Single-Cell Transcriptomic Comparison of Corneal Epithelial Responses to Temporal Limbal vs. Clear Corneal Incisions in Phacoemulsification

Abstract

Background: Surgical incision geometry influences corneal epithelial regeneration, yet the cellular and molecular responses distinguishing temporal limbal from clear corneal wounds remain poorly understood. Advances in single-cell RNA sequencing (scRNA-seq) enable precise delineation of transcriptional programs governing ocular surface repair. Objective: To characterize incision-type–specific epithelial regenerative mechanisms and identify key molecular pathways and cell populations driving corneal wound healing at single-cell resolution. Methods: Corneal epithelial samples obtained following temporal limbal and clear corneal incisions were analyzed using scRNA-seq. Data integration, clustering, pseudotime trajectory mapping, and transcription factor network inference were performed using Harmony, Monocle2, and pySCENIC. Differential pathway enrichment and metabolic profiling were assessed via hallmark gene set analysis. Results: A total of 36,217 high-quality cells were resolved into 20 transcriptionally distinct populations. A novel EGFR⁺/NOTCH1⁺ activated basal sub-cluster exhibited 35% higher organoid formation and upregulation of TP63, KLF6, and SOX9, defining it as a regenerative driver. Limbal incisions demonstrated dominant Wnt/FGF/TGF-β pathway enrichment, enhanced stemness, and elevated fatty acid metabolism (ES = 2.67), whereas clear corneal incisions showed IL6-mediated inflammatory activation. Regulatory network analysis identified ATF3, JUN, KLF6, TWIST1, and TP63 as core transcriptional regulators. Conclusion: Temporal limbal incisions preferentially activate Wnt-driven regenerative programs through an EGFR⁺/NOTCH1⁺ basal progenitor population, whereas clear corneal incisions elicit inflammation-predominant repair.