Differentiation of canine adipose mesenchymal stem cells into insulin-producing cells: comparison of different culture medium compositions

Abstract

The aim of this study was to differentiate canine adipose-derived mesenchymal stem cells (ADMSCs) into insulin-producing cells by using culture media with different compositions to determine the most efficient media. Stem cells isolated from the fat tissues close to the bitch uterus were distributed into 6 groups: (1) Dulbecco's modified Eagle medium (DMEM)–high glucose (HG), β-mercaptoethanol, and nicotinamide; (2) DMEM-HG, β-mercaptoethanol, nicotinamide, and exendin-4; (3) DMEM-HG, β-mercaptoethanol, nicotinamide, exendin-4, B27, nonessential amino acids, and l-glutamine; (4) DMEM-HG, β-mercaptoethanol, and nicotinamide (for the initial 8-d period), and DMEM-HG, β-mercaptoethanol, nicotinamide, exendin-4, B27, nonessential amino acids, l-glutamine, and basic fibroblast growth factor (for the remaining 8-d period); (5) DMEM-HG and fetal bovine serum; and (6) DMEM–low glucose and fetal bovine serum (standard control group). Adipose-derived mesenchymal stem cells from groups 1 to 5 gradually became round in shape and gathered in clusters. These changes differed between the groups. In group 3, the cell clusters were apparently more in numbers and gathered as bigger aggregates. Dithizone staining showed that groups 3 and 4 were similar in terms of the mean area of each aggregate stained for insulin. However, only in group 4, the number of insulin aggregates and the total area of aggregates stained were significantly bigger than in the other groups. The mRNA expression of PDX1, BETA2, MafA, and Insulin were also confirmed in all the groups. We conclude that by manipulating the composition of the culture medium it is possible to induce canine ADMSCs into insulin-producing cells, and the 2-staged protocol that was used promoted the best differentiation.