Controlled generation of human embryoids using optogenetics

利用光遗传学控制人类胚胎的产生

• 批准号: 10505751
• 负责人: Jianping Fu
• 金额: $ 18.22万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-08 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10505751
• 关键词: Agonist; Architecture; Assisted Reproductive Technology; Biological Process; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell physiology; Cells; Clinical; Congenital Abnormality; Coupled; Data Set; Development; Diagnosis; Ectoderm; Embryo; Embryonic Development; Emerging Technologies; Engineering; Environment; Event; Generations; Genes; Genetic; Genetic Engineering; Goals; Human; Human Development; Image; In Vitro; Knowledge; Lead; Ligands; Light; Light Cell; Lighting; Masks; Mathematics; Measurement; Methods; Microfluidic Microchips; Microfluidics; Modeling; Molecular; Optics; Pathologic; Pattern; Photosensitivity; Physiological; Prevention; Process; Property; Proteins; Protocols documentation; Publishing; Reproducibility; Research; Research Personnel; Resolution; Signal Pathway; Signal Transduction; Source; Stem Cell Development; Structure; System; Techniques; Technology; Time; Tissue Sample; Tissues; Variant; antagonist; base; bone morphogenetic protein 4; clinical application; drug testing; embryo tissue; fascinate; genetic manipulation; high reward; high risk; human pluripotent stem cell; improved; in vitro Model; in vivo; model development; morphogens; nonhuman primate; novel; optogenetics; programs; protein protein interaction; public health relevance; receptor; regenerative therapy; self organization; single-cell RNA sequencing; spatiotemporal; stem cell differentiation; stem cells; teratogenesis; tool; tool development; transcriptome; transcriptome sequencing

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.

项目概要 了解人类胚胎发育对改善辅助生殖具有巨大影响 技术、基于干细胞的再生疗法以及预防遗传性出生缺陷和 致畸。然而，由于物种的多样性，我们对人类胚胎发育的了解是有限的。 人类和其他常用哺乳动物模型之间存在差异，且可获取性有限 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， 基于干细胞的体外培养方法，允许多细胞结构自发发育，同时 简化实验系统并提高高质量实时成像的可访问性具有巨大的意义 价值。尽管前景广阔，但现有文化系统的固有差异极大地限制了它们的发展 基础机制研究和临床应用的应用。光遗传学方法利用 基因工程方法和光学技术来控制修饰细胞或组织的生物功能 表达光敏蛋白。光遗传学的精确时空分辨率，加上 正确设计的照明系统可以提供强大且多功能的解决方案来改善 干细胞衍生的人类发育模型的可控性和可重复性。 我们的初步研究表明，外源性 BMP4 刺激可有效驱动人类多能性 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 聚集体的刺激会产生人类胚胎样结构（或胚状体）。在这个提议中 研究，我们将进行一项探索性的、高风险但高回报的研究，以证明整合 光遗传学和干细胞衍生的人类胚胎模型。具体来说，我们将获得光遗传学 hPSC 系 使用 optoBMP 系统；并检查其细胞内BMP活性和羊膜分化潜能 在光线照射下。然后将利用完全表征的 optoBMP-hPSC 来生成同类中的第一个， 光遗传学人类胚胎系统。 这项研究如果成功，将首次证明人类是成功一代的 具有发育信号光遗传学扰动的胚状体。这非常重要，因为有至关重要的 现有胚状体培养系统普遍存在的问题：缺乏再现性和可控性，以及 对此，我们对指导其发展和自组织的过程缺乏了解。 将光遗传学工具纳入现有的胚状体培养物将为实验者提供精确的结果 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，以了解背后的机制 人类胚胎在正常和病理情况下的发育，并用它们作为遗传的目标 操纵或药物测试。