Single-Cell Transcriptomics Shows Cellular Heterogeneity, Intercellular Communication, and Extracellular Matrix Remodeling in Corneal Fibrosis In Vivo

Purpose: Corneal fibrosis is a common clinical condition post ocular trauma/infection affecting 7% of world's population. This study characterized fibroblast heterogeneity, cellular trajectories, intercellular communications, and extracellular matrix (ECM) protein remodeling in fibrotic cornea in vivo via single-cell RNA sequencing (scRNA-seq). Methods: Naïve and alkali-injured fibrotic corneas of New Zealand White rabbits were obtained under approved institutional animal use and care protocol. Multimodal in vivo eye imaging, single-cell RNA-sequencing, hematoxylin and eosin staining, and immunofluorescence analyzed corneas. Unsupervised clustering, subclustering of ECM remodeling cells, trajectory inference, CellChat-based intercellular communication mapping, and R-programming-generated transcriptomic atlas. Results: Fourteen transcriptionally distinct cell clusters were identified via canonical marker genes. Basal epithelium and ECM remodeling clusters acted as communication hubs whereas differentiated epithelium had limited participation. Subclustering the ECM remodeling niche identified four distinct stromal cell populations, quiescent-keratocytes, activated-keratocytes, progenitor-like-keratocytes, and myofibroblasts. Transcriptional trajectories organized these cellular states into a bifurcating continuum; one aligned to fibroblast activation and myofibroblast formation whereas the other aligned to quiescent-keratocytes or progenitor-like-keratocytes. Eight pathways (MIF, NECTIN3, HGFα, POSTN, NAMPT, TWEAK, EPH, and VEGFC) demonstrated strong ligand-receptor connectivity and their protein expression corroborated predicted signaling. Stage-specific enrichment revealed temporal dynamics within ECM-remodeling cells and continuous landscape of stromal subpopulations from quiescent-keratocytes to proliferative-fibroblasts/myofibroblasts and progenitor-like-cells followed by late dominance of ECM organization, adhesion, and collagen biosynthesis. The progression trajectory appeared bidirectional along pseudotime. Conclusions: This study provides an integrative transcriptomic framework of stromal cell heterogeneity, intercellular signaling, and ECM remodeling trajectories in rabbit cornea in vivo and advances mechanistic understanding of corneal repair.