Equine osteoarthritis-related microRNA analysis in extracellular vesicles from horse mesenchymal stromal cells: a preliminary investigation

Osteoarthritis (OA) represents the most common cause of retirement from athletic activities in horses. As current pharmacological treatments can alleviate clinical symptoms, but lack regenerative effects on damaged cartilages, extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have emerged as a promising alternative. A relevant EVs’ cargo is represented by microRNAs (miRNAs), significant post-transcriptional gene regulators that can fully or partially regulate mRNAs, and whose gene expression can be modulated by the surrounding extracellular microenvironment. As further research is needed to explore their role in EVs and how MSC preconditioning influences them, the aim of this study was to establish optimized protocols for miRNA extraction and RT-qPCR analysis for characterizing EVs secreted by horse MSCs, allowing the future assessment of miRNA expression changes under different extracellular environment conditions in the context of equine OA. Previously obtained MSCs derived from bone marrow and fat tissue (3 healthy horses), were cultured under standard conditions. EVs were isolated via ultracentrifugation and characterized for size distribution and concentration through Nanoparticle Tracking Analysis. In parallel, a literature analysis was performed to identify a panel of miRNAs linked to the inflammatory response of OA. MiRNAs were therefore extracted from isolated EVs and amplified through RT-qPCR, and the expression of miRNAs selected from literature was evaluated. MSC-derived EVs were successfully isolated, with a concentration mean of 2,57 x 1010 particles/mL and a size mean of 139,40 nm. Extracted miRNAs showed a concentration ranging from 0,705 to 4,48 ng/µL. Moreover, nine out of the 23 miRNAs linked to OA from literature were detected through RT-qPCR. This preliminary investigation demonstrates the presence of miRNAs associated with the inflammatory process of OA within extracellular vesicles derived from equine MSCs under standard culture conditions. Indeed, miRNAs were extracted and detected through RT-qPCR in all samples, thus allowing for testing the miRNA-panel across different culture conditions with the future aim of better understanding the role of EVs as mediators in the crosstalk between MSCs and target cells.