无精症患者非基因整合诱导多能干细胞系的建立

Generation of pluripotent stem cells using non-integrated approach in human azoospermia patients

目的利用非基因整合方法建立无精症患者的诱导多能干细胞系(iPSCs)。方法采用目前商品化的无血清完全培养基(TeSR?2)和胚胎干细胞培养基(Stem Adhere? Defined Matrix)限定培养体系,利用非基因整合的仙台病毒感染无精症患者外周血单个核细胞,建立了无精症患者的i PSCs系,应用免疫荧光、核型分析、拟胚体和畸胎瘤实验对iPSCs系的多能性、核型及分化能力进行鉴定。结果建立的iPSCs系具有人胚胎干细胞的特征。免疫荧光显示,干细胞多能性标志基因表达的八聚体结合转录因子4(OCT4)、性别决定基因相关蛋白2(SOX2)以及干细胞特异表面抗原阶段特异性胚胎抗原-4(SSEA-4)、肿瘤排斥抗原-1-60(TRA-1-60)均阳性;核型分析显示其具有正常的核型;拟胚体和畸胎瘤实验显示其在体外和体内具有向3个胚层分化的能力。结论利用非基因整合方法成功构建了无精症患者的诱导多能干细胞系。

Objective To establish induced pluripotent stem cells(iPSCs)in patients with azoospermia by nonintegratedapproach.Methods Using the commercially available serum-free medium(TeSR?2)and embryonic stem cellculture medium(Stem Adhere?Defined Matrix)to define the culture system,the iPSCs were established by using nonintegratedSendai virus infection in peripheral blood mononuclear cells of azoospermia patients.The immunofluorescence,karyotype analysis,embryoid body differentiation and teratoma formation were used to identify pluripotency,karyotype anddifferentiation ability of iPSCs.Results The established iPSCs showed the characteristics of human embryonic stem cells.Immunofluorescence analysis showed that octamer-binding transcription factor4(OCT4),SRY-related-box protein-2(SOX2),stage-specific embryonic antigen-4(SSEA-4)and tumor rejection antigen-1-60(TRA-1-60)were positive for theexpression of stem cell pluripotency markers.Karyotype analysis showed that they had normal karyotype.In addition,embryoid body and teratoma tests showed that the iPSCs had the ability to differentiate into three germ layers in vitro and invivo.Conclusion The induction of pluripotent stem cell line is successfully constructed by non-integrated approach inazoospermia patients.