Molecular Analysis of the Early Stages of Human Trophoblast Differentiation

项目总结（见说明）：项目II 很少有人知道的形成早期阶段的人滋养层（TB）的分化，特别是滋养外胚层（TE）的规范和形成的绒毛膜绒毛TB人口之间的步骤。最近，我们确定了早期妊娠人绒毛膜作为一个利基的细胞，共表达标记的多能性和结核病的命运决定因素。这一发现表明，绒毛膜是人类结核病祖细胞（TBPC）的来源。为了验证这一理论，我们从这种膜上建立了连续自我更新的TBPC系，并表明它们分化为成熟的人类TB群体-多核合胞体滋养层细胞和侵袭性细胞滋养层细胞。 在这里，我们建议使用这个新的细胞培养模型来分析TBPC分配，自我更新和分化。待测试的特定假设基于全局转录谱，其显示TBPC表达DNA结合蛋白的组合，所述DNA结合蛋白与人类TB发展有关或调节小鼠中TB或其他干细胞的命运。我们将测试 这些分子控制TB发育转变的假说是人类胎盘形成的关键组成部分（目的1）。这些结果将使我们对结核病分化的早期步骤有新的了解，这些步骤尚未在人类中进行研究。然后，我们将使用这些信息来研究先兆子痫（PE）的类似过程，这是一种人类妊娠并发症（例如，母体高血压、蛋白尿和水肿（胎儿宫内生长受限）与结核病分化不良有关。我们将检验这一假设，即观察到的缺陷可以部分地解释为控制TBPC命运的分子电路的扰动（目的2）。因此，这些实验解决了缺陷胎盘的分子基础，这有助于从不孕到PE的一系列疾病。 该项目显著受益于U-54的所有其他组件。通过项目III和核心B，我们将扩展TBPC的表征，包括microRNA分析和功能分析。在项目IV中，我们将探讨胎盘和蜕膜之间在蜕膜化基质分泌因子（子宫内膜异位症）方面的对话。我们将通过协助分析 卵母细胞分泌组和核心C通过提供专业知识的教育推广，我们获得了开发类似的计划。因此，这些协同相互作用扩展了本项目中提出的实验，并使我们能够研究导致不育的生殖生物学的其他重要方面。

PROJECT SUMMARY (See Instructions): PROJECT II Very little is known about the formative early stages of human trophoblast (TB) differentiation, in particular, the steps between trophectoderm (TE) specification and formation of the TB populations of the chorionic villi. Recently, we identified the early-gestation human chorion as a niche for cells that coexpressed markers of pluripotency and TB fate determinants. This finding suggested that the chorion is a source of human TB progenitor cells (TBPCs). To test this theory, we established lines of continuously self renewing TBPCs from this membrane and showed that they differentiated into the mature human TB populations¿multi-nucleated syncytiotrophoblasts and invasive cytotrophoblasts. Here we propose using this new cell culture model to analyze TBPC allocation, self-renewal and differentiation. The specific hypotheses to be tested are based on global transcriptional profiling, which showed that TBPCs expressed a combination of DNA binding proteins that have been implicated in human TB development or that regulate the fate of TBs or other stem cells in the mouse. Thus, we will test the hypothesis that these molecules control TB developmental transitions that are key components of human placentation (Aim 1). The results will give us new insights into early steps in TB differentiation that have yet to be studied in humans. Then we will use this information to study the analogous processes in preeclampsia (PE), a human pregnancy complication {e.g., maternal hypertension, proteinuria and edema ¿ intrauterine growth restriction) that is associated with faulty TB differentiation. We will test the hypothesis that the observed deficits can be explained, in part, by perturbations in the molecular circuitry that governs TBPC fate (Aim 2). Thus, these experiments address the molecular underpinnings of faulty placentation, which contributes to a spectrum of disorders ranging from infertility to PE. This project significantly benefits from all the other components of the U-54. With Project III and Core B we will expand our characterization of TBPCs to include microRNA profiling and functional analyses. With Project IV we will explore the dialogue between the placenta and the decidua in terms of decidualized stromal secreted factors (¿ endometriosis). We will contribute to Project I by assisting in the analysis of the oocyte secretome and to Core C by providing expertise in educational outreach that we gained by developing similar programs. Thus, these synergistic interactions expand on the experiments proposed in this project and allow us to study other important aspects of reproductive biology that contribute to infertility.