Extracellular Vesicles from Canine Mesenchymal Stem Cells-Isolation, Characterization and miRNA Definition Following...

Extracellular vesicles (EVs) have become a key area of research, as scientists study their role in various biological processes. These vesicles appear to play a key role in the use of mesenchymal signaling cells (MSCs, formerly known as mesenchymal stem cells) to treat various diseases, such as osteoarthritis (OA), and other degenerative conditions. In our experiments, we examined EVs formed by canine mesenchymal signaling cells (MSCs) to identify them according to current guidelines and define their content, particularly the microRNA (miRNA) they contain, for future research projects. After obtaining the EVs, we demonstrated via Western blotting and transmission electron microscopy that the nanoparticles visible in the nanotracking analysis were positive for CD9 and ALIX and positive for CD9 and CD81, respectively. Markers for nanoparticles that do not represent extracellular particles-tested here as cytochrome C for mitochondrial particles and histones for nuclear particles-were negative. Finally, we detected a total of 85 different miRNAs in the negative controls. To determine the potential influence of various cell stimulations intended to induce osteoarthritis (e.g., interleukin-1β stimulation) or a possible treatment (e.g., shockwave therapy), or the influence of ITS prior to extracellular vesicle extraction, we detected a total of 208 different miRNAs. These results demonstrate how canine EVs from MSCs can be detected in vitro and how the EVs' miRNA profile changes after stimulation of the producing cells. This information may provide valuable insight into the understanding and treatment of osteoarthritis. Additionally, we demonstrated that using ITS instead of FCS to produce EVs should be reconsidered due to the significant change in miRNA expression levels.