Abstract
Purpose :
The study of extracellular vesicles (EVs) in ocular field has increased in the last years. However, although most research is focused on the most abundant, small EVs (exosomes), the presence of other minority, larger EVs is also relevant. The aim of this study is to characterize and compare EVs from conjunctival epithelial cells (IM-ConEpiC), human conjunctival mesenchymal stromal cells (Conj-MSC), and adipose-tissue (AT)-MSCs, focusing on the different EV subpopulations and main protein cargo.
Methods :
IM-HConEpi cell line was used as a source to obtain conjunctival epithelial EVs, whereas Conj-MSCs were isolated from cadaveric donor bulbar conjunctival tissue, and AT-MSC from lipoaspirates from living donors with ethical approval. 48 h conditioned media were collected from each cell type and EVs were isolated by differential centrifugation and ultracentrifugation to generate 2K, 10K, and 100K EV fractions. The EV subpopulations in each fraction were analyzed by nanoflow cytometry using a Beckman Coulter CytoFLEX LX flow cytometer. The levels of the EV markers CD9, CD63, CD81 and CD147, in each fraction were compared by EV-Bead Conjugated Flow Cytometry. Protein cargo of 100K EV fractions were studied by mass spectrometry.
Results :
The majority of EVs had a violet side scatter (VSSC) in the range of 80-110nm polystyrene beads (180-500nm EVs by Rosetta system analyses), while the proportion of large EVs varied between the cells. Results revealed that all markers were present in the EV fractions, with CD9 and CD147 being spread across the 2K, 10K and 100K fractions while CD63 and CD81 were enriched in the 100K fractions relative to the 2K and 10K fractions. Mass spectrometry results showed the presence of a variety of proteins in the three cell types EVs, with some of them being especially relevant in ocular surface pathophysiology, such as thrombospondin 1 (more abundant in MSC-derived EVs than in epithelial EVs) or IL-23 receptor (more abundant in IM-HConEpiC-EVs).
Conclusions :
Human conjunctival EVs showed differences depending on cell origin (epithelial or stromal). Conj-MSC derived EVs are more similar to AT-MSC-derived EVs, whereas IM-HConEpiC-EVs showed several relevant differences. These results, along with our previous studies suggest an interesting potential therapeutic effect of Conj-MSCs derived EVs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.