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.

摘要 这是为回应资助机会公告PA-18-867而提交的修订后的R21提案 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存在于非编码DNA中，并被认为是 影响基因调控。这激发了人们寻找影响基因表达水平(EQTL)的变异的努力 在各种各样的成人组织中。然而，大多数与疾病相关的SNPs-尽管它们位于 假定是受调控的区域-尚未发现eQTL。造成这种情况的一个原因可能是尽管 在不同组织(如GTEx)中定位eQTL的大规模努力，我们仍然没有检测到基因 对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 那些出现在早期发展阶段的，由于实际或道德限制而无法接触到。因此， 基因发现的速度从根本上受到获得相关人体组织的限制。 人类成熟细胞可以转化为干细胞的发现是迈向 解决这个问题。诱导多能干细胞(IPSCs)提供了一种可再生的人类组织来源 从理论上讲，可以发育成任何类型的细胞。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IPSCs中获得的任何单个组织。 在干细胞生物学和新兴的单细胞技术的结合点上，有机会产生和 同时研究许多甚至大多数人类细胞类型，所有这些都在一个培养皿中进行。当在适当的地方生长时 在这种情况下，干细胞会自发形成被称为类胚体(EBS)的分化器官。单元格 在EBS内异步分化为起源于所有三个生殖层的细胞类型，包括 多能细胞、中间细胞和成熟细胞类型。通过将单细胞RNA测序(scRNA-seq)应用于细胞 在eBS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基因范围内 环境。EBS的使用还将使我们能够观察到细胞的转变和调控事件 在静态细胞培养中很明显。