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.

本研究的目的是通过使用不同成分的培养基来确定最有效的培养基，从而将犬脂肪来源的间充质干细胞 (ADMSC) 分化为产生胰岛素的细胞。从靠近母犬子宫的脂肪组织中分离出的干细胞分为6组：（1）Dulbecco改良Eagle培养基（DMEM）-高糖（HG）、β-巯基乙醇和烟酰胺；(2) DMEM-HG、β-巯基乙醇、烟酰胺和exendin-4；(3) DMEM-HG、β-巯基乙醇、烟酰胺、exendin-4、B27、非必需氨基酸和l-谷氨酰胺；(4) DMEM-HG、β-巯基乙醇和烟酰胺（用于最初的 8 天）和 DMEM-HG、β-巯基乙醇、烟酰胺、exendin-4、B27、非必需氨基酸、l-谷氨酰胺和碱性成纤维细胞生长因子（剩余 8 天期间）；(5) DMEM-HG和胎牛血清；(6) DMEM-低糖胎牛血清（标准对照组）。来自第 1 至第 5 组的脂肪间充质干细胞逐渐变圆并聚集成簇。这些变化在各组之间有所不同。在第 3 组中，细胞簇的数量明显更多，并且聚集成更大的聚集体。双硫腙染色显示，第 3 组和第 4 组在每个聚集体的胰岛素染色平均面积方面相似。然而，仅在第 4 组中，胰岛素聚集体的数量和染色的聚集体总面积显着大于其他组。PDX1、BETA2、MafA和Insulin的mRNA表达也在所有组中得到证实。