Abstract
Purpose :
Extracellular vesicles (EVs) are membranous structures released by cells. EVs carry a cargo of proteins, nucleic acids, and lipids that can mediate different biological functions. The purpose of this study is to characterize the protein and miRNA cargo of EVs isolated from human conjunctival epithelial and stromal cells.
Methods :
This study followed all ethical requirements. Three different human cell types were used: the immortalized human conjunctival epithelial cell line IM-HConEpiC, conjunctival mesenchymal stromal cells (Conj-MSCs) isolated from cadaveric donor tissue, and adipose tissue (AT)-MSCs obtained from lipoaspirates. EVs were isolated from cell culture conditioned media by differential ultracentrifugation. EV morphology was evaluated by atomic force microscopy (AFM) and size was determined by dynamic light scattering (DLS). EVs were characterized for CD9 and CD63 marker expression and quantified with the ExoELISA-Step kit (Immunostep). Protein cargo of EVs was analyzed by mass spectrometry using untargeted LC–MS/MSshotgun approach in TimsTOF Pro (Bruker). Micro RNAs (miRNA) were purified from EV samples and analyzed by Next Generation Sequencing (NGS). Three different samples from each cell type were studied.
Results :
EVs isolated from the three human cell types had round morphology observed by AFM, and co-expressed CD9 and CD63 markers. DLS analysis showed mean sizes of 330.1 ± 63.1 nm for IM-ConEpiC-EVs, 230.0 ± 37.0 nm for Conj-MSC-EVs, and 196.4 ± 28.6 nm for AT-MSC-EVs. Mass spectrometry revealed the presence of 1,892 proteins in the analyzed EVs. From them, 918 were found in the 3 cell types, whereas 300 were exclusively found in IM-HConEpiC-EVs, 19 in Conj-MSC-EVs, and 40 in AT-MSC-EVs. 1,111 mature miRNAs were detected in the EVs by NGS analysis. 312 miRNAS were present in the three cell type EVs, whereas 365 miRNAs were exclusively detected in IM-HConEpiC-EVs, 89 in Conj-MSC-EVs, and 87 in AT-MSC-EVs. Detected proteins and miRNAs are mainly involved in signal transduction, cell communication, metabolism, and cell growth.
Conclusions :
We carried out the first exhaustive analysis of conjunctival EVs cargo. Conjunctival epithelial cell derived EVs contain more proteins and miRNAs than stromal ones. Conj-MSC-EVs and AT-MSC-EVs share many characteristics that could imply that Conj-MSC-EVs might also have important therapeutic properties.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.