人类诱导多能干细胞体外定向分化为神经干细胞的方法  

Directed differentiation of human induced pluripotent stem cells into neural stem cells in vitro

作　　者：王乐[1] 马丹丹 徐盼 张茜[1] 崔勇[2] 叶放蕾[1] WANG Le;MA Dandan;XU Pan;ZHANG Qian;CUI Yong;YE Fanglei(Department of Otology,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052;Department of Otorhinolaryngology,Guangdong Academy of Medical Sciences&Guangdong Provincial People′s Hospital,Guangzhou 510080)

机构地区：[1]郑州大学第一附属医院耳科,郑州450052 [2]广东省医学科学院·广东省人民医院耳鼻咽喉科,广州510080

出　　处：《郑州大学学报（医学版）》2021年第5期619-622,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金项目(82002876);广东省自然科学基金项目(2015A030313535);河南省科技攻关项目(192102310115)。

摘　　要：目的:探讨人类诱导多能干细胞(hiPSC)体外定向分化为神经干细胞的方法。方法:通过无血清悬浮培养和贴壁培养促使hiPSC定向分化。以添加小分子化合物(A8301和DMH1)的无血清悬浮培养基培养的细胞为实验组,以不添加任何诱导剂的无血清悬浮培养基培养的细胞作为对照组。利用qRT-PCR及免疫荧光染色检测细胞中Pax6和Nestin的表达情况。结果:悬浮培养第3天时可形成类胚体;贴壁并更换贴壁培养基后,类胚体开始分化形成神经花环;第12天时神经花环形成最多。与对照组相比,实验组细胞Pax6和Nestin mRNA表达量增加(P<0.05),定向分化的hiPSC共同表达Pax6和Nestin蛋白。结论:HiPSC可通过无血清悬浮培养和贴壁培养形成神经干细胞,小分子化合物DMH1和A8301可更有效促进其分化。Aim:To investigate the method of human induced pluripotent stem cell(hiPSC)directed differentiation into neural stem cells in vitro.Methods:HiPSC were cultured in serum-free suspension medium to form embryoid bodies,which were then cultured in adherent medium to form neural rosettes containing neural stem cells.The hiPSC cultured with serum-free suspension medium supplemented with small molecular compounds(A8301,DMH1)were the experiment group and those cultured with serum-free suspension medium without any inducer were selected as control group.The expression levels of Pax6 and Nestin in hiPSC were analyzed by qRT-PCR and immunofluorescence staining.Results:Embryoid bodies were formed on the 3rd day with suspension culture.After the adherent culture,the embryoid bodies began to differentiate into the neural rosettes.The largest number of neural rosettes were formed on the 12th day.The expressions of Pax6 and Nestin mRNA in the experiment group were increased(P<0.05).Directionally differentiated hiPSC co-expressed Pax6 and Nestin proteins.Conclusion:HiPSC could differentiate into neural stem cells through serum-free suspension culture and adherent culture in vitro.And small molecular compounds(DMH1,A8301)can effectively promote the differentiation of human induced pluripotent stem cells into neural stem cells.

关 键 词：人类诱导多能干细胞 神经干细胞 诱导分化 A8301 DMH1 

分 类 号：R318[医药卫生—生物医学工程]