236 Assessment of the antifibrotic capacity of the secretome of equine adipose mesenchymal stem cells conditioned with PGE₂ in an in vitro model of endometrial fibrosis

Endometrosis is one of the main causes of infertility in mares, and is characterised by stromal- and periglandular fibrosis, endometrial atrophy, and reduction in the number of uterine glands, leading to irreversible degeneration of the endometrial tissue. The landmarks of the disease are overexpression of αSMA by local fibroblasts, the differentiation of fibroblasts into myofibroblasts, and increased production of extracellular matrix proteins. There is no cure for endometriosis; thus, new approaches aimed to modulate local inflammation and to stimulate tissue regeneration are needed. Cell therapies using mesenchymal stem cells (MSCs) are an attractive perspective, owed to their antifibrotic properties. We used the secretome of equine MSC primed with PGE₂ as antifibrotic inducer in an in vitro model of fibrosis to assess their antifibrotic potential. MSC from adipose tissue (AT-MSC) at passage 3 were preconditioned or not with 3 µM PGE₂ for 24 h. Conditioned medium (CM) or extracellular vesicles (EV) were collected from cultured MSC. In parallel, endometrial fibroblasts were induced to differentiate into myofibroblasts using 10 ng/mL of both TGF-β1 and TNFα for 24 h. AT-MSC and myofibroblasts were co-cultured in a Transwell system. Fully confluent MSC, CM of said cells or EV purified from the CM were placed in the top chamber; fully confluent myofibroblasts were seeded in the lower chamber. Co-culture was allowed for 48 h, after which total RNA/protein were extracted from myofibroblasts. RT-qPCR was used to detect the relative abundance of transcripts of αSMA, COL1A2, COL3A1, CTGF, MMP2, MMP9, TIMP 1, and TIMP2, while αSMA, COL1A2, and COL3A1 proteins were detected by Western blot. Statistics: ANOVA and Tukey post hoc with control as reference; P < 0.05. For treatments MSC, CM, and EV of PGE₂-treated cells, there was a decline in relative expression of αSMA, COL1A2, COL3A1, CTGF, TIMP 1, and TIMP2 in the myofibroblasts compared to control cells (myofibroblasts without PGE₂ preconditioned medium), while MMP2–9 levels were upregulated. Also, a decrease in αSMA, COL 1, and COL 3 protein production levels was observed in treated groups of myofibroblasts. PGE₂ drove the MSC secretome toward an antifibrotic response, thus representing a potential tool for the treatment of endometrial fibrosis.

This research was supported by FCYT REGULAR 1210349 and ANID grant 21201557 to LM.