Single cell transcriptomics of bulk and Chga-CreER marked intestinal epithelial cells.

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiating(ed) cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic activity predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic proxy for the prospective identification of facultative stem cells. Overall design: Single cell RNA sequencing on intestinal epithelial cells from a ChgaCreER-tdTomato::Rosa26LSL-eYFP mouse. After 5 daily 1mg tamoxifen injections, eYFP+ and bulk epithelial cells were sorted from small intestinal crypts and incubated with BioLegend Hashtag antibodies to distinguish sample origin.