Generating in vitro models of trophectoderm formation

生成滋养外胚层形成的体外模型

• 批准号: 10468196
• 负责人: Balaji M Rao
• 金额: $ 19.06万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-11 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10468196
• 关键词: Address; Area; Behavior; CDX2 gene; Cell Culture Techniques; Coculture Techniques; Complement; Derivation procedure; Development; Embryo; Endometrial; Epithelial; Ethics; Fetal Growth; Fetal Growth Retardation; First Pregnancy Trimester; Generations; Genetic; Herpes zoster disease; Human; Knock-out; Legal; Macaca; Mediating; Modeling; Molecular; Monkeys; Mus; Oocytes; Placenta; Pluripotent Stem Cells; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Reproducibility; Research; Research Design; Rodent Model; Role; Spontaneous abortion; Structure; Villous; Work; blastocyst; cell type; comparative; cost; cytotrophoblast; embryonic stem cell; human model; human pluripotent stem cell; implantation; improved; in vitro Model; in vivo; insight; knock-down; natural Blastocyst Implantation; nonhuman primate; novel therapeutics; sound; stem cell differentiation; stem cells; trophoblast; trophoblast stem cell

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.

项目概要/摘要 人类妊娠始于囊胚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。胚胎着床异常是 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，由于伦理和道德问题，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显着机制差异 常见啮齿动物模型和人类之间的关系。可以说，机械研究的最大障碍 胚胎植入的问题在于缺乏能够准确模仿的道德合理且实验稳健的模型 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 来自人类和非人类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 由于其高度的遗传相似性，对人类滋养外胚层的研究很有吸引力。体外模型 非人类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，不可能用人类胚胎来实现。此外，比较 对源自多能干细胞的人类和非人类灵长类动物滋养外胚层模型的分析将 提供对人类囊胚形成和胚胎植入的机制见解。中心前提 我们提出的工作之一是可以使用以下方法生成猴滋养外胚层形成的体外模型 多能干细胞。具体来说，我们假设我们可以从猕猴滋养层干细胞中获得 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 研究从猕猴多能干细胞中产生 CDX2+ 和 CDX2- 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囊胚样结构 称为胚泡。然而，探索人类母细胞形成的类似研究在伦理和法律上都是不可接受的。 有疑问。在目标 2 中，我们将研究猕猴多能干细胞的母细胞生成。我们 还将研究人类和人类的空囊胚样结构（称为滋养层）的生成 猕猴多能干细胞，最后，利用人和猕猴的体外模型，我们将探讨其作用 CDX2 在滋养外胚层形成中的作用。总的来说，我们的研究解决了可重复、 实验上可行且符合伦理道德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类灵长类动物体内研究， 并能够对人类滋养外胚层形成和胚胎植入进行机制研究。