Characterization of Extracellular Vesicle RNA-Cargo of Equine Mesenchymal Stromal Cells: Which Role in Tissue Regeneration?

Horse adipose-derived Mesenchymal Stromal Cells (E-Ad-MSCs) are extensively studied for their anti-inflammatory and pro-regenerative properties observed at muscular-skeletal structure level. Their biological effects seem linked, at least in part, to the release of nanometric extracellular vesicles (EV) containing trophic factors, cytokines and coding and regulatory RNAs. This study aimed at investigating RNA content within EV produced by E-Ad-MSC to explore their regenerative potential and to open new perspectives in cell-free therapeutic procedures. EVs were obtained from 4 horses via ultracentrifugation of the E-Ad-MSC conditioned medium. Resulting pellet was used for electron microscopy analysis (TEM and SEM) and small RNA sequencing via Illumina technology. TEM an SEM analyses reveled a mixture of shedding vesicles and exosomes as demonstrated by CD90 and Flotillin-1 reactivity with size ranged from 30 to 300 nm. Unique reads and the last Ensembl gene annotation (86) were used to characterize the exosome cargo: standard bioinformatics analyses (trimming, mapping and counting) were applied. One of the most abundant species of RNA cargo is represented by snoRNAs, a conserved group of small non-coding RNAs recently considered regulatory with miRNA-like functionality. These data represent a key step in the characterization of E-Ad-MSC secretome and shed light on the mechanisms of action cells-derived vesicles: not annotated sequences and non coding RNAs with new regulatory roles are the most abundant part of the cargo underlining the complex nature of their information content.