Molecular identity of human limbal heterogeneity involved in corneal homeostasis and privilege

Purpose: The corneal limbus maintains the homeostasis, immune and angiogenic privilege of cornea. This study aimed to depict the landscape of human limbal tissues by single-cell RNA sequencing (scRNA-seq). Methods: Single cells of human limbus collected from donor corneas were subjected to 10x scRNA-seq, followed by clustering cell types through the t-distributed stochastic neighbor embedding (t-SNE) and unbiased computational informatic analysis. Immunofluorescent staining was performed using human corneas to validate the analysis results. Results: 47,627 cells acquired from six human limbal tissues were collected and subjected to scRNA-seq. 14 distinct clusters were identified and 8 cell types were annotated with representative markers. In-depth dissection revealed three limbal epithelial cell subtypes and refined the X-Y-Z hypothesis of corneal epithelial maintenance. We further unveiled two cell states with higher stemness (TP63+ and CCL20+ cells), and two other differentiated cell states (GPHA2+ and KRT6B + cells) in homeostatic limbal stem/progenitor cells (LSPCs) that differ in transcriptional profiles. Cell-cell communication analysis revealed the central role of LSPCs and their bidirectional regulation with various niche cells. Moreover, comparative analysis between limbus and skin deciphered the pivotal contribution of limbal immune cells, vascular and lymphatic endothelial cells to corneal immune and angiogenic privilege. Conclusions: The human limbus atlas provided valuable resources and foundations for understanding corneal biology, disease and potential interventions.