Jiang Wei, Shi Yan, Zhao Dongxin, Chen Song, Yong Jun, Zhang Jing, Qing Tingting, Sun Xiaoning, Zhang Peng, Ding Mingxiao, Li Dongsheng, Deng Hongkui
Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing 100871, China.
Cell Res. 2007 Apr;17(4):333-44. doi: 10.1038/cr.2007.28.
The capacity for self-renewal and differentiation of human embryonic stem (ES) cells makes them a potential source for generation of pancreatic beta cells for treating type I diabetes mellitus. Here, we report a newly developed and effective method, carried out in a serum-free system, which induced human ES cells to differentiate into insulin-producing cells. Activin A was used in the initial stage to induce definitive endoderm differentiation from human ES cells, as detected by the expression of the definitive endoderm markers Sox17 and Brachyury. Further, all-trans retinoic acid (RA) was used to promote pancreatic differentiation, as indicated by the expression of the early pancreatic transcription factors pdx1 and hlxb9. After maturation in DMEM/F12 serum-free medium with bFGF and nicotinamide, the differentiated cells expressed islet specific markers such as C-peptide, insulin, glucagon and glut2. The percentage of C-peptide-positive cells exceeded 15%. The secretion of insulin and C-peptide by these cells corresponded to the variations in glucose levels. When transplanted into renal capsules of Streptozotocin (STZ)-treated nude mice, these differentiated human ES cells survived and maintained the expression of beta cell marker genes, including C-peptide, pdx1, glucokinase, nkx6.1, IAPP, pax6 and Tcf1. Thirty percent of the transplanted nude mice exhibited apparent restoration of stable euglycemia; and the corrected phenotype was sustained for more than six weeks. Our new method provides a promising in vitro differentiation model for studying the mechanisms of human pancreas development and illustrates the potential of using human ES cells for the treatment of type I diabetes mellitus.
人类胚胎干细胞的自我更新和分化能力使其成为生成用于治疗I型糖尿病的胰腺β细胞的潜在来源。在此，我们报告了一种新开发的有效方法，该方法在无血清系统中进行，可诱导人类胚胎干细胞分化为产生胰岛素的细胞。在初始阶段使用激活素A诱导人类胚胎干细胞向定形内胚层分化，通过定形内胚层标记物Sox17和Brachyury的表达进行检测。此外，全反式维甲酸（RA）用于促进胰腺分化，早期胰腺转录因子pdx1和hlxb9的表达表明了这一点。在含有碱性成纤维细胞生长因子（bFGF）和烟酰胺的DMEM/F12无血清培养基中成熟后，分化细胞表达胰岛特异性标记物，如C肽、胰岛素、胰高血糖素和葡萄糖转运蛋白2（glut2）。C肽阳性细胞的百分比超过15%。这些细胞分泌的胰岛素和C肽与葡萄糖水平的变化相对应。当将这些分化的人类胚胎干细胞移植到经链脲佐菌素（STZ）处理的裸鼠的肾囊中时，它们存活下来并维持β细胞标记基因的表达，包括C肽、pdx1、葡萄糖激酶、nkx6.1、胰岛淀粉样多肽（IAPP）、pax6和Tcf1。30%的移植裸鼠表现出明显的稳定血糖恢复；并且纠正后的表型持续超过六周。我们的新方法为研究人类胰腺发育机制提供了一个有前景的体外分化模型，并说明了使用人类胚胎干细胞治疗I型糖尿病的潜力。
