人包皮成纤维细胞来源的多能性干细胞的建立

Generation of induced pluripotent stem cells from human foreskin fibroblasts

目的探讨人包皮成纤维细胞重编程为诱导多能性干细胞(iPSCs)的方法。方法利用反转录病毒携带绿色荧光蛋白(GFP)作为指示,转导到人包皮成纤维细胞中,确定最佳的病毒感染剂量,应用经典的Yamanaka方法,联合小分子物质组蛋白去乙酰化酶抑制剂丙戊酸盐(VPA)诱导人包皮成纤维细胞为多能性干细胞。通过碱性磷酸酶(AKP)染色及免疫荧光细胞化学方法,检测人胚胎干细胞多能性标记物在所建立的诱导多能性干细胞(iPSCs)中的表达,RT-PCR检测拟胚体(EB)三胚层特异基因的表达。结果以GFP作为指示,建立了高效的反转录病毒感染体系,将人包皮成纤维细胞重编程为iPSCs;所建立的iPSCsAKP染色阳性,表达人胚胎干细胞多能性标记物OCT4、TRA-1-60和TRA-1-81,体外诱导分化后具有三胚层特异基因的表达;小分子物质VPA的添加并未显著提高重编程效率。结论在GFP所指示的最佳病毒感染剂量下,联合小分子物质VPA成功建立了人包皮成纤维细胞来源的iPSCs。

Objective To optimize the method for establishment of induced pluripotent stem cells from human foreskin fibroblasts. Methods We used GFP as an indicator to determine the optimized dose of the retrovirus during the reprogramming of human foreskin fibroblasts into human induced pluripotent stem cells （iPSCs） by Yamanaka＇ s classical strategy with the combination of the small-molecule compounds VPA, a histone deaeetylase inhibitor. The characteristics of established iPSCs were tested via alkaline phophatase （AP） staining, immunofluorescent detection of human embryonic stem （ES） cells surface antigens, and RT-PCR analysis of three germ layers specific gene expression in cell during differentiation in vitro. Results Using GFP expression as an indicator of the retrovirus transduction, we established a high efficient system to successfully reprogramme human foreskin fibroblasts into induced pluripotent stem ceils. The iPSCs showed positive for alkaline phosphatase, expressed high levels of human ES cells pluripotency markers such as OCT4, TRA-1-60 and TRA-1-81. RT-PCR analysis indicated that the germ layers differentiation related gene expression was detected in the embryonic body when derived from these hiPS cells. Conclusion Our results showed that we successfully generated hiPS cells from adult foreskin fibroblasts in the indication of GFP with the combination of the small-molecule compounds VPA. [