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

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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.