Modeling X-chromosome inactivation and reactivation during human development

Stem-cell-based embryo models generate much excitement as they offer a window into an early phase of human development that has remained largely inaccessible to scientific investigation. An important epigenetic phenomenon during early embryogenesis is the epigenetic silencing of one of the two X chromosomes in female embryos, which ensures an equal output of X-linked gene expression between the sexes. X-chromosome inactivation (XCI) is thought to be established within the first three weeks of human development, although the inactive X-chromosome is reactivated in primordial germ cells (PGCs) that migrate to the embryonic gonads. Here, we summarize our current understanding of X-chromosome dynamics during human development and comment on the potential of recently established stem-cell-based models to reveal the underlying mechanisms

ZMYM2 inhibits NANOG-mediated reprogramming.

Background: NANOG is a homeodomain-containing transcription factor which forms one of the hubs in the pluripotency network and plays a key role in the reprogramming of somatic cells and epiblast stem cells to naïve pluripotency.  Studies have found that NANOG has many interacting partners and some of these were shown to play a role in its ability to mediate reprogramming. In this study, we set out to analyse the effect of NANOG interactors on the reprogramming process. Methods: Epiblast stem cells and somatic cells were reprogrammed to naïve pluripotency using MEK/ERK inhibitor PD0325901, GSK3β inhibitor CHIR99021 and Leukaemia Inhibitory Factor (together termed 2i Plus LIF). Zmym2 was knocked out using the CRISPR/Cas9 system or overexpressed using the PiggyBac system. Reprogramming was quantified after ZMYM2 deletion or overexpression, in diverse reprogramming systems. In addition, embryonic stem cell self renewal was quantified in differentiation assays after ZMYM2 removal or overexpression. Results: In this work, we identified ZMYM2/ZFP198, which physically associates with NANOG as a key negative regulator of NANOG-mediated reprogramming of both epiblast stem cells and somatic cells. In addition, ZMYM2 impairs the self renewal of embryonic stem cells and its overexpression promotes differentiation. Conclusions: We propose that ZMYM2 curtails NANOG's actions during the reprogramming of both somatic cells and epiblast stem cells and impedes embryonic stem cell self renewal, promoting differentiation.This study was supported by the Wellcome Trust through a Wellcome Trust Fellowship to J.C.R.S. [101861], Wellcome Trust Studentship to M.L. [079249], and a core funding grant jointly with the Medical Research Council (MRC) to the Wellcome-MRC Cambridge Stem Cell Institute [079249]

Human Naive Pluripotent Stem Cells Model X Chromosome Dampening and X Inactivation

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 XaXaintermediate. 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 Xiabnormalities seen in primed hESCs, it may provide cells better suited for downstream applications

A genome-wide CRISPR-Cas9 knockout screen identifies essential and growth-restricting genes in human trophoblast stem cells

The recent derivation of human trophoblast stem cells (hTSCs) provides a scalable in vitro model system of human placental development, but the molecular regulators of hTSC identity have not been systematically explored thus far. Here, we utilize a genome-wide CRISPR-Cas9 knockout screen to comprehensively identify essential and growth-restricting genes in hTSCs. By cross-referencing our data to those from similar genetic screens performed in other cell types, as well as gene expression data from early human embryos, we define hTSC-specific and -enriched regulators. These include both well-established and previously uncharacterized trophoblast regulators, such as ARID3A, GATA2, and TEAD1 (essential), and GCM1, PTPN14, and TET2 (growth-restricting). Integrated analysis of chromatin accessibility, gene expression, and genome-wide location data reveals that the transcription factor TEAD1 regulates the expression of many trophoblast regulators in hTSCs. In the absence of TEAD1, hTSCs fail to complete faithful differentiation into extravillous trophoblast (EVT) cells and instead show a bias towards syncytiotrophoblast (STB) differentiation, thus indicating that this transcription factor safeguards the bipotent lineage potential of hTSCs. Overall, our study provides a valuable resource for dissecting the molecular regulation of human placental development and diseases

3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages

Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ( blastoids ) that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated in vitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages

Systematic Identification of Culture Conditions for Induction and Maintenance of Naive Human Pluripotency

Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, “naive” state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.Simons Foundation (Grant SFLIFE 286977)National Institutes of Health (U.S.) (Grant RO1-CA084198)National Science Foundation (U.S.). Graduate Research FellowshipJerome and Florence Brill Graduate Student Fellowshi

Molecular Criteria for Defining the Naive Human Pluripotent State.

Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.This study was supported by grants from the Simons Foundation (SFLIFE #286977 to R.J) and in part by the NIH (RO1-CA084198) to R.J., from the Swiss National Science Foundation and the European Research Council (KRABnKAP, No. 268721) to D.T. The work in J.R.E’s laboratory was supported by the Howard Hughes Medical Institute and Gordon and Betty Moore Foundation (GBMF3034) and the Mary K. Chapman Foundation. J.R.E is an Investigator of the Howard Hughes Medical Institute. T.W.T. is supported by a Sir Henry Wellcome Postdoctoral Fellowship (098889/Z/12/Z), J.P. by a Foundation Bettencourt Award and by the Association pour la Recherche sur le Cancer (ARC), M.I. by a postdoctoral training grant from the Fonds de la Recherche en Santé du Québec. R.J. is co-founder of Fate Therapeutics and an adviser to Stemgent.This is the final version of the article. It first appeared from Cell Press via http://www.cell.com/cell-stem-cell/abstract/S1934-5909(16)30161-

Promotion of Reprogramming to Ground State Pluripotency by Signal Inhibition

Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency—including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras—were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1–2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon