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

项目概要/摘要 人类胎盘在维持胎儿生长的适当环境方面起着重要作用，但仍然是一个重要的环境。 了解不多的器官，特别是在妊娠早期。在过去的几年里，单细胞的进展 分析和干细胞衍生终于应用于这个器官，导致显着扩大， 细胞异质性的知识，以及基于细胞的建模，滋养层，上皮细胞， 胎盘具体来说，多个研究小组已经建立了滋养层干细胞的衍生方案， (TSC)和滋养层类器官从早期妊娠的人胎盘，这使得分化的研究 分为功能性合体滋养细胞（STB）和绒毛外滋养细胞（EVT）。其他组织，包括我们自己的， 已经建立了将人类多能干细胞（hPSC）转化为真正的 TSC，允许正常和异常滋养层分化的建模。然而，最近的数据 表明原代TSC具有比最初描述更有限的分化潜能， 这与EVT的前体最一致，而不是真正的双电位“TSC”。事实上，虽然人类 滋养外胚层（TE）细胞和早期妊娠绒毛CTB（vCTB）共表达TP 63和CDX 2，原代TSC 缺乏CDX 2，而hPSC衍生的TSC在从TE向TSC的转变过程中丢失CDX 2。此外，胎盘 受21三体（T21）影响的妊娠显示出持续的vCTB层， 保留CDX 2表达超过第一个三个月;这是伴随着T21-CTB的异常，形成 体外STB，我们使用T21影响的hPSC重现的表型。我们假设CDX 2 + vCTB代表一种独特的滋养层祖细胞状态，可能更原始，和/或已改变 分化潜能本申请的目的是确定CDX 2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 与T21影响的胎盘中异常持续存在相关的细胞缺陷， 伴随胎盘功能障碍为此，我们建议使用CTB、TSC和 滋养层类器官，直接来源于正常和T21受影响的胎盘或多能干细胞，沿着 利用最先进的单细胞/单核转录组分析和互补功能测定， 实现这些目标。成功完成此提案将提供一个全面的分子 妊娠早期胎盘细胞滋养层异质性的功能评估， 不同的体外人类滋养层模型系统的能力和局限性，并提供见解 研究受21三体影响的妊娠中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.