Single-Cell RNA Transcriptome Helps Define the Limbal/Corneal Epithelial Stem/Early Transit Amplifying Cells and How Autophagy Affects This Population

Purpose: Single-cell RNA-sequencing (scRNA-seq) was used to interrogate the relatively rare stem (SC) and early transit amplifying (TA) cell populations in limbal/corneal epithelia from wild-type and autophagy-compromised mice. Methods: We conducted scRNA-seq on ocular anterior segmental tissue from wild-type and beclin 1-deficient (beclin1+/-) mice, using a 10X Gemomics pipeline. Cell populations were distinguished by t-distributed stochastic neighbor embedding. Seurat analysis was conducted to compare gene expression profiles between these two groups of mice. Differential protein expression patterns were validated by immunofluorescence staining and immunoblotting. Results: Unbiased clustering detected 10 distinct populations: three clusters of mesenchymal and seven clusters of epithelial cells, based on their unique molecular signatures. A discrete group of mesenchymal cells expressed genes associated with corneal stromal SCs. We identified three limbal/corneal epithelial cell subpopulations designated as stem/early TA, mature TA, and differentiated corneal epithelial cells. Thioredoxin-interacting protein and PDZ-binding kinase (PBK) were identified as novel regulators of stem/early TA cell quiescence. PBK arrested corneal epithelial cells in G2/M phase of the cell cycle. Beclin1+/- mice displayed a decrease in proliferation-associated (Ki67, Lrig1) and stress-response (H2ax) genes. The most increased gene in beclin1+/- mice was transcription factor ATF3, which negatively regulates limbal epithelial cell proliferation. Conclusions: Establishment of a comprehensive atlas of genes expressed by stromal and epithelial cells from limbus and cornea forms the foundation for unraveling regulatory networks among these distinct tissues. Similarly, scRNA-seq profiling of the anterior segmental epithelia from wild-type and autophagy-deficient mice provides new insights into how autophagy influences proliferation in these tissues.