Mesenchymal stromal cells and extracellular vesicles in equine osteoarthritis: do IL-1β and TNF-α influence their microRNA cargo?

Osteoarthritis (OA) represents the most common cause of retirement from athletic activities in horses. As current pharmacological treatments can alleviate clinical symptoms, with no effect on recovery of 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 whose 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, this study aimed to characterize EVs secreted by horse MSCs, investigating miRNA expression changes under different extracellular conditions mimicking the equine OA environment. Primary adipose tissue derived-MSCs were isolated from slaughtered horses and used to produce 6 pools of 3 animals each. MSC pools were cultured under standard conditions and exposed to pro-inflammatory stimuli (IL-1β, TNF-α). 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, showing no significant differences between untreated and treated MSCs for both concentration and size. Following normalization both according to a synthetic internal control and for total EV volume, 2 out of the 23 selected miRNAs resulted differentially expressed, while 5 showed significant changes only when normalizing for the synthetic internal control. These results provide valuable insights on the influence of different pro-inflammatory stimuli on MSC-derived EVs and their miRNA cargo. Extending the analysis to MSCs isolated from other sources (e.g. bone marrow) will allow to deepen our knowledge on the MSC behaviour when exposed to inflammatory milieu.