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and N.N (noa.novershtern@weizman.ac.il). PrefaceThe molecular mechanisms and signalling pathways that regulate the in vitro preservation of distinct pluripotent stem cell configurations, and their induction in somatic cells via direct reprogramming approaches, continue to constitute a highly exciting area of research. In this review, we provide an integrative synthesis on recent discoveries related to isolating unique naïve and primed pluripotent stem cell states with altered functional and molecular characteristics, and from different species. We overview pathways underlying pluripotent state transitions and interconversion in vitro and in vivo. We conclude by highlighting unresolved key questions, future directions and potential novel applications of such dynamic pluripotent cell states. IntroductionPluripotency describes cells that have the potential to give rise to cells from all three embryonic germ-layers and possibly to primordial germ cells (PGCs), but not extra-embryonic tissues 1 . While pluripotency is a transient state in vivo, pluripotent cells can be derived from different stages of early embryonic development and indefinitely maintained in an artificially induced self-renewal state in vitro, by supplementing exogenous cues 2 . Thus, it is important to stress that self-renewal is not a defining feature of pluripotency and is only transiently assembled during early development. Pluripotency is highly dynamic and evolves at different stages of pre-and post-implantation stages 3 . However, the self-renewal aspect is a highly useful in vitro artificial "engineering trick" 4 that has brought pluripotent cells to the front stage as a tool for tissue replacement, disease modelling and animal engineering technologies 5 .There are multiple pluripotent stem cell types that can be isolated from vertebrates, including rodents and human, typically annotated based on their donor cell-of-origin (Fig. 1). Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of developing pre-implantation mouse or human blastocysts [6][7][8] . Epiblast stem cells (EpiSCs) are isolated from mouse postimplantation epiblasts 9,10 , however equivalent derivations haven't been attempted with human embryos due to justified ethical complexities. Early rodent migrating PGCs can be converted in vitro into pluripotent ESC-like cells termed embryonic germ cells 11,12 . Mouse neonatal and adult spermatogonial stem cells can be reverted toward pluripotency and generate male germ stem cells (GSCs) [13][14][15] . The latter have the disadvantage of retaining only male imprint signature, which can increase tumorigenic potential 15 . Intriguingly, stable and validated EGs and GSCs have not been isolated from primates thus far 16,17 (Fig. 1).Somatic cell reprogramming provides alternative routes for isolating pluripotent cell types. Human and rodent somatic cells can be artificially reprogrammed into ESC-like cells following reprogramming via nuclear transfer, termed NT-ESCs [18][19][20] . Ten years ago, Yamanaka established d...

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods.

Summary Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro . Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.