Proteomic Insights into Human Limbal Epithelial Progenitor-Derived Small Extracellular Vesicles.

Limbal epithelial stem/progenitor cells (LEPC), supported by limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) within a specialized niche, are responsible for maintaining the corneal epithelium. Small extracellular vesicles (sEV) emerged as critical mediators of intercellular communication in various stem cell niches, yet their role in maintaining human limbal niche homeostasis remains poorly understood. In this study, tangential flow filtration and size exclusion chromatography were used to isolate sEV from LEPC-, LMSC- and LM-conditioned media. The isolated sEV from LEPC exhibited properties characteristic for sEV as confirmed by nanoparticle tracking analysis for size and concentration, by electron microscopy for morphology, and by western blot analysis of canonical EV markers including the cell-specific protein (cytokeratin 17/19). Quantitative and comparative proteomic profiling revealed distinct molecular signatures of LEPC-derived sEV, enriched in factors associated with keratinocyte development, extracellular matrix organization, and niche regulation. These findings suggest that LEPC-derived sEV may serve as important signaling mediators within the limbal niche microenvironment, though additional studies are needed to determine their specific functional roles in maintaining niche homeostasis.