Short preconditioning with TGFβ of equine adipose tissue-derived mesenchymal stem cells predisposes towards an anti-fibrotic secretory phenotype: A possible tool for treatment of endometrosis in mares.

Abstract

Endometrosis in mares is a disease resulting from chronic inflammation characterized by peri glandular fibrosis. There is no effective treatment so far, which opens the door for exploring the use of stem cells as a candidate. Transforming growth factor beta (TGF&#x3b2;) is crucial for the establishment and progression of fibrosis in mare's endometrosis. We aimed to develop regenerative approaches to treat endometrosis by using mesenchymal stem cells (MSC), for which understanding the effect of TGF&#x3b2; on exogenous MSC is crucial. We isolated and characterized equine adipose MSC from six donors. Cells were pooled and exposed to 10&#xa0;ng/ml of TGF&#x3b2; for 0, 4, and 24&#xa0;h, after which cells were analyzed for proliferation, migration, mesodermal differentiation, expression of fibrosis-related mRNAs, and prostaglandin E2 secretion. At 24&#xa0;h of exposition to TGF&#x3b2;, there was a progressive increase in the contraction of the monolayer, leading to nodular structures, while cell viability did not change. Exposure to TGF&#x3b2; impaired adipogenic and osteogenic differentiation after 4&#xa0;h of treatment, which was more marked at 24&#xa0;h, represented by a decrease in Oil red and Alizarin red staining, as well as a significant drop (p&#xa0;<&#xa0;0.05) in the expression of key gene regulators of differentiation processes (PPARG for adipose and RUNX2 for osteogenic differentiation). TGF&#x3b2; increased chondrogenic differentiation as shown by the upsurge in size of the resulting 3D cell pellet and intensity of Alcian Blue staining, as well as the significant up-regulation of SOX9 expression (p&#xa0;<&#xa0;0.05) at 4&#xa0;h, which reached a maximum peak at 24&#xa0;h (p&#xa0;<&#xa0;0.01), indicative of up-regulation of glycosaminoglycan synthesis. Preconditioning MSC with TGF&#x3b2; led to a significant increase (p&#xa0;<&#xa0;0.05) in the expression of myofibroblast gene markers aSMA, COL1A1, and TGF&#x3b2; at 24&#xa0;h exposition time. In contrast, the expression of COL3A1 did not change with respect to the control but registered a significant downregulation compared to 4&#xa0;h (p&#xa0;<&#xa0;0.05). TGF&#x3b2; also affected the expression of genes involved in PGEsynthesis and function; COX2, PTGES, and the PGEreceptor EP4 were all significantly upregulated early at 4&#xa0;h (p&#xa0;<&#xa0;0.05). Cells exposed to TGF&#x3b2; showed a significant upregulation of PGEsecretion at 4&#xa0;h compared to untreated cells (p&#xa0;<&#xa0;0.05); conversely, at 24&#xa0;h, the PGEvalues decreased significantly compared to control cells (p&#xa0;<&#xa0;0.05). Preconditioning MSC for 4&#xa0;h led to an anti-fibrotic secretory phenotype, while a longer period (24&#xa0;h) led to a pro-fibrotic one. It is tempting to propose a 4-h preconditioning of exogenous MSC with TGF&#x3b2; to drive them towards an anti-fibrotic phenotype for cellular and cell-free therapies in fibrotic diseases such as endometrosis of mares.