Transwell接触共培养促进单散iPSCs生长及分化
发布时间：2018-08-10 19:11
【摘要】：目的:观察Transwell接触共培养促进单散人诱导多能干细胞(induced pluripotent stem cells,iPSCs)生长及分化的作用。方法:将1~2代牛角膜内皮细胞(corneal endothelial cells,CECs)接种在Transwell小室底面培养8 h后,应用Accutase消化及40μm过滤处理获得单散iPSCs,将其接种到已有CECs的Transwell小室内共培养14 d,前3 d使用mTeSR1培养基,第4天开始用含10%胎牛血清的低糖DMEM培养基。分别进行实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction,qPCR)、免疫荧光、死活细胞染色及碱性磷酸酶(alkaline phosphatase,ALP)染色,对iPSCs多能特性表达及分化进行鉴定。设定单散iPSCs共培养组为实验组,常规培养iPSCs组为对照组(一),非共培养单散iPSCs组为对照组(二)。结果:培养牛CECs形态呈典型的六边形铺路石样外观。人iPSCs呈克隆样生长,共培养3 d后iPSCs贴壁呈单散细胞生长,免疫荧光检测未分化标志Nanog和Oct4呈阳性。qPCR检测Nanog、Oct4和Sox2 mRNA表达,实验组与对照组(一)比较差异无统计学意义(P0.05)。死活细胞染色显示,实验组死细胞明显减少,与对照组(二)比较差异有统计学意义(P0.01)。共培养14 d后,人iPSCs形态比较均一,呈多边形,体积增大,无明显克隆团块;ALP染色阴性;免疫荧光染色ZO-1、AQP1和CD31表达阳性,CD34和CD133表达阴性。qPCR检测Oct4、Nanog和Sox2 mRNA表达明显下调,与对照组(一)比较差异有统计学意义(P0.01)。结论:与牛CECs共培养可增强人单散iPSCs活性,使iPSCs形态上向内皮样细胞转化,表达部分CECs的标志。Transwell接触共培养模型可以促进单散iPSCs生长及分化。
[Abstract]:Aim: to observe the effect of co-culture of Transwell on the growth and differentiation of (induced pluripotent stem cells. Methods: bovine corneal endothelial cells (corneal endothelial cells) were cultured at the bottom of Transwell chamber for 8 h, then digested with Accutase and treated with 40 渭 m filtration. The cells were inoculated into the Transwell chamber of CECs for 14 days, and mTeSR1 medium was used for the first 3 days. On the 4th day, low sugar DMEM medium containing 10% fetal bovine serum was used. The multipotent expression and differentiation of iPSCs were identified by real-time fluorescence quantitative polymerase chain reaction (real-time fluorescence quantitative polymerase chain reaction- Q PCR), immunofluorescence, live and dead cell staining and alkaline phosphatase (alkaline) staining. Single powder iPSCs co-culture group was established as experimental group, conventional cultured iPSCs group as control group (1) and non co cultured single powder iPSCs group as control group (2). Results: the morphology of cultured bovine CECs showed typical hexagonal paving stone appearance. After 3 days of co-culture, human iPSCs grew as a single cell, and Nanog and Oct4 were positive for Nanog and Oct4. The expression of Nanog Oct4 and Sox2 mRNA in the experimental group was not significantly different from that in the control group (P0.05). The dead cells in the experimental group were significantly decreased compared with the control group (P0.01). After 14 days of co-culture, the morphology of human iPSCs was homogenous, polygonal, volume increased, and there was no significant negative staining of iPSCs, and the expression of CD34 and CD133 was negative in ZO-1AQP1 and CD31 by immunofluorescence staining. QPCR showed that the expression of Oct4Nangand Sox2 mRNA was down-regulated. Compared with the control group (1), the difference was statistically significant (P0.01). Conclusion: Co-culture with bovine CECs can enhance the activity of iPSCs and transform iPSCs into endothelium-like cells in morphology. Transwell contact co-culture model of partial CECs expression can promote the growth and differentiation of monodisperse iPSCs.
【作者单位】： 暨南大学附属第一医院眼科;暨南大学再生医学教育部重点实验室;暨南大学医学院眼科研究所;
【基金】：国家自然科学基金资助项目(No.81371689) 广东省自然科学基金资助项目(No.S2013010013391)
【分类号】：R329
本文编号：2175932
[Abstract]:Aim: to observe the effect of co-culture of Transwell on the growth and differentiation of (induced pluripotent stem cells. Methods: bovine corneal endothelial cells (corneal endothelial cells) were cultured at the bottom of Transwell chamber for 8 h, then digested with Accutase and treated with 40 渭 m filtration. The cells were inoculated into the Transwell chamber of CECs for 14 days, and mTeSR1 medium was used for the first 3 days. On the 4th day, low sugar DMEM medium containing 10% fetal bovine serum was used. The multipotent expression and differentiation of iPSCs were identified by real-time fluorescence quantitative polymerase chain reaction (real-time fluorescence quantitative polymerase chain reaction- Q PCR), immunofluorescence, live and dead cell staining and alkaline phosphatase (alkaline) staining. Single powder iPSCs co-culture group was established as experimental group, conventional cultured iPSCs group as control group (1) and non co cultured single powder iPSCs group as control group (2). Results: the morphology of cultured bovine CECs showed typical hexagonal paving stone appearance. After 3 days of co-culture, human iPSCs grew as a single cell, and Nanog and Oct4 were positive for Nanog and Oct4. The expression of Nanog Oct4 and Sox2 mRNA in the experimental group was not significantly different from that in the control group (P0.05). The dead cells in the experimental group were significantly decreased compared with the control group (P0.01). After 14 days of co-culture, the morphology of human iPSCs was homogenous, polygonal, volume increased, and there was no significant negative staining of iPSCs, and the expression of CD34 and CD133 was negative in ZO-1AQP1 and CD31 by immunofluorescence staining. QPCR showed that the expression of Oct4Nangand Sox2 mRNA was down-regulated. Compared with the control group (1), the difference was statistically significant (P0.01). Conclusion: Co-culture with bovine CECs can enhance the activity of iPSCs and transform iPSCs into endothelium-like cells in morphology. Transwell contact co-culture model of partial CECs expression can promote the growth and differentiation of monodisperse iPSCs.
【作者单位】： 暨南大学附属第一医院眼科;暨南大学再生医学教育部重点实验室;暨南大学医学院眼科研究所;
【基金】：国家自然科学基金资助项目(No.81371689) 广东省自然科学基金资助项目(No.S2013010013391)
【分类号】：R329
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