Generating in vitro models of trophectoderm formation

项目总结/摘要 人类妊娠开始于胚泡阶段滋养外胚层之间的特定相互作用 胚胎和子宫内膜上皮，随后由胚胎侵入上皮。随后 滋养外胚层产生胎盘的所有滋养层细胞类型。胚胎植入的主要障碍是 与许多妊娠并发症相关，包括流产、先兆子痫和胎儿生长受限。 然而，胚胎植入的分子机制仍然知之甚少，由于伦理和 对人类胚胎研究的法律的限制，以及植入的显著机制差异 和人类之间的差异。可以说，机械研究的最大障碍 缺乏道德上合理的和实验上可靠的模型来准确地模拟 人类滋养外胚层为了克服这一挑战，我们建议开发体外模型， 来自人和非人灵长类多能干细胞的滋养外胚层。非人类灵长类动物模型是 由于它们的高度遗传相似性，对人类滋养外胚层的研究很有吸引力。体外模型 非人灵长类动物将使细胞培养和基因敲除研究成为可能，而这些研究在猴子身上很难进行 由于伦理和法律的限制，人类胚胎是不可能的。此外，比较 对来源于多能干细胞的滋养外胚层的人和非人灵长类动物模型的分析将 为人类胚泡形成和胚胎植入提供了机制性的见解。中心前提 我们提出的工作的一个重要方面是，可以使用 多能干细胞具体来说，我们假设我们可以从哺乳动物中获得猕猴滋养层干细胞。 猕猴多能干细胞，类似于以前对人类多能干细胞的研究。在目标1中，我们 研究从猕猴多能干细胞产生CDX 2+和CDX 2-猕猴滋养层干细胞 细胞小鼠滋养层干细胞与多能干细胞共培养产生胚泡样结构 叫做类胚体。然而，探索人类类胚体形成的类似研究在伦理和法律上是不合理的。 可疑的。在目标2中，我们将研究从猕猴多能干细胞产生类胚体。我们 还将研究人类的空胚泡样结构（称为滋养球）的产生， 最后，利用人和猕猴的体外模型，我们将审问多能干细胞的作用 CDX 2在滋养外胚层形成中的作用。总的来说，我们的研究解决了对可重复的， 实验上可行，道德上合理的人类滋养外胚层模型。体外模型 本文提出的方法将补充受成本和复杂性限制的非人灵长类动物体内研究， 并且能够对人滋养外胚层形成和胚胎植入进行机理研究。

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.