Single-cell RNA sequencing of human embryonic stem cells reveals immediate and deferred transcriptomic responses to retinoic acid-mediated effects across cell subpopulations.

Identifying chemical-mediated molecular signatures may provide nuanced insights into early developmental processes, characterized by plasticity, rapid growth, and differentiation. This requires increased resolution of the multiple cell states and trajectories that are present at this stage. Cell-based models can facilitate higher-throughput evaluation and screening of potential developmental toxicants. In this study, we used single-cell RNA sequencing to characterize the transcriptomic signatures of the H9 human pluripotent stem cell (hPSC) line maintained in pluripotency and treated with 30 nM all-trans retinoic acid (RA), an essential vitamin A metabolite and potential developmental morphogen, for 6 and 72 h. In addition, we evaluated the molecular effects of RA on hPSCs over an undirected differentiation course for 14 days, to characterize RA-associated cell transition states. For both pluripotent (6- and 72-h) and undirected differentiation (14-day) exposure paradigms, transcriptional responses to RA were observed and compared to gastrulation markers of altered developmental trajectories. At 14 days in differentiation media, we observed cell clusters representing multiple cell types whose trajectory was altered by RA exposure. The overarching goal of this study was to contribute single cell resolution data that could be applied to future transcriptomic biomarkers for developmental toxicant/morphogen chemical screening.