敲低赖氨酸特异性去甲基化酶1(LSD1)促进人诱导性多能干细胞分化为产胰岛素细胞

Knockdown of LSD1 promotes differentiation of human induced pluripotent stem cells into insulin-producing cells in vitro

目的探索一种人诱导性多能干细胞(hiPSC)体外高效分化为产胰岛素细胞(IPC)的诱导方案。方法 RNAi技术敲低hiPSC赖氨酸特异性去甲基化酶1(LSD1)基因后,利用四步法诱导其体外分化为IPC。流式细胞术分析IPC分化效率,实时定量PCR检测细胞LSD1、POU5同源盒转录因子1(OCT4)、Y染色体性别决定区因子17(SOX17)、叉头盒蛋白A2 (FOXA2)、胰腺和十二指肠同源盒蛋白1 (PDX1)、配对盒转录因子4(PAX4)、PAX6、肝细胞核因子6 (HNF6)、肝细胞核因子1同源盒蛋白A(TCF1)、NK6同源盒蛋白1(NKX6. 1)、葡萄糖转运子2(GLUT2)、葡萄糖激酶(GK)、胰岛素及MAF b ZIP转录因子A(MAFA)的mRNA水平,免疫荧光技术检测细胞PDX1、胰岛素的表达和定位;双硫腙(DTZ)染色和透射电镜分别观察细胞内胰岛素的分泌和分泌颗粒分布情况; ELISA检测诱导后细胞胰岛素和C肽分泌量。结果 LSD1敲低组的IPC分化效率较对照组有明显提高,胰岛β细胞发育相关基因SOX17、PDX1、PAX4、胰岛素的mRNA表达显著上调。LSD1敲低组的IPC共表达成熟β细胞特异性蛋白PDX1和胰岛素。另外,这些IPC能感应葡萄糖刺激并以胰岛素分泌小泡形式释放胰岛素,胰岛素或C肽释放量约为天然胰岛细胞的1/6(而对照组仅为1/8)。结论敲低LSD1基因可以促进hiPSC体外高效分化为IPC。

Objective To investigate a protocol for the efficient differentiation of human induced pluripotent stem cells( hiPSCs) into insulin-producing cells( IPCs) in vitro. Methods Lysine-specific demethylase 1( LSD1) gene was knocked down in hiPSCs by RNAi. A four-step method was performed to induce the differentiation of hiPSCs into IPCs. The differentiation efficiency of IPCs was analyzed by flow cytometry. Real-time quantitative PCR was used to detect the mRNA levels of LSD1,OCT4,SOX17,FOXA2,PDX1,PAX4,PAX6,HNF6,TCF1,NKX6. 1,GLUT2,GK,insulin and MAFA. The expression and localization of PDX1 and insulin were determined by immunofluorescence technique. DTZ staining and transmission electron microscopy were used to observe the secretion and distribution of intracellular insulin-containing granules in IPCs. In addition,the yield of insulin and C-peptide of IPCs were tested by ELISA. Results Compared with the control,the LSD1 knockdown group showed a higher differentiation efficiency of IPCs and the mRNA expression of pancreatic islet β-cell developmentrelated genes SOX17,PDX1,PAX4 and insulin were significantly up-regulated. IPCs from the LSD1 knockdown group co-expressed mature β-cell specific markers PDX1 and insulin. In the LSD1 knockdown group,IPCs released insulin as secretory vesicles in response to glucose stimuli,and the yield of insulin or C-peptide reached 1/6 of adult human islets( only1/8 in the control group). Conclusion Knockdown of LSD1 can promote the efficient differentiation of hiPSCs into IPCs in vitro.