Department of Immunology, Shiraz Medical School, Shiraz University of Medical Sciences, Po Box 71345-1798, Shiraz, Iran.
Student Research Committee, Shiraz University of Medical Sciences, Po Box 71345-1798, Shiraz, Iran.
In Vitro Cell Dev Biol Anim. 2019 Jun;55(6):462-471. doi: 10.1007/s11626-019-00358-z. Epub 2019 May 20.
To produce insulin-producing cells (IPCs) from bone marrow mesenchymal stem cells (BM-MSCs) using a simple and cost effective method. During the initial 7 days of three-dimensional (3D) culture, BM-MSCs were cultured on 1% agar or agarose to form multicellular spheroids. Spheroids and spheroid-derived single cells (SS and SSC, respectively) were cultured in the absence of any proteinaceous growth factor in a simple specific medium for a further 7 d. The insulin content of the differentiated cells was evaluated at the mRNA and protein levels. Furthermore, the expression of pancreatic beta cells-related genes other than INS as well as the in vitro responses of IPCs to different glucose concentrations were investigated. Cellular clusters generated on agar and SS conditions (agar+SS-IPCs) stained better with beta cell specific stains and were more reactive to serum-containing insulin reactive antibodies compared with agarose-SS-IPCs. Gene expression analysis revealed that in comparison to agarose + SS-IPCs, agar+SS-IPCs expressed significantly higher levels of INS-1, INS-2, PDX-1, NKX6.1, and XBP-1. Of interest, agar+SS-IPCs expressed 2215.3 ± 120.8-fold more INS-1 gene compared to BM-MSCs. The expression of β-cell associated genes was also higher in agar+SS-IPCs compared to the agar+SSC-IPCs. Moreover, the expression of INS-1 gene was significantly higher in agar+SS-IPCs compared with agar+SSC-IPCs after culture in media with high concentration of glucose. Compared to the most expensive and time-consuming protocols, 3D culture of MSCs on agar followed by 2D culture of cellular clusters in a minimally supplemented high glucose media produced highly potent IPCs which may pay the way to the treatment of diabetic patients.
用简单且经济有效的方法从骨髓间充质干细胞（BM-MSCs）产生胰岛素产生细胞（IPC）。在三维（3D）培养的最初 7 天内，将 BM-MSCs 培养在 1%琼脂或琼脂糖上以形成多细胞球体。然后，将球体和源自球体的单细胞（分别为 SS 和 SSC）在没有任何蛋白质生长因子的情况下在简单的特定培养基中培养 7 天。通过实时聚合酶链反应和 Western blot 分析评估分化细胞的胰岛素含量。此外，还研究了除 INS 以外的胰腺β细胞相关基因的表达以及 IPC 对不同葡萄糖浓度的体外反应。在琼脂和 SS 条件下（琼脂+SS-IPC）生成的细胞簇用β细胞特异性染色剂染色更好，并且与琼脂糖-SS-IPC 相比，对含有血清的胰岛素反应性抗体更具反应性。基因表达分析表明，与琼脂糖+SS-IPC 相比，琼脂+SS-IPC 表达的 INS-1、INS-2、PDX-1、NKX6.1 和 XBP-1 水平明显更高。有趣的是，与 BM-MSCs 相比，琼脂+SS-IPC 表达 INS-1 基因的倍数增加了 2215.3±120.8 倍。与琼脂+SSC-IPC 相比，琼脂+SS-IPC 中β细胞相关基因的表达也更高。此外，在高浓度葡萄糖培养基中培养后，琼脂+SS-IPC 中 INS-1 基因的表达明显高于琼脂+SSC-IPC。与最昂贵和最耗时的方案相比，3D 培养 MSC 上的琼脂，然后在高葡萄糖补充培养基中二维培养细胞簇，可产生高效的 IPC，这可能为糖尿病患者的治疗开辟道路。
