Mechanisms of X chromosome inactivation during human trophoblast differentiation in vitro

项目摘要/摘要 在雌性胎盘哺乳动物的早期发育过程中，两条X染色体中的一条在胚胎细胞中被随机灭活，以使X连锁基因的表达与雄性平衡。虽然这一过程在胚胎分化的背景下已经被很好地描述，但仍然不清楚性染色体的剂量补偿是如何在包括胎盘在内的人类胚胎外组织中建立的。体外受精(IVF)出生的男婴出人意料地高，这被归因于滋养层血统中XCI的缺陷，突显了性染色体剂量补偿在人类早期发育中的临床意义。为了能够剖析XCI在人类滋养层发育过程中的早期机制，我们建议利用“幼稚”的人类多能干细胞(HPSCs)，它在植入前的胚胎中表现出多能细胞的转录和表观遗传学特征，包括在雌性细胞中存在两条活跃的X染色体。我们已经证明，幼稚的hPSCs可以很容易地分化为自我更新的人滋养层干细胞(HTSCs)，hTSCs可以进一步分化为特化的滋养层细胞类型和3D滋养层细胞器。在这里，我们将结合这些基于干细胞的滋养层细胞发育的2D和3D模型，以及表观基因组学和单细胞方法来研究XCI在人类滋养层细胞分化过程中的机制。目的1将在体外确定人滋养层细胞XCI的动力学和必要的调节因子。基于我们使用双等位X连锁报告系的初步研究和来自非人类灵长类胚胎的证据，我们假设在幼稚hPSCs中HTSC的诱导伴随着随机的XCI，并且在胚胎谱系中先于XCI。这种随机的XCI模式与小鼠胚胎外膜中父系X染色体印记失活形成对比，加强了对人类特有模型系统的需求。目的2研究X染色体剂量对hTSCs和hPSCs来源的滋养层器官的影响。基于体外受精过程中出生的男婴的增加以及滋养层系是第一个在非人类灵长类胚胎中经历XCI的事实，我们假设XCI是建立自我更新和双功能的人类滋养层祖细胞所必需的。我们将通过分离未能完成XCI的hTSCs并评估其向特化滋养层细胞类型和器质分化的潜力来检验这一假设。我们还将对具有XO(Turner综合征)和XXY(Klinefelter综合征)基因的hPSC进行HTSC衍生，以评估X染色体剂量对HTSC衍生和滋养层类器官侵袭的影响。拟议的研究提供了一个独特的体外模型系统，在其中调查表观遗传重编程错误如何导致胚胎植入和胎盘发育障碍。

PROJECT SUMMARY/ABSTRACT During early development of female placental mammals one of the two X chromosomes is randomly inactivated in embryonic cells to equalize the expression of X-linked genes with males. While this process has been well-described in the context of embryonic differentiation, it remains poorly understood how dosage compensation of sex chromosomes is established in human extraembryonic tissues, including the placenta. The clinical significance of sex chromosome dosage compensation during early human development is underscored by the unexpectedly high prevalence of male babies born by in vitro fertilization (IVF), which has been attributed to defects in XCI in the trophoblast lineage. To enable dissection of early mechanisms of XCI during human trophoblast development, we propose to leverage "naive" human pluripotent stem cells (hPSCs), which exhibit transcriptional and epigenetic features of pluripotent cells in pre-implantation embryos, including the presence of two active X chromosomes in female cells. We have shown that naive hPSCs can readily differentiate into self-renewing human trophoblast stem cells (hTSCs), which can further differentiate into specialized trophoblast cell types and 3D trophoblast organoids. Here, we will combine these stem-cell-based 2D and 3D models of trophoblast development with epigenomic and single cell approaches to investigate mechanisms of XCI during human trophoblast differentiation. Aim 1 will define the kinetics and essential regulators of XCI during human trophoblast specification in vitro. Based on our preliminary studies using a biallelic X-linked reporter line and evidence from non-human primate embryos, we hypothesize that hTSC induction in naive hPSCs is accompanied by random XCI and precedes XCI in embryonic lineages. This random XCI pattern contrasts with imprinted inactivation of the paternal X chromosome in the extraembryonic membranes of mice, reinforcing the need for human-specific model systems. Aim 2 will investigate the impact of X chromosome dosage on hTSCs and trophoblast organoids derived from naive hPSCs. Based on the increased prevalence of male babies born by IVF procedures and the fact that the trophoblast lineage is the first to undergo XCI in non-human primate embryos, we hypothesize that XCI is required for establishing self-renewing and bipotent human trophoblast progenitors. We will test this hypothesis by isolating hTSCs that have failed to complete XCI and evaluating their differentiation potential into specialized trophoblast cell types and organoids. We will also perform hTSC derivation on naive hPSCs with XO (Turner syndrome) and XXY (Klinefelter syndrome) genotypes to assess the impact of X chromosome dosage on hTSC derivation and trophoblast organoid invasion in co-culture assays with human endometrial cells. The proposed studies offer a unique in vitro model system in which to investigate how errors in epigenetic reprogramming contribute to disturbances in embryo implantation and placental development.