Generating in vitro models of trophectoderm formation

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PROJECT SUMMARY/ABSTRACT Human pregnancy begins with specific interactions between the trophectoderm layer of the blastocyst-stage embryo and the endometrial epithelium, followed by invasion of the epithelium by the embryo. Subsequently, the trophectoderm gives rise to all trophoblast cell types of the placenta. Abnormalities in embryo implantation are associated with many pregnancy complications including miscarriage, preeclampsia and fetal growth restriction. Yet, the molecular mechanisms underlying embryo implantation remain poorly understood due to ethical and legal constraints on research with human embryos, and significant mechanistic differences in implantation between common rodent models and humans. Arguably, the most significant impediment to mechanistic studies on embryo implantation is the lack of ethically sound and experimentally robust models that accurately mimic the human trophectoderm. To overcome this challenge, we propose to develop in vitro models of the trophectoderm from human and nonhuman primate pluripotent stem cells. Nonhuman primate models are attractive for studies on the human trophectoderm due to their high genetic similarity. In vitro models in nonhuman primates will enable cell culture and genetic knockout studies that are difficult to conduct with monkey embryos, and not possible with human embryos due to ethical and legal constraints. Furthermore, comparative analysis of human and nonhuman primate models of the trophectoderm derived from pluripotent stem cells will provide mechanistic insight into blastocyst formation and embryo implantation in humans. The central premise of our proposed work is that in vitro models for monkey trophectoderm formation can be generated using pluripotent stem cells. Specifically, we hypothesize that we can derive macaque trophoblast stem cells from macaque pluripotent stem cells, similar to previous studies with human pluripotent stem cells. In Aim 1, we will investigate the generation of CDX2+ and CDX2- macaque trophoblast stem cells from macaque pluripotent stem cells. Co-culture of mouse trophoblast stem cells with pluripotent stem cells generates blastocyst-like structures called blastoids. However, similar studies to explore formation of human blastoids is ethically and legally questionable. In Aim 2, we will investigate the generation of blastoids from macaque pluripotent stem cells. We will also investigate the generation of empty blastocyst like structures called trophospheres from human and macaque pluripotent stem cells, Finally, using human and macaque in vitro models, we will interrogate the role of CDX2 in trophectoderm formation. Overall, our research addresses the critical need for reproducible, experimentally accessible, and ethically sound models for the human trophectoderm. The in vitro models proposed herein will complement in vivo studies in nonhuman primates that are limited by cost and complexity, and enable mechanistic studies on human trophectoderm formation and embryo implantation.