A comparative study of endoderm differentiation in humans and chimpanzees

There is substantial interest in the genetic regulatory framework that is established in early human development, and in the evolutionary forces that shaped early developmental processes in humans. Progress in these areas has been slow because it is difficult to obtain relevant biological samples. Recent technological developments in the generation and differentiation of inducible pluripotent stem cells (iPSCs) provide the ability to develop in vitro models of early human and non-human primates developmental stages. We have previously established matched iPSC panels from humans and chimpanzees. Using these panels, we comparatively characterized gene regulatory changes through a four-day timecourse differentiation of iPSCs (day 1) into primary streak (day 2), endoderm progenitors (day 3), and definitive endoderm (day 4). As might be expected, we found that differentiation stage (in effect, cell type) is the major driver of variation in gene expression levels in our study, followed by species. We then identified thousands of differentially expressed genes between humans and chimpanzees in each differentiation stage. Yet, when we considered gene-specific dynamic regulatory trajectories throughout the timecourse, we found that 75% of genes, including nearly all known endoderm developmental markers, have conserved trajectories in the two species. Interestingly, we observed a marked reduction of both intra- and inter-species variation in gene expression levels in primitive streak samples compared to the iPSCs, with a recovery of variation in endoderm progenitors. The reduction in variation in gene expression levels at a specific developmental stage, paired with the high degree of conservation of temporal expression across species, is consistent with the dynamics of developmental canalization. Overall, we conclude that endoderm development in iPSC-based models are highly conserved and canalized between humans and our closest evolutionary relative. We used a panel of 6 human and 4 chimpanzee iPSC lines previously derived and characterized in the Gilad lab. We differentiated the iPSCs into definitive endoderm, a process that was completed over 3 days, and included replicates of cell lines over the differentiation process. We harvested RNA from iPSCs (day 0) prior to differentiation and subsequently every 24 hours to capture intermediate populations corresponding to primitive streak (day 1), endoderm progenitors (day 2), and definitive endoderm (day 3). Overall, we collected a total of 32 human samples and 32 chimpanzee samples. We assessed used RNA sequencing to estimate gene expression levels in all samples. There are a total of 64 samples (6 human cell lines + 2 technical replicates at 4 time points; 4 chimpanzee cell lines x 2 technical replicates at 4 time points.)