PROJECT SUMMARY/ABSTRACT The trophectoderm layer of the blastocyst stage embryo forms the epithelial cytotrophoblast (CTB), which gives rise to all trophoblast cell types in the placenta. A vital process during early human placental development is the differentiation of CTB to extravillous trophoblasts (EVTs), and subsequent invasion of uterine tissue by a subset of EVTs that have a mesenchymal phenotype. Abnormalities in trophoblast differentiation and invasion are im- plicated in placenta-related pathologies such as preeclampsia and abnormally invasive placenta. Yet, molecular mechanisms underlying CTB differentiation in the early gestation human placenta remain poorly understood due to restrictions on research with human embryos and fetal tissue, and significant differences between early pla- cental development in humans and animal models. The derivation of human trophoblast stem cells (hTSCs) from first trimester placentas and blastocyst stage embryos has provided a powerful tool for studying CTB differ- entiation to EVTs in vitro. However, current 2D culture systems do not provide insight into molecular mechanisms that regulate EVT differentiation of CTBs in vivo, even though they give rise to EVT cell types in vitro. Current systems do not incorporate critical cues for differentiation provided by the ECM, or they include non-physiological differentiation triggers that are not relevant in vivo. Since EVT differentiation involves both matrix degradation and cell migration, a 3D cell culture system incorporating ECM proteins found in vivo is necessary. We propose to construct synthetic matrices for use in a 3D cell culture model to investigate EVT differentiation of CTB. In conjunction with chemically defined media, these matrices will enable mechanistic studies on EVT differentiation of CTB in 3D culture. We will use human trophoblast stem cells (hTSCs) derived from primary placental samples to model the CTB. Two specific aims are proposed. In Aim 1, we will construct polymeric hydrogels based on gelatin methacryloyl (GelMA) with stiffness comparable to published values for the decidua basalis. We will in- vestigate the effect of incorporating laminin, fibronectin, collagen I, collagen IV, and combinations thereof in these matrices, on EVT differentiation and invasion in 3D culture. In Aim 2, we will develop a 3D co-culture system using synthetic matrices to investigate the effect of decidualized and non-decidualized endometrial stro- mal cells on EVT differentiation and invasion. In summary, our work addresses the critical need for defined in vitro experimental systems that provide a reasonable representation of in vivo physiology, and lays the founda- tion for future studies on molecular mechanisms underlying trophoblast differentiation and invasion.