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.