Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.
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Acknowledgements J.H.H. is supported by a gift from Ilana and Pascal Mantoux, and research grants from the European Research Council starting investigator grant (StG-2011-281906, for funding iPS cell experiments only), the Leona M. and Harry B. Helmsley Charitable Trust, the BIRAX (Britain Israel Research and Academic Exchange Partnership), The Sir Charles Clore Research Prize, the Israel Science Foundation (Bikura, ICORE (Israeli Centre of Research Excellence) and Regular researchprogram), the IsraelCancer ResearchFoundation, the ERANET E-Rare disease program, the Benoziyo Endowment fund, Fritz Thyssen Stiftung (used for human iPS cell experiments only), EMBO young investigator program, the Alon Foundation scholar award, a grant from E. A. and R. Drake, postdoctoral fellowships from ICRF and the Weizmann Dean fellowship award for A.A.M. We thank the embryologists of the Racine In Vitro Fertilization Laboratory at the Lis Maternity Hospital (A. Carmon, T. Cohen and N.M.Raz)for theirskilfulassistance.Wethankthe WeizmannInstitutemanagement for providing financial and infrastructural support.