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: 10651667
• 负责人: Yoav Gilad
• 金额: $ 16.4万
• 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/10651667
• 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; Process; Quantitative Trait Loci; Research Design; Resources; Sample Size; Sampling; Source; Stem cell pluripotency; Suggestion; System; Testing; Time; Tissues; Transformed Cell Line; Untranslated RNA; Variant; 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 这是一项经过修订的R21提案，以响应资金机会公告PA-18-867，“新颖 将遗传变异与功能和疾病联系起来的方法”。 与疾病相关的大多数遗传变异都位于非编码DNA中，被认为是 影响基因调节。这激发了识别影响基因表达水平（EQTL）的变体的努力 在各种成年组织中。但是，大多数与疾病相关的SNP-尽管它们位于 推定的监管区域 - 尚未发现是EQTL。原因之一可能是需求 大规模绘制eqtl的努力（例如，GTEX），我们尚未检查基因 细胞类型或最相关的疾病的调节。许多人体组织和细胞类型，特别是 由于实际或道德的约束，在早期发展中存在的人是无法访问的。那， 遗传发现的空间从根本上受到相关人体组织的限制。 发现成熟的人类细胞可以转化为干细胞的发现是迈向干细胞的重要一步 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 从理论上讲，可以发展成任何细胞类型。但是，实际上，发现如何生产可能需要数年 使用定向分化的IPSC的任何单个组织。 在干细胞生物学和新兴的单细胞技术的联系中，有机会产生和 简单地研究许多人，甚至大多数人的细胞类型。在适当的 条件是，干细胞构成了被称为胚胎体（EB）的类器官。细胞 在EBS内部，异步分化为源自所有三个细菌层的细胞类型，包括 多能，中间和成熟的细胞类型。通过将单细胞RNA-sequent（SCRNA-SEQ）应用于细胞 在EBS中，我们可以在多种细胞类型中共同识别EQTL，所有EQTL都在受控的遗传中 环境。 EBS的使用还将使我们能够观察细胞过渡和不属于的调节事件 静态细胞培养中的证据。