Development of iPSCs for comparative genomics in primates

摘要 这是该项目第二任期(5-8年)的竞争性续签申请。 确定人类和我们的灵长类近亲之间的基因调控差异是至关重要的 了解人类特有特征的分子基础。比较基因组学为我们提供了 识别物种特有的和保守的调控特征，这为我们提供了对遗传 基因调控中的适应体系结构。然而，伦理和实际的考虑排除了比较 对活灵长类，特别是猿的分子特征的研究。来自非人类类人猿的冷冻死后组织 很难获得，即使它们可用，它们也不是许多功能基因组的最佳模板 化验。因此，我们通常无法从足够多的个体中收集样本来定位和研究基因调控。 非人类类人猿中的基因位点；我们无法研究类人猿几个组织中的基因调控；我们无法 研究发育过程中基因调控的动态；而我们无法研究对 在进化和临床上相关的暴露。 在这个项目的第一阶段，我们建议通过建立一个诱导的比较小组来应对这一挑战 来自人类和黑猩猩的多能干细胞(IPSCs)。我们已经成功地做到了这一点，我们分享了 这些线路自由和不受限制地与30多个实验室(见支持函)进行比较 研究人员进行的功能基因组研究，否则他们将无法获得适当的样本 进行他们的研究。我们现在请求支持，以继续维护和分配这一社区资源， 我们将通过两种方式扩大这一领域。首先，我们将使用剩余的代码生成额外的黑猩猩iPSC行 我们在黑猩猩研究暂停之前收集的样本。我们预计这些线路很快就会变成 这是未来美国黑猩猩研究的唯一种群样本来源。第二，我们将使用 人类和黑猩猩的ipscs将开发一系列动态区分的有机体，我们也将分享这些 自由地允许其他团体有效地利用可比较的IPSC小组。这些产品的独特属性 动态分化类器官是指它们含有异步化分化的细胞。虽然他们不是 产生可用批量rna-seq数据进行有意义分析的纯细胞群体，单细胞rna-seq可以 被用来将有机物质分解成数十种细胞类型和发育阶段，使我们能够探索 在成人组织中观察不到的动态调控过程10。

Abstract This is a competitive renewal application for a second term of this project (years 5-8). Characterizing gene regulatory differences between humans and our close primate relatives is essential for understanding the molecular basis of human-specific traits. Comparative genomics provides us with the tools to identify both species-specific and conserved regulatory features, which has provided valuable insight into the genetic architecture of adaptation in gene regulation. However, ethical and practical considerations preclude comparative studies of molecular traits in live primates, particularly apes. Frozen post-mortem tissues from non-human apes are difficult to obtain, and even when they are available, they are not optimal templates for many functional genomic assays. Thus, we are generally unable to collect samples from enough individuals to map and study gene regulatory loci in non-human apes; we are unable to study gene regulation in more than a few tissues from apes; we are unable to study the dynamics of gene regulation during development; and we are unable to study regulatory responses to evolutionarily and clinically relevant exposures. In the first term of this project, we proposed to address this challenge by establishing a comparative panel of induced pluripotent stem cells (iPSCs) from humans and chimpanzees. We have successfully done this, and we have shared these lines freely and without restriction with more than 30 labs (see letters of support), facilitating comparative functional genomic studies by investigators who would not otherwise have been able to obtain appropriate samples to conduct their research. We now request support to continue to maintain and distribute this community resource, which we will expand in two ways. First, we will generate additional chimpanzee iPSC lines using the remaining samples we collected prior to the moratorium on chimpanzee research. We expect that these lines will soon become the only population-sample resource for future studies involving chimpanzees in the USA. Second, we will use human and chimpanzee iPSCs to develop a series of dynamically differentiating organoids, which we will also share freely to allow other groups to effectively utilize the comparative iPSC panel. The unique property of these dynamically differentiating organoids is that they contain asynchronously differentiating cells. While they do not produce pure cell populations that can be meaningfully analyzed with bulk RNA-seq data, single-cell RNA-seq can be used to deconvolve the organoids into dozens of cell types and developmental stages, allowing us to explore dynamic regulatory processes that cannot be observed in adult tissues10.