Automated minute scale RNA-seq of pluripotent stem cell differentiation reveals early divergence of human and mouse gene expression kinetics.

Author summary For regenerative medicine, a slow developmental clock operating in human pluripotent stem cells imposes severe obstacles to many pluripotent stem cell therapies. Producing the desired cell types with the necessary level of physiological maturity in clinically-relevant time frames will be essential if the therapy is aimed at being patient-specific. Thus, understanding what controls species-specific rates of differentiation and identifying ways to accelerate it would have a broad impact on improving the therapeutic value of human pluripotent stem cells. In this paper, we measured the immediate-early gene expression responses to neural differentiation signals in mouse and human pluripotent stem cells. We quantitatively summarized the gene expression patterns using a flexible model that allowed us to identify shared trends and dynamic changes across species. We found that responses occurred equally early across species, but expression patterns were prolonged in human cells and the rate of upregulation in gene expression was significantly faster in mouse cells. Our results represent a significant contribution in furthering the knowledge of early developmental transcriptional programs. Pluripotent stem cells retain the developmental timing of their species of origin in vitro, an observation that suggests the existence of a cell-intrinsic developmental clock, yet the nature and machinery of the clock remain a mystery. We hypothesize that one possible component may lie in species-specific differences in the kinetics of transcriptional responses to differentiation signals. Using a liquid-handling robot, mouse and human pluripotent stem cells were exposed to identical neural differentiation conditions and sampled for RNA-sequencing at high frequency, every 4 or 10 minutes, for the first 10 hours of differentiation to test for differences in transcriptomic response rates. The majority of initial transcriptional responses occurred within a rapid window in the first minutes of differentiation for both human and mouse stem cells. Despite similarly early onsets of gene expression changes, we observed shortened and condensed gene expression patterns in mouse pluripotent stem cells compared to protracted trends in human pluripotent stem cells. Moreover, the speed at which individual genes were upregulated, as measured by the slopes of gene expression changes over time, was significantly faster in mouse compared to human cells. These results suggest that downstream transcriptomic response kinetics to signaling cues are faster in mouse versus human cells, and may offer a partial account for the vast differences in developmental rates across species.