Isolation, Characterization and Proteomic Profiling of Small Extracellular Vesicles derived from Limbal Niche Cells

Abstract

Purpose : Extracellular vesicles (EV), specifically small extracellular vesicles (sEV), serve as mediators in intercellular communication across diverse cell types, contributing to cell division and survival to maintain homeostasis within various stem cell niches, including the limbal stem cell niche. However, the understanding of sEV content and their interactions within the limbal stem cell niche remains limited.

Methods : Fluorescent-activated cell sorting was employed to isolate limbal niche cells i.e limbal mesenchymal stromal cells (LMSC) and limbal melanocytes (LM) from organ-cultured corneal-limbal tissue. Subsequently, sEV were isolated from conditioned media using a combination of tangential flow filtration and size exclusion chromatography based on size and elution time. The isolated sEV underwent characterization through transmission electron microscopy, nanoparticle tracking analysis, and western blot analysis. Further, the sEV proteome was analyzed using multiplex bead arrays and quantitative mass spectrometry. The preservation of sEV functional characteristics was assessed by their internalization by limbal epithelial progenitor cells (LEPC) using flow cytometry and confocal microscopy.

Results : The isolated sEV exhibited typical characteristics, including a size distribution peaking at 135 nm and typical EV morphology. They displayed classical EV markers such as CD63, CD81, CD9 and Alix, in addition to cell-specific markers CD90 for LMSC- and Melan-A for LM-sEV. Label-free quantitative proteomic analysis identified 511 proteins in LM-sEV and 466 proteins in LMSC-sEV. Bioinformatics analysis revealed a shared function in extracellular matrix (ECM) deposition, with LMSC-sEV showing involvement in collagen ECM remodeling and cell-matrix adhesion, while LM-sEV were implicated in bioprocesses like cellular pigmentation and development. Moreover, fluorescently labeled sEV were detected in perinuclear compartments of LEPC.

Conclusions : The characterization of LM- and LMSC-sEV protein cargo underscore the diverse roles of sEV in regulation of cells and ECM within the limbal stem cell niche, shedding light on their potential contributions to tissue remodeling and homeostasis.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.