Single-cell RNA sequencing of murine limbal epithelia reveals Gas1 as a novel stem/progenitor cell marker for the corneal epithelium

The corneal epithelium is endowed with a rare population of stem cells that reside within the limbus, a circumferential transition zone that partitions the cornea from the conjunctiva, thus referred to as limbal epithelial stem cells (LESC). Despite the surge in investigations using single-cell RNA sequencing (scRNA-seq) of the ocular surface, a unifying marker(s) that distinguish these cells from their progeny is yet to be identified. We used a keratin (K)-14-driven lineage-tracing system and SmartSeq-2 single-cell transcriptomics in 5-60-week-old mice to interrogate the identity of limbal epithelia. Four cell clusters were identified, derived from both Confetti ⁺ and Confetti ⁻ cells (clusters 0–3), with cluster 3 designated to harbor progenitor cells. We found one gene of interest in cluster 3, growth arrest-specific gene 1 ( Gas1 ) coding for a cell-surface protein. PCR, flow cytometry and immunofluorescence disclosed this gene to be rarely expressed in limbal epithelial cells. Gas1 was also co-expressed with K14 in both young and old mice and upregulated following a mild mechanical debridement injury to the central cornea. The cell-surface expression of this antigen can be used to identify, extract and enrich progenitor cells for downstream molecular investigations and for generating better-quality cell-based grafts to treat severe corneal disease.