Single-cell Transcriptomic Profiling Reveals Novel Stem Cell Markers for Corneal Limbal Epithelium Homeostasis

Sox9 is a well-established marker of skin epithelial hair follicle stem cells and has been observed in the corneal limbus of Sox9-EGFP transgenic mice. Using Sox9-EGFP as a reference marker, this study characterized the transcriptomic atlas of corneal limbal epithelial cells during homeostasis and identified novel markers of limbal stem cells (LSCs) by single-cell RNA sequencing (scRNA-seq). A total of 21,066 viable cells were profiled, revealing 13 distinct cell clusters with unique molecular signatures. The cluster enriched with EGFP-positive cells was identified as the putative LSCs population during corneal epithelial homeostasis. Comparative analysis of corneal LSCs and early differentiated cells revealed several differentially expressed genes (DEGs) strongly associated with stemness-related pathways, including Gabrp, Fmo1, Fmo2, and Alcam, which were identified as potential novel LSC markers. These genes were exclusively expressed in the limbus, as confirmed by quantitative PCR (qPCR), Western blotting, and immunofluorescence staining. This study delineated the heterogeneous cellular composition of the limbal epithelium and uncovered several novel candidate markers that may be critical for maintaining corneal epithelial homeostasis.