Mechanisms of X chromosome inactivation during human trophoblast differentiation in vitro

项目总结/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随机失活，以使X连锁基因的表达与雄性相同。虽然这一过程在胚胎分化的背景下得到了很好的描述，但对性染色体的剂量补偿如何在人类胚外组织（包括胎盘）中建立仍知之甚少。通过体外受精（IVF）出生的男性婴儿的意外高患病率强调了性染色体剂量补偿在早期人类发育期间的临床意义，这归因于滋养层谱系中XCI的缺陷。为了能够在人类滋养层发育期间解剖XCI的早期机制，我们提出利用"幼稚"人类多能干细胞（hPSC），其在植入前胚胎中表现出多能细胞的转录和表观遗传特征，包括在雌性细胞中存在两个活性X染色体。我们已经表明，幼稚hPSC可以容易地分化成自我更新的人滋养层干细胞（hTSC），其可以进一步分化成专门的滋养层细胞类型和3D滋养层类器官。在这里，我们将结合联合收割机这些干细胞为基础的2D和3D模型的滋养层发育与表观基因组和单细胞的方法来研究机制XCI在人类滋养层分化。目的1将明确XCI在体外培养人滋养层细胞特化过程中的动力学和重要调控因子。基于我们使用双等位基因X连锁报告基因系的初步研究和来自非人灵长类动物胚胎的证据，我们假设幼稚hPSC中的hTSC诱导伴随着随机XCI并且在胚胎谱系中先于XCI。这种随机XCI模式与小鼠胚外膜中父系X染色体的印记失活形成对比，加强了对人类特异性模型系统的需求。目的2将研究X染色体剂量对hTSC和来源于幼稚hPSC的滋养层类器官的影响。基于通过IVF程序出生的男性婴儿的患病率增加以及滋养层谱系在非人灵长类动物胚胎中首先经历XCI的事实，我们假设XCI是建立自我更新和双能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并评估其分化为专门的滋养层细胞类型和类器官的潜力来测试这一假设。我们还将在具有XO（特纳综合征）和XXY（克兰费尔特综合征）基因型的幼稚hPSC上进行hTSC衍生，以评估X染色体剂量对与人子宫内膜细胞共培养测定中的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，其中调查表观遗传重编程中的错误如何导致胚胎植入和胎盘发育的障碍。

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.