Synthetic matrices for studies on trophoblast differentiation in 3D culture

项目总结/摘要 胚泡期胚胎的滋养外胚层形成上皮细胞滋养层（CTB）， 上升到胎盘中的所有滋养层细胞类型。人类胎盘早期发育的一个重要过程是 CTB分化为绒毛外滋养层细胞（EVT），随后由一个亚群侵入子宫组织， 具有间充质表型的EVT。滋养细胞分化和侵袭的异常是不正常的。 在胎盘相关的病理学如先兆子痫和异常侵入性胎盘中复杂。然而，分子 妊娠早期人类胎盘中CTB分化的潜在机制仍然知之甚少， 对人类胚胎和胎儿组织研究的限制，以及早期胚胎和胎儿组织之间的显著差异， 人类和动物模型的中枢发育。人滋养层干细胞（hTSCs）的来源 从妊娠早期胎盘和胚泡阶段胚胎中提取的DNA为研究CTB差异提供了有力的工具， 体外诱导EVT。然而，目前的2D培养系统不能提供对分子机制的深入了解 其在体内调节CTB的EVT分化，即使它们在体外产生EVT细胞类型。电流 系统不包括ECM提供的分化的关键线索，或者它们包括非生理性的 在体内不相关的分化触发物。由于EVT分化涉及基质降解 和细胞迁移，因此，结合体内发现的ECM蛋白的3D细胞培养系统是必要的。我们提出 构建用于3D细胞培养模型的合成基质，以研究CTB的EVT分化。在 结合化学成分确定的培养基，这些基质将使EVT分化的机制研究成为可能 CTB在3D文化中的作用我们将使用来自原代胎盘样本的人滋养层干细胞（hTSCs） 来模拟CTB提出了两个具体目标。在目标1中，我们将构建基于以下的聚合物水凝胶： 甲基丙烯酰明胶（GelMA），其硬度与已发表的底蜕膜值相当。我们会- 研究将层粘连蛋白、纤连蛋白、胶原蛋白I、胶原蛋白IV及其组合掺入 这些基质，在EVT分化和3D培养中的侵袭。在目标2中，我们将开发3D共培养 系统使用合成矩阵来研究蜕膜化和非蜕膜化子宫内膜stroke的影响， mal细胞对EVT分化和侵袭的影响。总之，我们的工作解决了在 体外实验系统，提供了体内生理学的合理代表，并奠定了基础， 为进一步研究滋养层细胞分化和侵袭的分子机制提供参考。

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.