2种不同诱导方法体外诱导人多潜能干细胞分化成大脑基底内侧神经节隆起区神经前体细胞的差异  

Comparison of Inducing Human Pluripotent Stem Cells to Differentiate into Medial Ganglionic Eminence Neural Progenitor Cells in vitro with Two Culture Methods

作　　者：韩肖华 陈潞婷 李娟[1] 李丹萍 陈红[1] HAN Xiao-hua;CHEN Lu-ting;Li Juan;Li Dan-ping;Chen Hong(Department of Rehabilitation Medicine,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China;Department of Reha-bilitation Medicine,General Hospital of the Yangtze River Shipping,Wuhan 430019,China)

机构地区：[1]华中科技大学同济医学院附属同济医院康复医学科,武汉430030 [2]长江航运总医院康复医学科,武汉430019

出　　处：《神经损伤与功能重建》2021年第10期565-568,共4页Neural Injury and Functional Reconstruction

基　　金：国家自然科学基金(No.81774404)。

摘　　要：目的:比较单层贴壁培养法和拟胚体(EB)悬浮培养法诱导人多潜能干细胞分化为大脑基底内侧神经节隆起区(MGE)神经前体细胞分化的效率,并探讨不同浓度的SHH通路激动剂SAG对分化为MGE神经前体细胞纯度的影响,为细胞移植治疗探索优化的分化方案。方法:分别采用单层贴壁诱导法和EB悬浮法诱导人多潜能干细胞分化为神经上皮细胞,通过加入不同浓度的SAG使其腹侧化,最终诱导成表达NKX2.1的MGE前体细胞并进行计数。比较2种不同的分化方案和不同浓度的SAG对MGE神经前体细胞纯度的影响。结果:在单层贴壁诱导法中,当SAG浓度为0μM、0.1μM、0.5μM、1μM、2μM和3μM时,NKX2.1阳性细胞率分别为0%、(48.3±11.5)%、(78.6±2.7)%、(77.6±4.0)%、(69.0±7.5)%和(67.7±7.9)%;SAG浓度为0.5μM和1μM时诱导分化获得的MGE神经前体细胞纯度显著高于其他浓度组(P<0.001或0.05)。当SAG浓度为0.5μM时,在EB悬浮培养法中NKX2.1的阳性细胞率为(74.8±6.5)%,与单层贴壁诱导法差异无统计学意义(P>0.05)。采用单层贴壁诱导法时,神经球和神经元出现的时间点均要早于采用EB悬浮培养法。结论:采用单层贴壁诱导法,0.5μM的SAG可能是人多潜能干细胞分化培养为MGE神经前体细胞的更优化方案。Objective:To compare the different effects of inducing human pluripotent stem cells to differentiate into medial ganglionic eminence(MGE)neural progenitor cells with monolayer adherent culture and embryoid bodies(EB)culture methods and to explore the effect of different concentrations of SAG(SHH signaling agonist)on the purity of neural progenitor cells differentiated into MGE.Methods:Human pluripotent stem cells were dif-ferentiated into neuro epithelial cells using monolayer adherent culture and EB culture methods.Varying concentra-tions of SAG were used to induce ventralization.The cells were ultimately induced to become NKX2.1-expressing MGE neuro progenitor cells and then counted.The effect of the two culture methods and the different concentra-tions of SAG on the purity of MGE neural progenitor cells were investigated.Results:In the monolayer adherent culture method,the positive expression of NKX2.1 was 0%in 0μM SAG,(48.3±11.5)%in 0.1μM SAG,(78.6±2.7)%in 0.5μM SAG,(77.6±4.0)%in 1μM SAG,(69.0±7.5)%in 2μM SAG,and(67.7±7.9)%in 3μM SAG.The purity of differentiated MGE neural progenitor cells induced by 0.5μM and 1μM SAG was superior to those in-duced by other concentrations(P<0.001 or 0.05).Using the EB culture method with 0.5μM SAG,the positive ex-pression of NKX2.1 was(74.8±6.5)%and showed no significant difference from the monolayer adherent culture method(P>0.05).Neurosphere and neuron formation occurred earlier when using the monolayer adherent culture method than when using the EB culture method.Conclusion:To obtain MGE neural progenitor cells from human pluripotent stem cells for transplantation treatment,the monolayer adherent culture method with 0.5μM SAG may be optimal.

关 键 词：单层贴壁培养法 拟胚体悬浮培养法 人多潜能干细胞 大脑基底内侧神经节隆起区 神经前体细胞 SAG浓度 

分 类 号：R741[医药卫生—神经病学与精神病学] R741.02[医药卫生—临床医学]