Mechanisms of X chromosome inactivation during human trophoblast differentiation in vitro

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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.