Generating in vitro models of trophectoderm formation

项目摘要/摘要 人类妊娠始于囊胚期滋养外胚层之间的特定相互作用 胚胎和子宫内膜上皮，然后胚胎侵入上皮。随后， 滋养外胚层形成胎盘的所有滋养层细胞类型。胚胎植入中的异常有 与许多妊娠并发症有关，包括流产、先兆子痫和胎儿生长受限。 然而，由于伦理和伦理的原因，胚胎植入的分子机制仍然知之甚少。 对人类胚胎研究的法律限制，以及植入方面的重大机制差异 在普通的啮齿动物模型和人类之间。可以说，机械论研究的最大障碍 关于胚胎植入的问题是缺乏伦理上合理的和实验上可靠的模型来准确模拟 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 人和非人灵长类多能干细胞的滋养外胚层。非人灵长类动物模型是 对人类滋养外胚层的研究很有吸引力，因为它们具有很高的遗传相似性。体外模型的建立 非人灵长类动物将使细胞培养和基因敲除研究成为可能，而这在猴子身上是很难进行的 胚胎，由于伦理和法律的限制，人类胚胎是不可能的。此外，比较 多能干细胞来源的滋养外胚层的人和非人灵长类动物模型分析 提供对人类胚泡形成和胚胎植入的机械洞察力。中心前提 我们建议的工作之一是可以用来建立猴子滋养外胚层形成的体外模型 多能干细胞。具体地说，我们假设我们可以从猕猴滋养层干细胞 猕猴多能干细胞，类似于之前对人类多能干细胞的研究。在目标1中，我们将 猕猴多能干细胞分化为CDX2+和CDX2-滋养层干细胞的研究 细胞。小鼠滋养层干细胞与多能干细胞共培养形成囊胚样结构 叫做胚泡。然而，类似的探索人类胚泡形成的研究在伦理上和法律上都是合法的。 有问题。在目标2中，我们将研究猕猴多能干细胞产生囊胚的过程。我们 还将研究人类和人类的空囊胚样结构的产生 猕猴的多能干细胞，最后，利用人类和猕猴的体外模型，我们将审问其作用 CDX2在滋养外胚层中的表达。总体而言，我们的研究解决了对可重现的、 人类滋养外胚层的实验可及且伦理合理的模型。体外模型 这里提出的建议将补充在非人类灵长类动物体内的研究，这些研究受到成本和复杂性的限制， 并使人类滋养外胚层形成和胚胎植入的机制研究成为可能。

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.