Controlled generation of human embryoids using optogenetics

项目摘要 了解人类胚胎发育对改善辅助生殖有巨大影响 技术、干细胞再生疗法和预防遗传性出生缺陷， 致畸作用然而，我们对人类胚胎发育的认识是有限的，由于激烈的物种 人类与其他常用哺乳动物模型之间存在分歧，并且可及性有限 用于研究目的的人类/非人类灵长类动物胚胎组织样本。为了克服这些困难， 基于干细胞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是巨大的 值尽管有很大的希望，但现有文化体系的固有差异极大地限制了它们的发展。 应用于基础机理研究和临床应用。光遗传学方法利用 基因工程方法和光学技术来控制细胞或组织的生物学功能， 来表达光敏蛋白。光遗传学精确的时空分辨率， 适当设计的照明系统，可以提供一个强大的和多功能的解决方案，以改善 干细胞衍生的人类发育模型的可控性和再现性。 我们的初步研究表明，外源性BMP 4刺激有效地驱动人类多能性 干细胞（hPSC）分化为羊膜外胚层样细胞（AMLC）;和局部外源性BMP 对hPSC聚集体的刺激产生人胚胎样结构（或胚状体）。在此提出的 我们将进行一项探索性、高风险但高回报的研究，以证明 光遗传学和干细胞衍生的人类胚胎模型。具体地，我们将获得光遗传学hPSC系， 用optoBMP系统检测细胞内BMP活性和羊膜分化潜能 在光照下。然后将利用完全表征的optoBMP-hPSC来产生，这是同类中的第一个， 人胚状体系统 这项研究如果成功，将首次证明人类的成功一代。 具有发育信号的光遗传学扰动的胚状体。这一点非常重要，因为 在现有的胚状体培养系统中普遍存在的问题：缺乏再现性和可控性， 这是因为我们对指导它们发展和自我组织的过程缺乏了解。 将光遗传学工具应用到现有的胚状体培养物中将为实验者提供精确的 对关键发展信号的时空控制。改进的再现性和实验性 一致性将极大地促进有意义的机制研究，以了解 在正常和病理情况下的人类胚胎发育，并将其用作遗传学的靶点， 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.