人胚胎干细胞体外定向诱导分化胰岛细胞移植治疗非肥胖-联合免疫缺陷糖尿病小鼠

Pancreatic islets differentiated from human embryonic stem cells correct hyperglycemia in non-obese diabetic/severe combined immunodeficient mice

目的探讨人胚胎干细胞(hESs)体外定向诱导分化胰岛细胞移植治疗非肥胖-联合免疫缺陷(NOD/SCID)糖尿病小鼠的可行性。方法体外分四阶段诱导hESs定向分化为胰岛细胞:第一阶段,予以活化素A(activin A)、渥曼青霉素(wortmannin)诱导分化形成定型内胚层;第二阶段,予以全反式维甲酸(RA)、NOGGIN、碱性成纤维细胞生长因子(bFGF)诱导胰腺细胞定向分化;第三阶段,予以表皮生长因子(EGF)扩增胰腺祖细胞;第四阶段,予以尼克酰胺(nicotinamide)、唾液素4(exendin-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)的表达;四阶段分化成熟的胰岛细胞体外检测胰岛素释放反应并植入链脲菌素(STZ)诱导形成的NOD/SCID糖尿病小鼠一侧附睾脂肪垫内,观察血糖变化。结果诱导第四阶段14天时hESs出现胰高糖素荧光表达;20天时细胞出现PDX-1和C肽共表达;22天形成的成熟胰岛细胞出现Glut-2和胰岛素的阳性表达;流式鉴定胰岛素阳性细胞占17.1%,C肽阳性细胞占3.8%;体外检测有葡萄糖刺激的胰岛素释放反应。分化成熟胰岛细胞约(3～5)×106植入NOD/SCID糖尿病小鼠体内可以逆转其高血糖至少8周。结论体外定向诱导hESs分化形成的胰岛细胞植入NOD/SCID糖尿病小鼠附睾脂肪垫内可以逆转其高血糖。

Objective To investigate whether pancreatic progenitors differentiated from human embryonic stem （hES） cells could correct hyperglycemia in non-obese diabetic（NOD）/severe combined immunodeficient（SCID） mice or not. Methods Pancreatic islets derived from hES cells line YT1 according to the optimized four-stage differentiation protocol in a chemical-defined culture system were observed. In the first stage,activin A and wortmannin were utilized to induce definitive endoderm formation. In the second stage, the differentiated endoderm cells were treated with all- trans retinoic acid（RA） ,NOGGIN and basic fibroblast growth factor（bFGF） to induce pancreatic specialization. In the third stage,pancreatic progenitors were induced by epidermal growth factor（EGF）-regulated expansion. Finally,in the fourth stage, a cocktail of factors was utilized to induce mature insulin-producing cells. The differentiated human pancreatic islet cells of （3-5）; 106 were then transplanted into one of the epididymal fat pads（EFP） of NOD/SCID mice. Graft survival and function were measured by blood glucose levels. Results The differentiated hES cells obtained by the four-stage approach comprised nearly 17.1 ; insulin-positive cells and 3.8 ; C-peptide positive as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing ceils co-expressed mature ; cell-specific markers such as PDX1, insulin,C-peptide and glucagon. After implantation into EFP of NOD/SCID mice, hES cell-derived pancreatic islets corrected hyperglycemia for an least eight weeks. Conclusion Human terminal differentiation pancreatic islet cells can correct hyperglycemia in NOD/SCID mice.