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. These cells are thus referred to as limbal epithelial stem cells. Despite the surge in investigations using single-cell RNA sequencing (scRNA-seq) of the ocular surface, a unifying marker(s) that distinguishes 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- to 60-week-old mice to interrogate the identity of limbal epithelia. These results were then validated using flow cytometry, immunofluorescence, and a central corneal injury model. Four cell clusters were identified, derived from both Confetti + and Confetti - cells (clusters 0-3), with cluster 3 designated as harboring progenitor cells. We focused on one gene of interest in cluster 3, growth arrest-specific gene 1 ( Gas1 ), which codes for a cell-surface protein. PCR, flow cytometry, and immunofluorescence revealed that this gene is expressed in a rare population of limbal epithelial cells. Gas1 was also coexpressed with K14 in both young and old mice and upregulated after a mild mechanical debridement injury to the central cornea. The cell-surface expression of this protein 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.