Controlled generation of human embryoids using optogenetics

项目摘要 了解人类胚胎发育对改进辅助生殖有巨大影响 技术，以干细胞为基础的再生疗法，以及预防遗传出生缺陷和 畸形症。然而，由于极端物种的存在，我们对人类胚胎发育的了解有限。 人类与其他常用哺乳动物模型之间的差异以及有限的可及性 用于研究目的的人类/非人类灵长类胚胎组织样本。为了克服这些困难， 基于干细胞的体外培养方法，允许多细胞结构的自发发育 简化实验系统和提高高质量实时成像的可及性具有巨大的意义 价值。尽管有很大的希望，但现有文化体系的内在差异显著限制了他们的 应用于基础力学研究和临床应用。光遗传方法利用 控制修饰细胞或组织生物功能的基因工程方法和光学技术 表达光敏蛋白。光遗传学的精确时空分辨率，加上 经过适当设计的照明系统，可以提供强大而通用的解决方案来改善 干细胞人类发育模型的可控性和重复性。 我们的初步研究表明，外源性BMP4刺激有效地驱动了人类的多能性 干细胞(HPSCs)分化为羊膜外胚层样细胞(AMLCs)；定位外源性BMP 对hPSC聚集体的刺激会产生类似人类胚胎的结构(或类胚胎)。在本建议中 研究，我们将进行一项探索性、高风险但高回报的研究，以证明 光遗传学和干细胞来源的人类胚胎模型。具体地说，我们将得到光遗传hPSC线 应用光骨形成蛋白系统，检测其细胞内BMP活性和羊膜分化潜能 在光照下。然后，将利用完全表征的光骨形成蛋白-hPSCs来产生第一个此类细胞， 光遗传人类胚胎系统。 这项研究如果成功，将第一次展示人类成功的一代 具有发育信号的光遗传扰动的胚状体。这一点非常重要，因为有至关重要的 现有胚状体培养系统中普遍存在的问题：缺乏重复性和可控性，以及 对此，我们缺乏对引导它们发展和自我组织的过程的了解。 将光遗传学工具整合到现有的胚胎培养中将为实验者提供精确的 对关键发展信号的时空控制。改进的重复性和实验性 一致性将极大地促进有意义的机械论研究，以了解其背后的机制 人类胚胎在正常和病理情况下的发育，并将其作为遗传的靶点 操纵或药物测试。

Project Summary Understanding human embryonic development has tremendous impact on improving assisted reproductive technologies, stem cell-based regenerative therapies, and the prevention of genetic birth defects and teratogenesis. However, our knowledge about human embryonic development is limited due to drastic species divergences between humans and other commonly used mammalian models and limited accessibility to human/non-human primate embryonic tissue samples for research purposes. To overcome these difficulties, stem cell-based in vitro culture methods that allow spontaneous development of multicellular structures while simplifying the experimental system and improving accessibility for high-quality live imaging are of tremendous value. Despite the great promise, the inherent variations of existing culture systems significantly limit their applications for fundamental mechanistic research and clinical applications. Optogenetic approaches utilize genetic engineering methods and optical technologies to control biological functions of cells or tissues modified to express photosensitive proteins. The precise spatiotemporal resolution of optogenetics, coupled with properly engineered illumination systems, can offer a powerful and versatile solution to improve the controllability and reproducibility of stem-cell derived human development models. Our preliminary studies reveal that exogenous BMP4 stimulation efficiently drives human pluripotent stem cells (hPSCs) to differentiate into amniotic ectoderm-like cells (AMLCs); and localized exogenous BMP stimulation to hPSC aggregates generates human embryonic-like structures (or embryoids). In this proposed research, we will pursue an exploratory, high-risk but high-reward study to demonstrate the integration of optogenetics and stem cell-derived human embryo models. Specifically, we will derive optogenetic hPSC lines using an optoBMP system; and examine their intracellular BMP activities and amniotic differentiation potentials under light exposure. Fully characterized optoBMP-hPSCs will then be utilized to generate, the first of its kind, optogenetic human embryoid system. This research, if successful, will demonstrate, for the first time, the successful generation of human embryoids with optogenetic perturbation of developmental signals. This is very significant, as there are crucial issues pervading in existing embryoid culture systems: a lack of reproducibility and controllability and, coupled to this, our lack of understanding of the processes that guide their development and self-organization. Incorporating optogenetic tools into existing embryoid cultures will offer the experimenter precise spatiotemporal control over the key development signals. The improved reproducibility and experimental consistency will greatly facilitate meaningful mechanistic studies to understand mechanisms that underlie human embryonic development in normal and pathological situations, and to use them as targets for genetic manipulation or drug testing.