159 Transforming growth factor β priming of horse adipose mesenchymal stem cells stimulates antifibrotic cargo in their secreted extracellular vesicles

Therapeutic properties of mesenchymal stem cells (MSC) can be enhanced by cytokine priming. We were interested in reverting fibrosis linked to endometrosis using MSC or their extracellular vesicles (EVs) and their cargo of regulatory molecules. This cargo of EVs differs according to the environment of the cells; therefore, we hypothesised that MSC subjected to transforming growth factor (TGF)β profibrotic preconditioning secrete EVs with antifibrotic cargo, which could reverse the production of the main fibrotic cellular marker smooth muscle actin (αSMA) and thus the differentiation to myofibroblasts in an in vitro model of mare endometrosis. In experiment 1, MSC tested in our laboratory were exposed to TGFβ (10 ng mL⁻¹) for 0, 1, 4, 12, and 24 h. Control MSC (naïve) were not exposed, an additional group was treated with 10 μM of SB431542, a selective inhibitor of TGFβ receptor-I, alongside TGFβ. All MSC were cultured in Dulbecco’s modified Eagle medium with 1% fetal bovine serum depleted of EVs. At the indicated time points, medium (5 mL) was collected, EVs were separated by ultracentrifugation, quantified using nanoparticle tracking, and confirmed by western blot for CD9, CD63, and CD81. The cargo of fibrotic-related miRNAs was evaluated using quantitative PCR for antifibrotic miRNAs (mir-29, mir-122, mir-214, and mir-486) and profibrotic miRNAs (mir-192 and mir-199). The fold change of expression variation (FC) was calculated. In experiment 2, endometrial fibroblasts from three different mares were challenged for 24 h with a fibrotic cocktail composed of IL1, IL6, TNF, and TGFβ (10 ng mL⁻¹ of each) or mock (PBS) as previously set in the laboratory. After this, endometrial fibroblasts were incubated with 1 × 10⁹ EVs mL⁻¹ collected after 48 h of culture of MSC of experiment 1 exposed for 4 h to TGFβ. The cellular mRNA was isolated and αSMA expression tested using ΔΔCT method. An ANOVA and Tukey post hoc test were conducted using as reference naïve (experiment 1) or mock (experiment 2) with a significance of P < 0.05. In experiment 1, TGFβ treatment for 4 h increased expression of antifibrotic miRNAs (FC = 2.1; P < 0.05), 24-h exposure increased mir-192 and mir-199 expression (12.3 FC; P < 0.05). In SB431542 treatment, 4 h of exposure yielded 3.2 FC increase of mir-29 and mir-122 (P < 0.01), while 24-h treatment raised expression of mir-199 (FC of 5.4; P < 0.05). In nontreated naïve cells, there was a 2.5 FC increase of mir-29 expression at 4 h and dramatic increase (FC = 22; P < 0.01) of profibrotic miRNAs. In experiment 2, EVs from experimental groups produced an inhibitory effect on αSMA expression in the endometrial fibroblasts (FC −0.4; P < 0.05). No such effect was detected for the naïve MSC-derived EVs. The treatment of MSC with TGFβ + SB431542 induced the most notable aSMA inhibition compared with the mock group. We propose that the miRNA cargo of EVs from MSC-treated cells exerted an antifibrotic action on induced fibrotic endometrial fibroblasts. Although proteins might also be involved, they were not studied here. This points to a key role of modulating the TGFβ pathway as potential tool for acellular therapies of endometrosis.

This study was funded by FONDECYT 1210349, VRID 219.153.027-INV.