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.

项目摘要/摘要在女性胎盘哺乳动物的早期发育过程中，两种X染色体之一在胚胎细胞中随机灭活，以均衡X连锁基因与雄性的表达。尽管该过程在胚胎分化的背景下被很好地描述了，但它仍然不足以了解性染色体的剂量补偿是在包括胎盘在内的人类外囊性组织中建立的。人类早期发育期间性染色体剂量补偿的临床意义突显了由体外受精（IVF）出生的男性婴儿出乎意料的高患病率（IVF），这已归因于滋养细胞中的XCI缺陷。为了解剖人类滋养细胞发育过程中XCI的早期机制，我们建议利用“天真”的人类多能干细胞（HPSC），这些干细胞（HPSC）在胚胎前胚胎前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跃的X染色体。我们已经表明，天真的HPSC可以很容易地分化为自我更新的人滋养细胞干细胞（HTSC），这些干细胞可以进一步分化为专业的滋养细胞类型和3D滋养细胞类器官。在这里，我们将结合这些基于干细胞的滋养细胞发育的2D和3D模型与表观基因组和单细胞方法，以研究人类滋养细胞分化过程中XCI的机制。 AIM 1将在体外人类滋养细胞规范期间定义XCI的动力学和基本调节剂。基于我们的初步研究，使用双重X连锁的记者系和非人类灵长类动物胚胎的证据，我们假设幼稚的HPSC中的HTSC诱导伴随着随机XCI，并且先于胚胎谱系中的XCI。这种随机的XCI模式与小鼠外胚膜膜中父亲X染色体的灭绝形成对比，从而增强了对人类特异性模型系统的需求。 AIM 2将研究X染色体剂量对源自幼稚HPSC的HTSC和滋养细胞器官的影响。基于IVF手术所生的男性婴儿的患病率的增加，以及滋养细胞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核滋养肉体滋养型祖细胞所必需的。我们将通过隔离未完成XCI并评估其分化潜力的HTSC来检验这一假设，并将其分化潜力评估为专门的滋养细胞类型和类器官。我们还将在XO（Turner综合征）和XXY（KlineFelter综合征）基因型上对NAIVE HPSC进行HTSC推导，以评估X染色体剂量对HTSC衍生物和滋养细胞器官侵袭与人内膜细胞的X染色体剂量对HTSC衍生物和滋养细胞器官侵袭的影响。拟议的研究提供了一种独特的体外模型系统，在其中研究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干扰。