共培养环境中兔角膜内皮细胞诱导人iPSCs分化

Mixed culture of human iPSCs with rabbit corneal endothelial cells induces differentiation of iPSCs

目的:观察人诱导多能干细胞(induced pluripotent stem cells,iPSCs)与兔角膜内皮细胞(cornealendothelial cells,CECs)共培养前后的形态学变化,检测iPSCs向CECs分化前后部分标志蛋白表达的变化,为研究iPSCs向CECs的分化提供实验依据。方法:分离培养兔CECs并传代,同时使用无饲养层细胞法培养扩增人脐带源iPSCs,Western blotting对其进行鉴定;用量子点对iPSCs进行标记示踪,以不同密度比例建立iPSCs与CECs的共培养模式,用原子力显微镜(atomic force microscopy,AFM)结合倒置显微镜观察共培养前后iPSCs的形态学变化,免疫荧光法检测iPSCs分化前后CD34、CD133、CD31和水通道蛋白1(AQP1)蛋白表达的变化。结果:培养扩增的兔CECs形态为六边形,呈典型的铺路石样外观;iPSCs呈克隆样生长,Western blotting检测Oct4、Nanog和Sox2多能性标志蛋白均呈阳性;10 nmol/L量子点标记的iPSCs以1/4悬液与融合60%的兔CECs共培养为最佳模式;AFM结合倒置显微镜观察到共培养后iPSCs体积增大,胞浆增多,核浆比减小,细胞膜表面可见颗粒状突起物,膜表面粗糙度增加;免疫荧光法检测分化前iPSCs的CD34、CD133、CD31和AQP1表达均呈阴性,共培养2周后iPSCs的CD34、CD133和CD31表达阴性,AQP1表达阳性。结论:与兔CECs混合共培养后人iPSCs不仅从形态学上向内皮样细胞方向转化,同时表达角膜内皮细胞标志蛋白AQP1。

To investigate the morphology and protein expression of human induced pluripotent stem ceils （iPSCs） in the mixed -euhure environment with rabbit corneal endothelial cells （CECs） and to provide the experimental basis and the mechanism of iPSC differentiation into CECs. METHODS： Primary rabbit CECs were isolated with trypsin and subcultured. The human iPSCs were cultured and amplified by a feeder - free method and their characteristics were evaluated by Western blotting, iPSCs labeled with quantum dots of appropriate concentration were used to establish mixed - culture model with rabbit CECs. The morphology of iPSCs was evaluated by atomic force microscopy （AFM） and inverted mi- croscopy. The protein expression of CD31, CD34, CD133 and aquaporin 1 （AQP1） in iPSCs was tested by the method of im- munofluorescence. RESULTS： The rabbit CECs were hexagonal and showed a typical cobblestone appearance, iPSCs grew in the cloning form, and 3 pluripotent proteins Oct4, Nanog and Sox2 were expressed positively. 1/4 suspension of iPSCs la- beled with l0 nmol/L quantum dots and 60% confluence of rabbit CECs made best mixed culture for each other. Under AFM and inverted microscope, the volume of iPSC became bigger and the nuclear - cytoplasm ratio was decreased after 7 days of mixed culture. Some granular protrusions of the membrane were observed and the surface roughness of the cell membrane in- creased. The protein expression of CD31, CD34, CD133 and AQP1 in iPSCs was negative, while AQP1 was detected after mixed cuhure for 2 weeks. CONCLUSION ： iPSCs morphologically change to endothelial - like cells after mixed culture with rabbit CECs and express the marker protein AQP1 of CECs at the same time.