脐带间充质细胞来源诱导性多潜能干细胞的建立和鉴定

Generation of human induced pluripotent stem cells from umbilical cord matrix cells

目的建立和鉴定脐带间充质细胞（UMC）来源的诱导性多潜能干细胞（iPS）。方法以逆转录病毒作为Sox2、KIf4、Oct4、c—Myc基因转移载体，将UMC细胞编程为iPS细胞。应用RT-PCR检测细胞基因表达量，鉴定外源编程基因是否整合到iPS细胞核内，分析细胞核型，对细胞进行碱性磷酸酶染色和免疫荧光染色，体内分化畸胎瘤和体外分化拟胚体实验，对建立的iPS细胞进行鉴定。结果（1）iPS细胞形态学上类似于胚胎干细胞（ES）。（2）iPS与ES细胞内源多能基因（Nanog、Oct-4、Rexl、Sox-2）表达谱相类似，外源编程基因（Sox-2、KIf4、Oct-4、c—Myc）表达发生沉默。（3）外源编程基因已被插入到iPS细胞核内。（4）iPS细胞核型正常，碱性磷酸酶活性增高，表达Es细胞特异性膜蛋白（SSEA3、SSEA4），质蛋白（TRA-1-60、TRA-1—81），核蛋白（Nanog）。（5）iPS细胞在体内能分化为畸胎瘤，在体外能分化为拟胚体。结论iPS细胞类似于Es细胞具有多向分化潜能。

Objective To generate of human induced pluripotent stem cells from umbilical cord matrix cells（UMC）. Methods Sox2 and Klf4 and Oct4 and c-Myc were transfected into UMC cells with retrovirus, and then UMC cells was reprogrammed to iPS cells. Gene expression was confirmed with RT - PCR and the integration was confirmed with cell karyotype, iPS cells were further validated with cell alkaline phosphatase detection and immunofluorescence staining, differentiating into teratomas in vivo and embryoid bodies in vitro. Results iPS cells were similar to embryonic stem cells （ES）. The expression of Nanog, Oct4, Rexl and Sox2 in iPS cells were higher than UMC cells, and Sox2, Klf4, Oct4, c-Myc silenced in iPS ceils. Exogenous genes were inserted into the nucleus of iPS cells, which was confirmed by 1% agarose gel electrophoresis, iPS cell karyotype was normal, alkaline phosphatase activity increased, ES cell-specific proteins, including SSEA3, SSEA4, TRA-1-60, TRA-1-81 and Nanog, were expressed, iPS cells were differ- entiated into a teratoma in vivo and embryoid bodies in vitro. Conclusions iPS cells were similar to ES cells, which had pluripotency.