Enhanced adipogenic differentiation of bovine bone marrow-derived mesenchymal stem cells

Abstract

Until now, the isolation and characterisation of bovine bone marrow-derived mesenchymal stem cells (bBM-MSCs) have not been established, which prompted us to optimise the differentiation protocol for bBM-MSCs. In this study, bBM-MSCs were freshly isolated from three six-month-old cattle and used for pluripotent or phenotypic characterisation by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometer. Initially, the cells were adherent to plastic surfaces and exhibited spindle-like morphology. The cells expressed pluripotent as well as typical MSC markers. In addition, bBM-MSCs were differentiated into chondrocytes and osteocytes, but less efficiently into adipocytes. Accordingly, we conducted this study to establish the optimal adipogenic differentiation protocol under specific culture conditions. For this purpose, we formulated the basal differentiation medium using low-glucose Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) foetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin (P/S), 1 µM dexamethasone, 0.5 mM indomethacin and 0.5 mM 3-isobutyl-l-methylxanthine, and added with three levels of insulin at 10, 15 and 20 µg/mL as insulin is the key adipogenesis inducer. The level of differentiation was evaluated by Oil Red O staining and by analysing the expression of adipocyte lineage-specific genes by a quantitative real time RT-PCR. In this study, we found that the bBM-MSCs were effectively differentiated into adipocytes as manifested by the presence of Oil Red O-stained lipid droplets. The mRNA levels of adipocyte-specific genes in the differentiated cells were highly expressed as compared with the non-differentiated bBM-MSCs. In conclusion, we successfully isolated and characterised bBM-MSCs with multipotent and differentiation potential. Additionally, enhanced in vitro adipogenic differentiation protocol for bBM-MSCs was established.