Decoding cellular stress states for toxicology using single-cell transcriptomics

Single-cell transcriptomics (SCTr) offers a powerful platform for decoding the complexities of cellular stress responses to chemical exposures, capturing rare and transient subpopulations that can be obscured in bulk measurements. This study employs SCTr to profile the responses of HepaRG liver cells to various stress-inducing chemicals, providing detailed insights into adaptive stress response pathways (SRPs), encompassing the response to unfolded proteins, oxidative stress, heat shock, and DNA damage. By leveraging SCTr, we identify distinct cellular subpopulations and elucidate their transcriptomic profiles, linking specific gene expression patterns to biological mechanisms underlying stress responses. Our findings demonstrate the utility of SCTr in discerning the dose-dependent effects of chemicals at a granular level, highlighted by key differences between pseudo-bulk and single-cell analysis. Chemicals such as troglitazone, brefeldin A, rotenone, and tunicamycin induced unique transcriptional signatures associated with specific stress response pathways. Single-cell connectivity mapping with signatures of SRPs and cell fates, including autophagy and apoptosis, revealed clusters of cells with adaptive and potentially adverse phenotypes. These SCTr data have implications for identifying toxicological and disease mechanisms involving adaptive stress pathways. The data can also provide unique insights into toxicological processes by beginning to resolve adaptive chemical exposures, where cells recover, from those that are adverse from which cells do not recover, offering potentially new approaches for chemical safety assessments. Overall design: We profiled reference perturbagens known to induce stress responses, including the unfolded protein response (UPR), oxidative stress response (OSR), heat-shock response (HSR), response to hypoxia (HPX), and DNA damage response (DDR), in the differentiated HepaRG human liver cell model at three different concentrations. HepaRG cells were dissociated and fixed for single-cell transcriptomic (SCTr) assessments. Here, vehicle (DMSO) treated cells were profiled with 10X Genomics Chromium Next GEM Single Cell 3' Reagent Kits v3.1.