卵巢早衰患者来源的诱导多能干细胞的建系、鉴定及诱导分化

Generation,identification and differentiation of primary ovarian insufficiency patient-derived iPSCs

目的:建立和鉴定卵巢早衰(又称原发性卵巢功能不全,primary ovarian insufficiency,POI)患者来源的诱导多能干细胞(induced pluripotent stem cells,i PSCs),研究其向原始生殖细胞分化的潜能。方法:将表达Oct4、Sox2、c-Myc、Klf4和Lin28基因的p EB-C5和表达SV40 T抗原的p EB-Tg质粒转染POI患者的外周血单个核细胞,将其重编程为i PSCs并检测其干细胞多能性特性。添加转化生长因子β1(transfroming growth factor-β1,TGF-β1)和骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)后,应用real-time PCR和Western blot方法分别检测原始生殖细胞标志物的mRNA及蛋白表达水平。结果:通过碱性磷酸酶染色、细胞免疫荧光、拟胚体及畸胎瘤形成检测,证明了POI患者外周血来源的i PSCs可成功向3胚层细胞分化并保持多能性。添加TGF-β1和BMP4后,诱导组原始生殖细胞特异性标志物干细胞生长因子受体(stem cell growth factor receptor,c-Kit)、发育多能性相关蛋白3(developmental pluripotency-associated 3,STELLA/DPPA3)和DEAD盒多肽4(DEAD box polypeptide 4,VASA/DDX4)的mRNA水平明显升高(P<0.05),VASA/DDX4蛋白表达水平亦明显增加(P<0.05)。结论:POI患者外周血单个核细胞在体外可被成功诱导成无外源性基因整合的i PSCs,并且具有多能分化特性,添加TGF-β1和BMP4后可向原始生殖细胞分化。

AIM： To generate and identify primary ovarian insufficiency（ POI） patient-derived induced pluripotent stem cells（ iP SCs） and to explore their differentiation potential to primordial germ cells. METHODS： Plasmid pE B-C5 expressing reprogramming factors Oct4,Sox2,c-Myc,Klf4 and Lin28,and plasmid pE B-Tg expressing SV40 T antigen,were transfected into peripheral blood mononuclear cells（ PBMCs） derived from POI patients at the same time.PBMCs were reprogrammed into iP SCs,and the pluripotency of the cells was identified. After supplementation of transforming growth factor-β1（ TGF-β1） and bone morphogenetic protein 4（ BMP4）,the mRNA and protein expression of primordial germ cell markers was detected by real-time PCR and Western blot,respectively. RESULTS： iP SCs derived from the PBMCs of POI patient differentiated into 3-germ layer cells and maintained pluripotency by the detection of alkaline phosphatase staining,immunofluorescence,embryoid body and teratoma formation. After addition of TGF-β1 and BMP4,the primordial germ cell markers,including stem cell growth factor receptor（ c-Kit）,developmental pluripotency-associated 3（ STELLA/DPPA3） and DEAD box polypeptide 4（ VASA/DDX4） were increased at mRNA level（ P 0. 05）,and VASA / DDX4 was also up-regulated at protein level in induced group. CONCLUSION： PBMCs of POI patient are reprogrammed into integration-free iP SCs in vitro and maintain pluripotency. They differentiate into primordial germ cells byadding TGF-β1 and BMP4.