No Cell Left Behind: Using Embryoid Bodies to Understand Human Biology

不遗余力：利用胚胎体来了解人类生物学

• 批准号: 10427990
• 负责人: Yoav Gilad
• 金额: $ 28.7万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427990
• 关键词: Acute; Adult; Affect; Autopsy; Biological; Cardiac Myocytes; Cell Culture Techniques; Cell Line; Cells; Collection; Complex; Controlled Environment; DNA; Data; Development; Developmental Process; Disease; Embryo; Environment; Ethics; Event; Funding Opportunities; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Diseases; Genetic Variation; Genomics; Genotype-Tissue Expression Project; Germ Layers; Goals; Hepatocyte; Human; Human Biology; Human body; In Vitro; Individual; Learning; Left; Maps; Measures; Modeling; Neurons; Nucleic Acid Regulatory Sequences; Organoids; Phenotype; Preparation; Problem Solving; Process; Quantitative Trait Loci; Regulation; Research Design; Resources; Sample Size; Sampling; Source; Stem cell pluripotency; Suggestion; System; Testing; Time; Tissues; Untranslated RNA; Variant; cell transformation; cell type; design; differentiation protocol; directed differentiation; disorder risk; experimental study; genetic variant; genome wide association study; human disease; human tissue; induced pluripotent stem cell; inter-individual variation; novel strategies; power analysis; response; single cell technology; single-cell RNA sequencing; stem cell biology; stem cell differentiation; stem cells; temporal measurement; theories; trait; unethical

Abstract This is a revised R21 proposal submitted in response to funding opportunity announcement PA-18-867, “Novel Approaches for Relating Genetic Variation to Function and Disease”. Most of the genetic variants that are associated with disease lie within non-coding DNA and are thought to affect gene regulation. This has inspired efforts to identify variants that affect gene expression levels (eQTLs) in a wide range of adult tissues. However, most disease-associated SNPs – though they are located in putatively regulatory regions – have not been found to be eQTLs. One reason for this could be that despite large-scale efforts to map eQTLs in diverse sets of tissues (e.g, GTEx), we still have not yet examined gene regulation in the cell types or states most relevant for disease. Many human tissues and cell types, especially those that are present in early development, are inaccessible due to practical or ethical constraints. Thus, the pace of genetic discovery is fundamentally limited by access to relevant human tissues. The discovery that mature human cells can be transformed into stem cells was an important step toward solving this problem. Induced pluripotent stem cells (iPSCs) provide a renewable source of human tissue that can, in theory, develop into any cell type. In practice, however, it can take years to discover how to produce any single tissue from iPSCs using directed differentiation. At the nexus of stem cell biology and emerging single-cell technologies, there is an opportunity to generate and study many, or even most, human cell types simultaneously, all within a single dish. When grown in the proper conditions, stem cells form spontaneously differentiating organoids known as embryoid bodies (EBs). Cells within EBs differentiate asynchronously into cell types originating from all three germ layers, including pluripotent, intermediate, and mature cell types. By applying single-cell RNA-sequencing (scRNA-seq) to cells within EBs, we can jointly identify eQTLs across a multitude of cell types, all within a controlled genetic environment. The use of EBs will also allow us to observe cellular transitions and regulatory events that are not evident in static cell culture.

抽象的 这是针对资助机会公告 PA-18-867“Novel 将遗传变异与功能和疾病联系起来的方法”。 大多数与疾病相关的遗传变异都存在于非编码 DNA 中，并且被认为 影响基因调控。这激发了人们努力识别影响基因表达水平（eQTL）的变异 广泛存在于成人组织中。然而，大多数与疾病相关的 SNP——尽管它们位于 假定的监管区域——尚未发现 eQTL。造成这种情况的原因之一可能是尽管 大规模努力在不同的组织中绘制 eQTL（例如 GTEx），但我们仍然没有检查基因 与疾病最相关的细胞类型或状态的调节。许多人体组织和细胞类型，特别是 由于实际或道德限制，早期开发中存在的那些是无法实现的。因此， 基因发现的速度从根本上受到相关人体组织获取的限制。 成熟的人体细胞可以转化为干细胞的发现是朝着这一目标迈出的重要一步。 解决这个问题。诱导多能干细胞 (iPSC) 提供了可再生的人体组织来源， 理论上，可以发育成任何细胞类型。然而，在实践中，可能需要数年时间才能发现如何生产 使用定向分化技术从 iPSC 中提取任何单个组织。 在干细胞生物学和新兴单细胞技术的结合中，有机会产生和 在一个培养皿中同时研究多种甚至大多数人类细胞类型。当在适当的环境中生长时 在一定条件下，干细胞会形成自发分化的类器官，称为类胚体（EB）。细胞 EB 内异步分化为源自所有三个胚层的细胞类型，包括 多能、中间和成熟细胞类型。通过对细胞应用单细胞 RNA 测序 (scRNA-seq) 在 EB 中，我们可以联合识别多种细胞类型的 eQTL，所有这些都在受控的遗传范围内 环境。 EB 的使用还将使我们能够观察细胞转变和调控事件，而这些是无法观察到的。 在静态细胞培养中很明显。