利用尿液细胞诱导建立血友病A患者特异性多潜能干细胞

摘要

Objective To generate hemophilia A (HA) patient-specific inducible pluripotent stem cells (iPSCs) and induce endothelial differentiation.Methods Tubular epithelial cells were isolated and cultured from the urine of HA patients.The iPSCs were generated by forced expression of Yamanaka factors (Oct4, Sox2, c-Myc and Klf4) using retroviruses and characterized by cell morphology, pluripotent marker staining and in vivo differentiation through teratoma formation.Induced endothelial differentiation of the iPSCs was achieved with the OP9 cell co-culture method.Results Patient-specific iPSCs were generated from urine cells of the HA patients, which could be identified by cell morphology, pluripotent stem cell surface marker staining and in vivo differentiation of three germ layers.The teratoma experiment has confirmed that such cells could differentiate into endothelial cells expressing the endothelial-specific markers CD144, CD31 and vWF.Conclusion HA patient-specific iPSCs could be generated from urine cells and can differentiate into endothelial cells.This has provided a new HA disease modeling approach and may serve as an applicable autologous cell source for gene correction and cell therapy studies for HA.%目的 通过逆转录病毒方法诱导建立两例血友病A(hemophilia A,HA)患者特异性诱导多潜能干细胞(inducible pluripotent stem cells,iPSCs)并定向分化为内皮细胞,为HA的发病机制以及细胞和基因治疗研究提供理想的细胞来源.方法 收集培养血友病A患者尿液细胞,使用携带Oct4、Sox2、c-Myc和Klf4等4种因子的逆转录病毒感染尿液细胞,诱导出HA患者特异性的诱导多潜能干细胞;通过形态学、干细胞多潜能表面标志物免疫荧光检测,成畸胎瘤实验鉴定iPSCs;iPSCs与OP9细胞共培养分化为内皮细胞.结果 成功建立了HA患者特异性iPSCs,其形态与人胚胎干细胞相似,细胞表面标志物免疫荧光检测均符合干细胞基因表达特征,成畸胎瘤实验显示其可在体内随机分化为三胚层组织;将iPSCs定向分化为内皮细胞,细胞表面标志物CD144、CD31、vWF免疫荧光均为阳性.结论 尿液细胞可以重编程得到血友病A患者特异性iPSCs,并能定向分化为内皮细胞,为血友病A研究提供细胞模型,也为后续基因修正后的细胞治疗提供了自体化的细胞来源.