Single cell transcriptomics of Autophagy high and low 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. Single cell RNA sequencing of intestinal epithelial cells with high and low autophagic flux. Cells were stained with the autophagic vesicle tracker dye, CytoID, and the brightest (CytoID_High) and dimmest (CytoID_Low) 15% of cells were sorted out and processed for sequencing. CytoID cells from high and low cells were pooled from N=2 mice with each mouse identified using BioLegend Hashtag antibodies.