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目的探讨体外诱导人胚胎干细胞(hESs)定向分化为胰岛细胞的可行性。方法体外分三阶段诱导hESCs定向分化为胰岛细胞。第一阶段:予以活化素-A、渥曼青霉素诱导分化形成定型内胚层;第二阶段:采用维甲酸(RA)、NOGGIN、碱性成纤维细胞生长因子(bFGF)诱导胰腺细胞定向分化;以EGF扩增胰腺祖细胞;第三阶段:采用尼克酰胺、唾液素4、bFGF及骨形成蛋白(BMP4)促进胰岛细胞成熟;观察诱导各阶段细胞形态变化,免疫荧光法鉴定胰十二指肠同源异型盒基因(PDX-1)、胰高糖素、胰岛素、C肽、葡萄糖转运子2(glut-2)表达。结果诱导14 d时hESs出现胰高糖素荧光表达;20 d时细胞出现PDX-1和C肽共表达;22 d形成的成熟胰岛细胞出现glut-2和胰岛素的阳性表达;胰岛素阳性细胞占17.1%,C肽阳性细胞占3.8%。结论体外可简单高效的诱导hESs定向分化为成熟胰岛细胞。
Objective To investigate the feasibility of in vitro differentiation of human embryonic stem cells (hESs) into islet cells. Methods The hESCs were induced to differentiate into islet cells in three phases in vitro. In the first phase, definitive differentiation was induced by activin-A and wortmannin to form definitive endoderm. In the second stage, pancreas cells were induced to differentiate with RA, NOGGIN and basic fibroblast growth factor (bFGF) EGF to amplify pancreatic progenitor cells; Phase III: Nicotinamide, sialin 4, bFGF and bone morphogenetic protein (BMP4) were used to promote islet cell maturation; morphological changes were observed at different stages of induction; pancreas duodenum was identified by immunofluorescence (PDX-1), glucagon, insulin, C-peptide, and glut-2 expression. Results The glucagon expression of hESs was observed on the 14th day. PDX-1 and C peptide were co-expressed on day 20, while the expression of glut-2 and insulin was observed on the 22th day. Insulin-positive cells accounted for 17.1 %, C peptide positive cells accounted for 3.8%. Conclusion The hESs can be induced to differentiate into mature islet cells easily and efficiently in vitro.