each figure plotting RNA-seq data and in Supplementary Table 2. Source data of all repeats of all experiments are provided in Supplementary Table 5. For figure panels showing representative images of morphologies or immunostainings, images from additional independent repeats are available at Figshare (https://doi.org/10.6084/m9.figshare.c.4250195). All other data supporting the findings of this study are available from the corresponding author upon reasonable request.
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The authors thank M. Montagner and S. Dupont for critical reading of the manuscript, and the Martello Laboratory for discussions and suggestions. Thanks are also given to the following: the Smith Laboratory for sharing reset H9 cells and plasmids; Miltenyi Biotec for providing mmRNAs; A. Rosa and R. De Santis for their help with the neuronal differentiation of niPSCs; and A. Manfredi, TIGEM NGS and the Bioinformatics Core for their technical support on library generation and data processing. The authors are indebted to P. Brun for providing primary skin fibroblasts. G.M.’s Laboratory is supported by grants from the Giovanni Armenise–Harvard Foundation, the Telethon Foundation (TCP13013) and an ERC Starting Grant (MetEpiStem). D.C.’s Laboratory is supported by grants from the Giovanni Armenise–Harvard Foundation, the Telethon Foundation, the Rita Levi Montalcini programme from MIUR and an ERC Starting Grant (CellKarma). C.R.’s Laboratory is supported by the Italian Association for Cancer Research (IG17185). M.J.Z. is supported by a BMBF eMed grant (01ZX1504) and the Max Planck Society. N.E.’s Laboratory is supported by grants from the University of Padova (TRANSAC and PRAT), the CaRiPaRo Foundation, the Telethon Foundation (GGP15275), an Oak Foundation Award (W1095/OCAY-14-19) and the NIHR GOSH BRC. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.