Trophoblast progenitor heterogeneity and function in normal and Trisomy 21-affected placentae

项目摘要/摘要 人类胎盘在维持胎儿生长的适当环境中起着重要作用，但仍是一种 知之甚少的器官，特别是在怀孕早期。在过去的几年里，单细胞的进展 分析和干细胞提取最终被应用于这个器官，导致了显著的扩张，两者 了解细胞异质性以及基于细胞的建模，滋养层细胞，滋养层上皮细胞 胎盘。具体地说，多个小组已经建立了滋养层干细胞的衍生方案 (TSC)和滋养层细胞类器官，这为分化研究提供了可能 分为功能性合体滋养细胞(STB)和绒毛外滋养细胞(EVT)。其他团体，包括我们自己的， 已经建立了将人类多能干细胞(HPSC)转化为真性干细胞的可重复使用的方案 TSC，允许对正常和异常滋养细胞分化进行建模。然而，最近的数据 提示初级TSC的分化潜力比最初描述的更有限，并有一个 这与EVT的前驱最为一致，而不是真正的双潜能“TSC”。事实上，虽然人类 滋养外胚层细胞和早孕绒毛CTB共表达TP63和CDX2，原发TSC 缺乏CDX2，hPSC来源的TSC在从TE向TSC的转变过程中丢失CDX2。此外，胎盘 来自21三体(T21)受影响的妊娠显示持续的vCTB层，有相当大的比例 在妊娠早期保留CDX2的表达；伴随T21-CTB的异常而形成 STB在体外，这是我们用T21影响的hPSC概括的一个表型。我们假设CDX2 VCTB代表一种独特的滋养层祖细胞状态，这种状态可能更原始，和/或已经改变 分化潜力。本申请的目的是识别CDX2在TE建立中的作用， 维持滋养层祖细胞状态和TSC分化潜能，并表征 T21感染的胎盘中与其异常持续相关的细胞缺陷和 伴发胎盘功能障碍。为此，我们建议使用CTB、TSC和 滋养层类器官，直接来自正常和T21感染的胎盘或多能干细胞，沿着 利用最先进的单细胞/单核转录组图谱和互补功能分析 实现这些目标。这项提议的成功完成将提供一个全面的分子 和早孕胎盘细胞滋养细胞异质性的功能评估，确定 不同体外人滋养细胞模型系统的能力和局限性，并提供见解 探讨21三体影响妊娠胎盘功能障碍的机制。

Project Summary/Abstract The human placenta plays a major role in maintaining the proper environment for fetal growth, but remains a poorly-understood organ, particular during early gestation. Over the past few years, advances in single cell analysis and stem cell derivation have finally been applied to this organ, resulting in significant expansion, both of knowledge of cellular heterogeneity as well as of cell-based modeling, of trophoblast, the epithelial cells of the placenta. Specifically, multiple groups have established protocols for derivation of trophoblast stem cells (TSC) and trophoblast organoids from early gestation human placenta, which allow for study of differentiation into functional syncytiotrophoblast (STB) and extravillous trophoblast (EVT). Other groups, including our own, have established reproducible protocols for conversion of human pluripotent stem cells (hPSC) into bona fide TSC, allowing for modeling of both normal and abnormal trophoblast differentiation. However, recent data suggest that primary TSC have a more limited differentiation potential than originally described, with a profile that is most consistent with precursors to EVT, rather than a truly bipotential “TSC.” In fact, while human trophectoderm (TE) cells and early gestation villous CTB (vCTB) co-express TP63 and CDX2, primary TSC lack CDX2, and hPSC-derived TSC lose CDX2 during the transition from TE to TSC. In addition, placentas from Trisomy 21 (T21)-affected pregnancies show a persistent vCTB layer, with a significant proportion retaining CDX2 expression beyond first trimester; this is accompanied by an abnormality of T21-CTB to form STB in vitro, a phenotype which we have recapitulated using T21-affected hPSC. We hypothesize that CDX2+ vCTB represents a distinct trophoblast progenitor state, one that is possibly more primitive, and/or has altered differentiation potential. The goal of this application is to identify the role of CDX2 in establishment of TE, maintenance of a trophoblast progenitor state, and TSC differentiation potential, and characterize the cellular defects associated with its abnormal persistence in T21-affected placentae and the accompanying placental dysfunction. To this end, we propose to use a combination of CTB, TSC, and trophoblast organoids, derived directly from normal and T21-affected placentas or pluripotent stem cells, along with state-of-the-art single cell/single nucleus transcriptome profiling and complementary functional assays to address these goals. Successful completion of this proposal will provide a comprehensive molecular and functional assessment of cytotrophoblast heterogeneity in early gestation placenta, identify capacities and limitations of different in vitro human trophoblast model systems, and offer insights into mechanisms of placental dysfunction in pregnancies affected by Trisomy 21.