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Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (Xi) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative XaXa intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets Xi abnormalities seen in primed hESCs, it may provide cells better suited for downstream applications.

Author(s): Alejandro De Los Angeles [1, 2, 3]; Francesco Ferrari [4]; Yuko Fujiwara [1, 2, 3]; Ronald Mathieu [5]; Soohyun Lee [4]; Semin Lee [4]; Ho-Chou Tu [1, 2, 3]; [...]

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeo-domain-containing protein NANOG (1,2), which has an essential role in [...]

Pluripotency is a developmental ground state that can be recreated by direct reprogramming. Establishment of pluripotency is crucially dependent on the homeodomain-containing transcription factor Nanog. Compared with other pluripotency-associated genes, however, Nanog shows relatively low sequence conservation. Here, we investigated whether Nanog orthologs have the capacity to orchestrate establishment of pluripotency in Nanog-/- somatic cells. Mammalian, avian and teleost orthologs of Nanog enabled efficient reprogramming to full pluripotency, despite sharing as little as 13% sequence identity with mouse Nanog. Nanog orthologs supported self-renewal of pluripotent cells in the absence of leukemia inhibitory factor, and directly regulated mouse Nanog target genes. Related homeodomain transcription factors showed no reprogramming activity. Nanog is distinguished by the presence of two unique residues in the DNA recognition helix of its homeodomain, and mutations in these positions impaired reprogramming. On the basis of genome analysis and homeodomain identity, we propose that Nanog is a vertebrate innovation, which shared an ancestor with the Bsx gene family prior to the vertebrate radiation. However, cephalochordate Bsx did not have the capacity to replace mouse Nanog in reprogramming. Surprisingly, the Nanog homeodomain, a short sequence that contains the only recognizable conservation between Nanog orthologs, was sufficient to induce naive pluripotency in Nanog-/- somatic cells. This shows that control of the pluripotent state resides within a unique DNAbinding domain, which appeared at least 450 million years ago in a common ancestor of vertebrates. Our results support the hypothesis that naive pluripotency is a generic feature of vertebrate development. [ABSTRACT FROM AUTHOR]

The cytokine leukemia inhibitory factor (Lif) sustains self-renewal of mouse embryonic and induced pluripotent stem cells by activating Jak kinase and the transcription factor Stat3. Here we investigate whether Jak/Stat3 may also contribute to induction of pluripotency. EpiSCs derived from postimplanta- tion embryos express low levels of Lif receptor and Stat3. We introduced into EpiSCs a Jak/Stat3 acti- vating receptor (GY118F) responsive to granulocyte colony stimulating factor (Gcsf). On transfer to ground state culture, in which MAPK signaling and glycogen synthase kinase are inhibited, Gcsf induced transcriptional resetting and functional re- programming. Activation of a tamoxifen-regulatable fusion, Stat3ERT2, also converted EpiSCs into chimera-competent iPSCs. We exploited GY118F to increase Jak/Stat3 activity during somatic cell re- programming. Incompletely reprogrammed cells derived from neural stem cells or fibroblasts responded to Gcsf with elevated frequencies of progression to ground state pluripotency. These findings indicate that Jak/Stat3 participate directly in molecular reprogramming and that activation of this pathway is a limiting component. [ABSTRACT FROM AUTHOR]

RNAi is a powerful tool for interrogating gene function in ES cells. Combining the high penetrance of a microRNA-embedded shRNA (shRNA-mir) cassette with a locus-defined, inducible expression strategy, we developed a system for RNAi in mouse ES cells. An shRNA-mir cassette is targeted near the constitutively active HPRT locus under a tetracycline (tet)-regulatable promoter through Cremediated site-specific recombination. The major advantage of this system is that the shRNA-mir cassette can be targeted to a precise locus, allowing for control of shRNA-mir expression in an inducible fashion. Induction of an shRNA-mir directed against the pluripotency factor, Nanog, resulted in the loss of self-renewal and differentiation to parietal endoderm-like cells, which can be rescued by the introduction of an RNAi-immune version of Nanog cDNA. Knockdown efficiency can be enhanced by using multiple shRNA-mir hairpins against the target gene, which was further validated by knocking down two additional ES cell factors. This site-directed, virus-free, and tet-inducible RNAi system, designated as SDVFi RNAi in our study, presents an efficient option for controlled gene silencing in ES cells. [ABSTRACT FROM AUTHOR]

Embryonic stem (ES) cells are pluripotent and of therapeutic potential in regenerative medicine. Understanding pluripotency at the molecular level should illuminate fundamental properties of stem cells and the process of cellular reprogramming. Through cell fusion the embryonic cell phenotype can be imposed on somatic cells, a process promoted by the homeodomain protein Nanog, which is central to the maintenance of ES cell pluripotency. Nanog is thought to function in concert with other factors such as Oct4 (ref. 8) and Sox2 (ref. 9) to establish ES cell identity. Here we explore the protein network in which Nanog operates in mouse ES cells. Using affinity purification of Nanog under native conditions followed by mass spectrometry, we have identified physically associated proteins. In an iterative fashion we also identified partners of several Nanog-associated proteins (including Oct4), validated the functional relevance of selected newly identified components and constructed a protein interaction network. The network is highly enriched for nuclear factors that are individually critical for maintenance of the ES cell state and co-regulated on differentiation. The network is linked to multiple co-repressor pathways and is composed of numerous proteins whose encoding genes are putative direct transcriptional targets of its members. This tight protein network seems to function as a cellular module dedicated to pluripotency. [ABSTRACT FROM AUTHOR]

Prmt5, an arginine methyltransferase, has multiple roles in germ cells, and possibly in pluripotency. Here we show that loss of Prmt5 function is early embryonic-lethal due to the abrogation of pluripotent cells in blastocysts. Prmt5 is also up-regulated in the cytoplasm during the derivation of embryonic stem (ES) cells together with Stat3, where they persist to maintain pluripotency. Prmt5 in association with Mep50 methylates cytosolic histone H2A (H2AR3me2s) to repress differentiation genes in ES cells. Loss of Prmt5 or Mep50 results in de-repression of differentiation genes, indicating the significance of the Prmt5/Mep50 complex for pluripotency, which may occur in conjunction with the leukemia inhibitory factor (LIF)/Stat3 pathway. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is a hypertensive pregnancy disorder with increased risk of maternal and fetal morbidity and mortality. Abnormal extravillous trophoblast (EVT) development and function is considered to be the underlying cause of PE, but has not been previously modeled in vitro . We previously derived induced pluripotent stem cells (iPSCs) from placentas of PE patients and characterized abnormalities in formation of syncytiotrophoblast and responses to changes in oxygen tension. In this study, we converted these primed iPSC to naïve iPSC, and then derived trophoblast stem cells (TSCs) and EVT to evaluate molecular mechanisms underlying PE. We found that primed (but not naïve) iPSC-derived PE-EVT have reduced surface HLA-G, blunted invasive capacity, and altered EVT-specific gene expression. These abnormalities correlated with promoter hypermethylation of genes associated with the epithelial-mesenchymal transition pathway, specifically in primed-iPSC derived PE-EVT. Our findings indicate that abnormal epigenetic regulation might play a role in PE pathogenesis., Competing Interests: M.H. has initiated a process for filing a patent for the protocol used. T.W.T. is a consultant for Stately Bio, Inc., (© 2024 The Authors.)