224 Analysis of the antifibrotic capacity of equine mesenchymal stem cell secretome from adipose and endometrial origin conditioned with PGE₂ on myofibroblast

Endometrosis is one of the main causes of infertility in mares. The hallmarks of endometrosis are the conversion of stromal fibroblasts to myofibroblasts, overexpression of smooth muscle actin (αSMA), and increased synthesis of extracellular matrix proteins, accompanied by an imbalance in the production of MMP2–9/TIMP 2–1. There is no cure for endometrosis at present; thus, new therapeutical approaches are welcome. Mesenchymal stem cells (MSC) are a promising option. Here we used the secretome of equine MSC (eMSC) in an in vitro model of fibrosis. The main goal was to assess the antifibrotic potential of the secretome of eMSC in the cellular model. Adipose (AT-eMSC) or endometrial eMSC (ET-eMSC) at passage three were preconditioned or not with 3 µM PGE₂ for 24 h in 1% extracellular vesicle (EV)-free serum. Conditioned medium (CM) was collected and spun (800g; 8 minutes) to discard cells or debris. Extracellular vesicles were isolated from the conditioned medium by ultracentrifugation and ultrafiltration (cutoff of 100 kDa). In parallel, endometrial fibroblasts were induced to differentiate into myofibroblasts by a cocktail of 10 ng mL⁻¹ each of TGFβ1, TNFα, IL-1β and IL-6 for 24 h. The eMSC and myofibroblasts were co-cultured in a Transwell system, and fully confluent eMSC were placed in the top chamber in depleted medium (EV-free; condition 1), the CM (condition 2) or EV (condition 3). Fully confluent myofibroblasts were seeded in the lower chamber and the co-culture was allowed for 48 h, after which myofibroblasts were subjected to total RNA/protein extraction. Gene expression of mRNAs was determined by RT-qPCR. Genes assayed were αSMA, COL1A2, COL3A1, CTGF, MMP2, MMP9, TIMP1 and TIMP2. The reverse-transcription quantitative PCR was analysed by analysis of variance and a post-Tukey post hoc test using GraphPad Prism 6.0 software with a level of significance P-value < 0.05. Gelatinase qualitative activity in myofibroblasts and cultured media was determined by gelatin zymography for MMP-9 and MMP-2. In all the experimental groups, PGE₂ treatment of cells triggered a significant down-regulation (P < 0.05) of the relative expression of COL1A2, COL3A1 and CTGF in the myofibroblasts, compared with control group (myofibroblasts without PGE₂ preconditioned medium). The expression of αSMA that did not change significantly. Conversely, MMP2 and MMP9 levels were up-regulated, as judged by pPCR (P < 0.05) as well as their activity (zymography assays, though not quantified, there was no activity in the control group). There was no effect of the type of cells (AT or ET) on the mentioned results. It is in concordance with the described antifibrotic properties of PGE₂, which seems to guide the eMSC secretome toward an antifibrotic response, independent of the origin of the MSC. We did not attempt to quantify PGE₂ in the samples; thus, we cannot rule out that some PGE₂ remained after pre-conditioning. In conclusion, pre-conditioning eMSC with PGE₂ and subsequent use of the cell’s secretome could improve the antifibrotic response of endometrial cells. Due to the ease of collecting CM versus isolation of EV, it could be potentially formulated in a way that can be infused into the endometrium of mares with endometrial fibrosis for its treatment.

Research was funded by FCYT Regular 1210349 and ANID grant 21201557.