Optimizing the Adipogenic Induction Protocol Using Rosiglitazone Improves the Physiological Parameters and Differentiation Capacity of Adipose Tissue-Derived Mesenchymal Stem Cells for Horses, Sheep, Dogs, Murines, and Humans

SIMPLE SUMMARY: Mesenchymal stem cell investigation has become an interesting scientific field in regenerative medicine and organ transplantation, particularly for chronic incurable disorders and massive tissue loss. Under suitable laboratory conditions, stem cells are able to renew themselves but also differentiate in bone, fat, and cartilage, which can be used to replace either damaged or lost tissue in veterinary medicine. Improper stimulation protocols cause low cell viability, death, and the failure of tissue repair. We used fat formation as a model to understand the cell specification requirements and to establish a common protocol to improve the cell activity and specification capacity for several mammalian species including humans. Thus, we replaced a widely used fat formation protocol referred to as 3-isobutyl-1-methylxantine and indomethacin with rosiglitazone. The results show that using 1–5 µM of rosiglitazone sufficiently improves the cell viability parameters, prevents cell death, and enhances fat formation compared to the traditional 3-isobutyl-1-methylxantine and indomethacin protocol. We found differences between the amount of fat formation and the level of the fat-regulating genes between species. This report provides essential information regarding the selection of a stem cell specification program and the requirement for each species for the further reproducibility and clinical targeting of obesity and metabolic disorders in veterinary medicine. ABSTRACT: The investigation of adipose tissue-derived mesenchymal stem cells (ASCs) has received considerable interest in regenerative medicine. A nontoxic adipogenic induction protocol valid for cells of different mammalian species has not been described. This study aims to establish an adipogenic differentiation protocol suitable for horses, sheep, dogs, murines, and human cells. An optimized rosiglitazone protocol, consisting of 5% fetal calf serum in Dulbecco’s Modified Eagle’s Medium, 10 μg/mL insulin, 0.55 μg/mL transferrin, 6.8 ng sodium selenite, 1 μM dexamethasone, and 1–5 μM of rosiglitazone, is compared to the 3-isobutyl-1-methylxantine (IBMX) protocol, where rosiglitazone was replaced with 0.5 mM IBMX and 0.2 mM indomethacin. Cell viability, cytotoxicity, a morphometric analysis of the lipid, and the expression of adipogenic markers for 14 days were assessed. The data revealed that using 5 µM of rosiglitazone promotes the adipogenic differentiation capacity in horse, sheep, and dog cells compared to IBMX induction. Meanwhile, marked reductions in the cell viability and cell number with the IBMX protocol were detected, and rosiglitazone increased the cell number and lipid droplet size, prevented apoptosis, and upregulated FABP-4 and Leptin expression in the cells of most of the species. Our data revealed that the rosiglitazone protocol improves the adipogenesis of ASCs, together with having less toxicity, and should be considered for cell reproducibility and clinical applications targeting obesity.