Systematic Analysis of Stromal-Epithelial Crosstalk Reveals the FGF21-FGFR3 Axis as a Key Driver of Corneal Wound Healing

Purpose: Corneal epithelial defects are a significant clinical problem and pose a severe threat to vision. However, their underlying mechanisms remain largely elusive. Here, we used single-cell RNA sequencing (scRNA-seq) and constructed cell-cell communication networks between corneal stromal cells (CSCs) and epithelial cells (CEpCs) to elucidate the pathological mechanism underlying corneal epithelial wound repair. Methods: Corneal tissues from normal and corneal epithelium defect mice were subjected to scRNA-seq. Comprehensive bioinformatic analyses, including unsupervised clustering, differential gene expression, functional enrichment, pseudotime, and RNA velocity analysis, were conducted to illustrate CSC heterogeneity. CellChat was used to identify the signaling crosstalk between CEpCs and CSCs. Mechanistic studies using co‑culture, FGFR3 perturbation, FGF21 manipulation, functional assays, and bulk RNA‑seq were performed to dissect the FGF21-FGFR3 axis in CEpCs and CSCs. Results: Unbiased clustering of the scRNA-seq data revealed eight distinct CSC subpopulations. Cell-cell communication mapping and immunofluorescence validation revealed that CSCs actively engaged CEpCs via the FGF21-FGFR3 axis. CEpCs co-cultured with FGF21‑overexpressing CSCs showed greater wound closure and EdU uptake. FGF21 stimulated ERK/AKT phosphorylation, promoted wound healing and proliferation, affects that were blunted by dabogratinib or FGFR3 knockdown and rescued by FGFR3 re‑expression. Functional experiments demonstrated that Fgf21 knockdown significantly delayed corneal wound healing, whereas topical administration of FGF21 eye drops promoted corneal epithelial wound repair in vivo. Bulk RNA-seq further revealed an FGF21-responsive transcriptional program involving extracellular matrix remodeling, cell adhesion, migration-related processes, and MAPK signaling. Conclusions: Our study revealed a novel interaction between CSCs and CEpCs mediated by the FGF21-FGFR3 axis. Exogenous FGF21 promotes the wound healing of CEpCs, suggesting a promising novel therapeutic strategy for corneal epithelial repair.