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

Abstract

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.

Keywords: Exosomes; Limbal epithelial progenitor cells; Limbal melancoytes; Limbal mesenchymal stromal cells; Limbal stem cell niche; Proteomics; Small extracellular vesicles.

Fig. 1

Enrichment and quantification of small extracellular vesicles (sEV): (A) Fluorescence-activated cell sorting (FACS) image showing the selection and separation of cells into CD90⁺CD117⁻ (LMSC), CD90⁻CD117⁺ (LM), and CD90⁻CD117⁻ (LEPC) populations. (B) Phase contrast images showing the morphological characteristics of the cell types: small cuboidal epithelial phenotype of limbal epithelial progenitor cells (LEPC); large, flattened, smooth bodies with multiple dendrites of limbal melanocytes (LM); spindle-shaped morphology with prominent nucleoli of limbal mesenchymal stromal cells (LMSC). C) Phase contrast images of LEPC before incubation in conditioned media (CM) (i), 24 h post incubation (ii), and on the day of CM harvesting (iii). D) Nanoparticle tracking analysis-showing particle counts in LEPC-sEV fractions (F1-F8) from conditioned and unconditioned media. Data presented as min to max whisker box plots (n = 14). E) The comparative analysis of particles produced/million cells of LEPC, LMSC and LM. Data presented as min to max whisker box plots (n = 14). F) Size distribution of LEPC-sEV and LM-sEV and LMSC-sEV particles in fraction 3 (F3) measured by nanoparticle tracking analysis. Data presented as min to max whisker box plots (n = 14/cell type). G) Particle-to-protein ratio analysis indicating the highest purity in fraction 3 (F3) for LEPC-sEV. Data are presented as min-to-max whisker box plots (n = 14)

Fig. 2

Characterization of small extracellular vesicles (sEV): (A) Western blot analysis of EV-specific markers in fractions F1-F8, void volume V0 and flow through FT from LEPC-sEV. Enrichment of EV markers (Alix, CD63, CD9, CD81) were predominantly observed in F3 fraction. The absence of the endoplasmic reticulum marker calnexin (CANX) and residual bovine serum albumin (BSA) across all fractions indicates a lack of cellular contamination and serum residue. Limbal epithelial progenitor markers CK17/19 and epithelial major syndecan, syndecan 1, were detected in F2-F4, with the highest levels in F3. Uncropped Western blot images are provided in Supplementary Figures – S2-S7. (B) Transmission electron microscopy (TEM) images of F3 fractions showing classical EV morphology. (C) Size distribution of LEPC-sEV in F3 based on TEM and Cryo TEM analysis. Data presented as min to max whisker box plots (n = 3). D) Cryo TEM images of F3 fraction showing sEV of varying sizes with classical EV morphology (black arrows) and intact membranes. White arrow showing an ice contamination

Fig. 3

Proteomic characterization of small extracellular vesicles (sEV): (A) Box plot showing the number of proteins identified in LEPC-sEV (n = 5) and the number of proteins commonly detected in at least three samples. B) Heatmap displaying unbiased clustering of protein intensity profiles from different biological samples based on LFQ intensities. C) Heatmaps showing the protein intensity profiles of (i) plasma membrane, endosomal and cell-specific proteins; (ii) cytosolic proteins; (iii) apolipoproteins and cellular contaminants in LEPC-sEV samples. (D) Top 10 enriched Gene Ontology (GO) terms for Biological Process, Molecular Function, Cellular Component and KEGG pathways in LEPC-sEV

Fig. 4

Comparative proteomic profiling of limbal epithelial progenitor cell derived small extracellular vesicles (sEV), limbal melanocyte (LM)-sEV and limbal mesenchymal stromal cell (LMSC) derived –sEV. (A) Venn diagram showing the number of unique and shared proteins identified among LEPC-sEV, LM-sEV and LMSC-sEV. B) Gene ontology analysis highlighting the unique biological processes, cellular components, molecular functions, and KEGG pathways enriched in proteins found exclusively in LEPC-sEV. (B) Heatmaps showing selected protein clusters uniquely present in LEPC-sEV, categorized as follows keratin profiling (i), s100 calcium binding proteins (ii), serpin family proteins (iii) and desmosomal related family proteins (iv). C) Heatmaps showing the selected ECM-related proteins including collagens, laminins and other glycoproteins (i), proteoglycans (ii), growth factors (iii) and tissue remodeling proteins. (C) Western blot validation showing: Fibronectin (FN1) in all three populations, with predominant expression in LM-and LMSC-sEV samples; SPARC in all three populations; transferrin predominantly expressed in LEPC-sEV; LAMC2 restricted to LEPC-sEV populations. Uncropped versions of the Western blot are shown in Supplementary Fig. 8