Wharton’s jelly mesenchymal stromal/stem cell–derived conditioned medium effect on equine endometrial cell viability

Endometritis is the major cause of reduced fertility in mares, with a consequent measurable economic loss. Mesenchymal stromal/stem cells (MSCs) and their derivates gained extremely high attention in recent years for their promising e!ects in the treatment of in#ammation (Carrade et al. 2012 Cell Med. 4, 1–12). Their application could help in the modulation of the immune in#ammatory response, providing regeneration and remodeling of injured tissue. The aim of this preliminary study was to evaluate in vitro the e!ect of Wharton’s jelly (WJ) MSC-derived conditioned medium (CM) on equine endometrial cells with lipopolysaccharide (LPS)-induced in#ammation. Equine WJMSCs were isolated from 3 samples, pooled and then frozen at passage (P) 3. The WJMSCs were thawed and maintained in culture medium (Dulbecco’s Modi"ed Eagle Medium (DMEM) + 10% fetal bovine serum) until 80%–90% con#uence, and then the culture medium was replaced with serum-free Ringer lactate solution. The CM was collected after 24 h of starvation and concentrated 10 times by centrifugation at 4000g for 30 minutes at 13°C using 3-kDa molecular weight cuto! "lter units and then stored at !80°C until use. Endometrial cells were obtained from 3 diestrus mare uteri. Cells at P1 were plated in 24-well plates at a density of 20 000 cells/well. Four di!erent conditions were tested: DMEM standard complete medium (CTRL), DMEM with the addition of (1) 10% of CM (CM), (2) 10 ng mL!1 LPS (LPS) and (3) LPS combined with CM (LPS + CM). Cell viability, apoptosis and necrosis were assessed after 6, 12 and 24 h of incubation, using acridine orange and propidium iodide #uorescent staining. Data were analysed by analysis of variance and Tukey’s test. Results (mean ± s.d.) of 3 replicates are reported in Table 1. At each incubation time, CM gave similar results to the CTRL for all parameters. After 6 h of incubation, LPS increased the percentage of necrotic cells, while co-treatment with CM prevented the detrimental e!ect. After 12 h of incubation, LPS decreased cell viability and increased apoptosis and necrosis compared with the CTRL and CM groups, while the co-treatment LPS + CM gave intermediate results. In conclusion, these preliminary results suggest that equine WJMSCs with cytokines, chemokines, growth factors and microvesicles may in part mitigate the LPS-induced inflammation on endometrial cells, promoting further investigations on WJMSC-CM characterisation and its mechanisms of action.