Furthermore, Kim et al. ASCs and improve their chondrogenic differentiation at its early stage using immunofluorescence, transmission and scanning electron microscopy, real-time PCR, and flow cytometry. Obtained results indicated that 5-azacytidine and resveratrol modulated mitochondrial dynamics, autophagy, and ER stress, leading to the enhancement of chondrogenesis in metabolically impaired ASCs. Therefore, pretreatment of these cells with 5-azacytidine and resveratrol may become a necessary intervention before clinical application of these cells in order to strengthen their multipotency and therapeutic potential. 1. Introduction Metabolic syndrome in humans (MetS) and horses (EMS) is usually more and more frequently diagnosed endocrine disorder all over the world, especially in well-developed countries [1, 2]. It occurs as a result of diet based on carbohydrate overload along with limited physical activity and genetic predisposition [1C3] and is characterized by fasting hyperleptinemia and hyperinsulinemia. Although obesity in MetS is recognized as a diagnostic factor, recent data suggests that severe obesity is not required for EMS diagnosis CP 31398 2HCl [4]. Finally, MetS and EMS culminate in vascular dysfunction, which in the course of MetS leads to the development of cardiovascular diseases and in EMS to which make them a stylish tool in cell-based therapies [20]. What is more, they exert a wide range of immunomodulatory effects due to CP 31398 2HCl the inhibition of CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells and activation of regulatory T cells (Treg) [21]. Additionally, ASCs promote macrophages polarization into immunosuppressive M2 type, which supports their application in the treatment of proinflammatory diseases, including metabolic syndrome [22]. We have also shown that ASCs are effective in the treatment of musculoskeletal disorders in small and large animals [23, 24]. Proregenerative properties of ASCs are partially explained by secretion of extracellular microvesicles (ExMVs) which improve intercellular signaling and support tissue regeneration [25, 26]. ExMVs contain a broad spectrum of cytokines, adipokines, hormones, and soluble growth factors that play a pivotal role in tissue regeneration [27]. Recently, ASC-derived ExMVs have been shown to contain high levels of proteins related to chondrogenic differentiation, including vascular endothelial growth factor B (VEGFB), hypoxia-inducible factor-1(HIF-1pretreatment of ASC derived from EMS horses (ASCEMS) with 5-azacytidine (AZA) and resveratrol (RES) may become distinct form of cellular pharmacotherapy able to CP 31398 2HCl reverse phenotype and improve multipotency of deteriorated cells. Our previous study revealed that application of AZA reversed the cytophysiological impairment of aged ASCs by epigenetic modifications and reduction of oxidative stress [29]. AZA treatment increased the mRNA levels of ten-eleven translocation methylcytosine dioxygenases (TET) and the B-cell lymphoma 2 (BCL-2)/bcl-2-like protein 4 (BAX) ratio, resulting in improved ASCs’ viability. On the other hand, RES, a natural polyphenol, has been shown to play a critical role in the regulation of cell fate and longevity the activation of 5 AMP-activated protein kinase (AMPK), forkhead box O3 (FOXO-3), and sirtuin-1 (SIRT1) genes [30]. In addition to its antioxidant activity, RES has been shown to reduce the CP 31398 2HCl inflammatory response and increase mitochondrial biogenesis by upregulating eNOS, which is associated with the SIRT1 pathway [31, 32]. In this study, CP 31398 2HCl we evaluated the chondrogenic differentiation potential of ASCEMS treated with the combination of AZA and RES. We examined the expression of genes and levels of proteins involved in the formation of extracellular matrix, oxidative stress, autophagy, mitochondrial biogenesis, and dynamics. 2. Materials and Methods All reagents used in this experiment were purchased from Sigma-Aldrich (Poland), unless indicated otherwise. 2.1. Classification of Animals Horses were age-matched (mixed FGF3 sex, 9C14 years; mean SD,.