Equine endometrial organoids treated with embryo-derived mesenchymal stem cell-secreted extracellular vesicles as a treatment for endometritis

A healthy uterine environment is essential for normal mare fertility and is critical for ensuring a positive financial return on breeding in the equine industry. Approximately 20% of Thoroughbred mares undergo persistent breeding-induced endometritis (PBIE), creating a uterine environment that is unfavorable for pregnancy. Despite extensive in vivo studies, the exact mechanisms causing endometritis and the most efficacious strategies for preventing or resolving PBIE are unknown. Organoids are physiologically-relevant 3D cell cultures that allow disease processes to be studied at the cellular level and eliminate confounding factors present in vivo. A potential therapeutic for PBIE is extracellular vesicles (EVs) secreted by embryo-derived mesenchymal stem cells (EDMSCs). EVs are lipid-bound nanoparticles containing proteins, mRNAs, and miRNAs that are naturally secreted by cells for intercellular communication. We hypothesized that EVs secreted by EDMSCs have anti-inflammatory properties to prevent PBIE. Our objectives were i) to assess EVs produced by EDMSCs for the presence of anti-inflammatory miRNAs via PCR and ii) to assess transfer of the anti-inflammatory miRNAs from EVs to endometrial organoids stimulated for inflammationwith frozen-killed sperm (FKS) or lipopolysaccharide (LPS). Results demonstrated the presence of reported anti-inflammatory miRNAs miR-10a, miR-21, miR-24, miR-145, and miR-146a in EVs secreted by three different EDMSC cell lines. Then, endometrial organoids (n=3 mares) were co-cultured with EDMSC EVs or PBS (control) for 24 h and subsequently exposed to two inflammatory stimuli (FKS or LPS). After 12h of exposure to inflammatory stimuli, organoids were washed and assessed for the presence of anti-inflammatory miRNAs by PCR (data was normalized using U6 snRNA). MiR-10a and miR-24 trended to be increased in organoids treated with EDMSC EVs compared to PBS controls in groups stimulated with either FKS or LPS. Furthermore, uptake of EVs by the organoids was corroborated by the co-culture of organoids with fluorescently-labelled (PKH26) EVs. EV uptake was confirmed by presence of a fluorescence signal in recipient organoid cells. While studies are ongoing, data suggests that EDMSC EVs contain anti-inflammatory miRNAs that may be delivered to endometrial cells to prevent PBIE in mares. Funding was provided by the Grayson-Jockey Club Research Foundation.