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

摘要 这是一份修订后的R21提案，以响应PA-18-867“小说”的资助机会公告。 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位于非编码DNA中， 影响基因调控。这激发了人们努力鉴定影响基因表达水平的变异（eQTL） 在成人组织中广泛存在。然而，大多数疾病相关的SNP-尽管它们位于 puppectin调控区-尚未发现eQTL。其中一个原因可能是，尽管 尽管我们大规模地绘制了不同组织（例如GTEx）中的eQTL，但我们仍然尚未检查基因 调节与疾病最相关的细胞类型或状态。许多人体组织和细胞类型，特别是 由于实际或道德上的限制，在早期发展中就存在的那些因素是无法获得的。因此 基因发现的速度从根本上受到获取相关人体组织的限制。 成熟的人类细胞可以转化为干细胞的发现，是人类向干细胞研究迈出的重要一步。 解决这个问题。诱导多能干细胞（iPSC）提供了人类组织的可再生来源， 从理论上讲，可以发展成任何类型的细胞。然而，在实践中，可能需要数年才能发现如何生产 使用定向分化从iPSC获得任何单个组织。 在干细胞生物学和新兴的单细胞技术的联系中，有机会产生并 在同一个培养皿中同时研究多种甚至大多数人类细胞类型。当生长在适当的 在某些条件下，干细胞自发形成分化的类器官，称为胚状体（EB）。细胞 在EB中，细胞异步分化成来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。通过将单细胞RNA测序（scRNA-seq）应用于细胞， 在EBs中，我们可以联合鉴定多种细胞类型的eQTL，所有这些都在一个受控的遗传内， 环境EB的使用还将使我们能够观察细胞的转换和调节事件， 在静态细胞培养中明显。

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.