Project Summary/Abstract Little is known about structure-function relationships in the human placenta. As pathologists, we can view the placenta only after delivery, and correlate our findings to clinical and prenatal course, as well as neonatal outcome. At best, however, we have lesions which correlate with disease, but have yet to be validated, either directly, through molecular analysis, or indirectly, through manipulation and functional analysis of in vitro models. Placental dysfunction, manifested clinically as preeclampsia (PE) with or without fetal growth restriction (FGR), is associated with histopathologic lesions of maternal vascular malperfusion (MVM) and fetal vascular malperfusion (FVM). These lesions differentially affect the three trophoblast compartments: cytotrophoblast (CTB, the putative stem cell), syncytiotrophoblast (STB, the cell type responsible for gas/nutrient exchange), and extravillous trophoblast (EVT, the invasive cell type at the maternal-fetal interface). Nevertheless, our understanding of these lesions is limited to morphology and immunolocalization of a few markers. Over the past few years, multiple groups, including ours, have used scRNAseq to characterize cellular heterogeneity within both normal and diseased placentae; however, these studies remain mostly descriptive, lacking both spatial context of the molecular data and functional evaluation of the distinct cell types. We therefore propose to apply novel technologies, including digital spatial profiling (DSP) and tissue decellularization followed by extracellular matrix (ECM)-specific mass spectrometry, as discovery-based approaches to better characterize specific MVM and FVM lesions at the molecular level. We will then use primary term CTB, to reproducibly model these phenotypes and to perform functional validation as part of a targeted evaluation approach. We will test the hypothesis that late gestation CTB respond to specific cell- and ECM-derived signals to proliferate and/or differentiate into either STB or EVT, thus identifying potential regenerative capacity in this unique transient organ. We will also probe the cellular and ECM origins of PE-associated placental dysfunction, testing the hypothesis that this disease originates from alterations in ECM composition and paracrine signaling from both placental and decidual (maternal) cells. Successful completion of this proposal will establish a detailed cellular and matrix atlas of the human placenta, with validated structure-function relationships, laying the groundwork for probing placental regenerative capacity in the setting of placental dysfunction.