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Mechanisms of X chromosome inactivation during human trophoblast differentiation in vitro

人滋养层体外分化过程中X染色体失活的机制

基本信息

批准号：
10727675
负责人：
Thorold Theunissen
金额：
$ 42.76万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-10 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10727675
关键词：
3-Dimensional Alleles Biological Assay Biological Models Biological Process CRISPR/Cas technology Cells Clonal Expansion Coculture Techniques Data Defect Derivation procedure Development Dissection Dosage Compensation (Genetics)Embryo Endometrial Epigenetic Process Epithelial Cells Event Exhibits Female Fertilization in Vitro Fluorescent in Situ Hybridization Generations Genes Genetic Genetic Transcription Genotype High Prevalence Human Human Development Immunofluorescence Immunologic Impairment In Vitro Inner Cell Mass Invaded Investigation Kinetics Klinefelter&apos s Syndrome Link Mediating Molecular Mus National Institute of Child Health and Human Development Organoids Pattern Phenotype Placenta Placentation Population Prevalence Procedures Process RNA Reporter Reporting Sex Chromosomes Sex Ratio Specific qualifier value Syncytiotrophoblast Testing Tissues Trees Turner&apos s Syndrome Villous X Chromosome X Inactivation Yolk Sac blastocyst blastomere structure cell type clinically significant dosage early pregnancy embryo cell embryo membrane embryo tissue epigenomics genome editing histone modification human model human pluripotent stem cell human stem cells implantation imprint in utero in vitro Model insight interest male natural Blastocyst Implantation nonhuman primate novel placental mammal progenitor self-renewal sex chromosome aneuploidy stem cell based approach stem cell model stem cells three-dimensional modeling transcriptomics trophoblast trophoblast stem cell

项目摘要

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.

项目概要/摘要在雌性胎盘哺乳动物的早期发育过程中，两条 X 染色体之一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，但人们对性染色体的剂量补偿是如何在人类胚胎外组织（包括胎盘）中建立的知之甚少。体外受精 (IVF) 出生的男婴的患病率出人意料地高，这突显了人类早期发育过程中性染色体剂量补偿的临床意义，这归因于滋养层谱系中 XCI 的缺陷。为了能够剖析人类滋养层发育过程中 XCI 的早期机制，我们建议利用“原始”人类多能干细胞 (hPSC)，它在植入前胚胎中表现出多能细胞的转录和表观遗传特征，包括雌性细胞中存在两条活性 X 染色体。我们已经证明，初始 hPSC 可以很容易地分化为自我更新的人类滋养层干细胞 (hTSC)，后者可以进一步分化为专门的滋养层细胞类型和 3D 滋养层类器官。在这里，我们将把这些基于干细胞的滋养层发育的 2D 和 3D 模型与表观基因组和单细胞方法结合起来，研究人类滋养层分化过程中 XCI 的机制。目标 1 将定义 XCI 在体外人类滋养层规范过程中的动力学和基本调节因子。根据我们使用双等位基因 X 连锁报告系的初步研究以及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，并且先于胚胎谱系中的 XCI。这种随机 XCI 模式与小鼠胚胎外膜中父本 X 染色体的印记失活形成对比，增强了对人类特异性模型系统的需求。目标 2 将研究 X 染色体剂量对 hTSC 和源自初始 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，以及滋养层谱系是第一个在非人类灵长类动物胚胎中经历 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 hTSC 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检验这一假设。我们还将对具有 XO（特纳综合征）和 XXY（克兰费尔特综合征）基因型的初始 hPSC 进行 hTSC 衍生，以评估在与人子宫内膜细胞共培养测定中 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，用于研究表观遗传重编程的错误如何导致胚胎植入和胎盘发育的干扰。