Development of iPSCs for comparative genomics in primates

摘要 这是该项目第二个任期（5-8年）的竞争性续期申请。 描述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 确定物种特异性和保守的调控特征，这为遗传学提供了有价值的见解。 基因调控中的适应结构。然而，道德和实际的考虑排除了比较， 对活体灵长类动物，特别是猿类的分子特征的研究。非人类猿类的冷冻死后组织 它们很难获得，即使它们是可用的，它们也不是许多功能基因组的最佳模板。 测定。因此，我们通常无法从足够的个体中收集样本来绘制和研究基因调控。 基因座在非人类猿类;我们无法研究基因调控超过几个组织从猿类;我们无法 研究发育过程中基因调控的动态;我们无法研究对 进化和临床相关的暴露。 在这个项目的第一个学期，我们建议通过建立一个比较小组来应对这一挑战。 人类和黑猩猩的多能干细胞（iPSC）。我们成功地做到了这一点，我们分享了 这些线免费和不受限制的超过30个实验室（见支持信），便于比较 由研究者进行功能基因组研究，否则他们无法获得适当的样本 进行他们的研究。我们现在请求支持继续维护和分发这一社区资源， 我们将从两个方面进行扩展。首先，我们将使用剩余的基因组产生更多的黑猩猩iPSC系。 我们在黑猩猩研究暂停之前收集的样本。我们预计这些线路将很快成为 唯一的人口样本资源，为未来的研究涉及黑猩猩在美国。第二，我们将使用 人类和黑猩猩的iPSC开发一系列动态分化的类器官，我们也将分享 自由地允许其他组有效地利用比较iPSC组。这些独特的属性 动态分化的类器官的另一个特征是它们含有异步分化的细胞。虽然他们不 产生可以用批量RNA-seq数据进行有意义分析的纯细胞群体，单细胞RNA-seq可以 用于将类器官分解为数十种细胞类型和发育阶段，使我们能够探索 在成人组织中无法观察到的动态调节过程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.