Zhou Shuyan, Li Furong, Li Yang, Zhang Cui, Sun Yao, Zhang Genbao
Department of Pathophysiology, Wannan Medical College, Wuhu 241002; Key Laboratory of Stem Cell and Cellular Therapy, Second Clinical Medical College, Shenzhen People's Hospital, Jinan University, Shenzhen 518020, China.
Key Laboratory of Stem Cell and Cellular Therapy, Second Clinical Medical College, Shenzhen People's Hospital, Jinan University, Shenzhen 518020, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Aug;34(8):718-724.
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 development-related 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 (only 1/8 in the control group). Conclusion Knockdown of LSD1 can promote the efficient differentiation of hiPSCs into IPCs in vitro.
目的 研究一种在体外将人诱导多能干细胞（hiPSCs）高效分化为胰岛素分泌细胞（IPCs）的方案。方法 通过RNA干扰在hiPSCs中敲低赖氨酸特异性去甲基化酶1（LSD1）基因。采用四步法诱导hiPSCs分化为IPCs。通过流式细胞术分析IPCs的分化效率。采用实时定量PCR检测LSD1、OCT4、SOX17、FOXA2、PDX1、PAX4、PAX6、HNF6、TCF1、NKX6.1、GLUT2、GK、胰岛素和MAFA的mRNA水平。采用免疫荧光技术检测PDX1和胰岛素的表达及定位。采用DTZ染色和透射电子显微镜观察IPCs中含胰岛素颗粒的分泌及分布情况。此外，采用ELISA检测IPCs中胰岛素和C肽的产量。结果 与对照组相比，LSD1敲低组的IPCs分化效率更高，胰岛β细胞发育相关基因SOX17、PDX1、PAX4和胰岛素的mRNA表达显著上调。LSD1敲低组的IPCs共表达成熟的β细胞特异性标志物PDX1和胰岛素。在LSD1敲低组中，IPCs在葡萄糖刺激下以分泌囊泡的形式释放胰岛素，胰岛素或C肽的产量达到成人胰岛的1/6（对照组仅为1/8）。结论 敲低LSD1可促进hiPSCs在体外高效分化为IPCs。
