Abstract
Subcutaneous fat tissue is an accessible and abundant source of multipotent stem cells for cell therapy in regenerative medicine. Successful trilineage differentiation is required to define the stemness features of the obtained mesenchymal cells, and adipogenesis is a part of it. Since indomethacin is bound to serum albumin, replacing foetal bovine serum (FBS) with horse serum (HS) in adipogenic induction protocols would suppress its cytotoxic effect and reveal a better adipogenic potential in equine MSCs. The equine subcutaneous adipose-derived stem cells (ASCs) were separately induced in adipogenesis by three different concentrations of 3-isobutyl-1-methylxanthine, IBMX (0.5 mM; 0.25 mM and 0.1 mM) and indomethacin (0.1 mM; 0.05 mM and 0.02 mM) for 48 h. In contrast to the IBMX, indomethacin in all concentrations caused dramatic cellular detachment. Further, the same induction concentrations were used in FBS and HS conditions for adipogenic induction. The MTT assay revealed that the culture media supplemented with HS raised cellular vitality by about 35% compared to those cultured in FBS. Based on those results, an adipogenic cocktail containing indomethacin (0.05 mM) and IBMX (0.5 mM), supplemented with HS and FBS, respectively, was applied for 18 days. The adiponectin gene expression was significantly up-regulated in HS-supplemented media since established changes in PPAR-gamma were insignificant. The tri-lineage differentiation was successful, and a cross-sectional area of adipocytes was performed. The albumin concentration was higher in HS than in FBS. In conclusion, our study revealed that HS is an appropriate supplement in induced adipogenesis since it probably suppresses the indomethacin-related cytotoxic effect and increases adipogenic ability in equine subcutaneous ASCs.