hiPSCs向多巴胺能神经前体细胞的定向分化对细胞增殖和迁移的影响

Effects of hiPSCs directed differentiation into dopaminergic neural progenitors on proliferationand migration

目的探讨人诱导性多能干细胞(hiPSCs)向多巴胺能神经前体细胞(DAPs)的定向分化对细胞增殖和迁移的影响,为优化诱导分化方案和细胞移植治疗提供新思路。方法hiPSCs的诱导分化分为实验组(Exp)和对照组(Cont)。实验组的诱导分化方案是在基础诱导培养基上添加3个信号通路的激活剂或抑制剂,对照组则仅使用基础诱导培养基。通过实时荧光定量PCR(qPCR)和免疫荧光检测诱导分化第0、7、14天的细胞多能性标志物NANOG、OCT4和SOX2,细胞增殖标志物KI67,神经干细胞(NSCs)标志物NESTIN,底板标志物FOXA2和DAPs标志物EN1、LMX1A、MSX1的表达;CCK8和划痕实验分别检测细胞增殖和迁移能力。结果qPCR结果显示,在诱导分化过程中,对照组和实验组的多能性基因NANOG的表达均下降,且实验组下降趋势更明显(D7:P=0.0019,D14:P=0.0001)。与对照组相比较,实验组第7天和第14天NESTIN、EN1、LMX1A和MSX1的mRNA水平表达均明显增加(NESTIN:P=0.0043;EN1:P=0.0002、LMX1A:P=0.0142、MSX1;P=0.0233)。免疫荧光染色结果表明,两组未诱导的hiPSCs均大量表达NANOG、OCT4、SOX2和KI67;第7天,实验组细胞大量表达FOXA2/NESTIN双阳性,对照组细胞大多仅表达NESTIN阳性;第14天,实验组大量表达EN1、LMX1A、MSX1,而对照组表达较少;CCK8增殖实验结果显示,第7天和14天时,实验组的450nm吸光值均高于对照组(D7:P=0.0430;D14:P=0.0001);划痕实验结果显示,在第7天时,对照组与实验组的划痕愈合趋势的差异无统计学意义(P>0.05),而第14天时,对照组相对于实验组的24 h划痕愈合趋势明显增加(P=0.0023)。结论诱导分化方案添加的通路激活剂或抑制剂,可以促进hiPSCs向DAPs的分化,在总体水平能够促进细胞增殖,而抑制细胞分化后期的迁移能力。

Objective To explore the effect of the directed differentiation of human induced pluripotent stem cells(hiPSCs)into dopaminergic neural progenitors(DAPs)on cell proliferation and migration,and to provide ideas and methods for optimizing the induction and differentiation scheme.Methods The induced differentiation of hiPSCs was divided into experimental group(Exp)and control group(Cont).In the experimental group,activators or inhibitors of the three signaling pathways were added to the basal induction medium,while in the control group,only basal induction medium was used.At day 0,day 7 and day 14 during the differentiation process,real-time fluorescence quantitative PCR(qPCR)and immunofluorescence were used to detect the expression of pluripotent markers NANOG,OCT4 and SOX2;cell proliferation marker KI67;neural stem cell marker NESTIN;floor marker FOXA2;DAPs markers EN1,LMX1A and MSX1.The proliferation ability of the cells was detected by CCK8 assay,and the migration ability of the cells was detected by wound scratch assay.Results QPCR results showed that the expression of pluripotent gene NANOG decreased in both the control group and the experimental group,andwas more obvious in the experimental group(D7:P=0.0019,D14:P=0.0001);Compared with the control group,mRNA levels of NESTIN,EN1,LMX1A and MSX1 in the experimental group were signifcantly increased on day 7 and 14(NESTIN:P=0.0043;EN1:P-0.0002;LMX1A:P=0.0142;MSX1:P=0.0233,respetively).Immunofuorescence staining results showed that most of the uninduced hiPSCs expressed NANOG,0CT4,S0X2 and KI67;On day 7,F0XA2/NESTIN double positive cells were mostly observed in the experimental group,while NESTIN positive cells mostly in the control group;On day 14,EN1 LMXIA.MSX1 was highly expressed in the experimental group,while less expressed in the control group.CCK8 proliferation assay showed that the 450nm absorbance value of the experimental group was higher than that of the control group(D7:P=0.0430;DI4:P=0.0001).Scratch test results showed that on day 7,there was no signifcant dfference in the healing area between the control group and the experimental group(P>0.05);On the 14th day,compared with the experimental group,the 24 h scratch healing trend was significantly increased in the con-trol group(P=0.0023).Conclusion The addition of pathway activators or inhibitors in the induction differentiation scheme can promote the diferentiation of hiPSCs into DAPs,promote cell proliferation at the overall level,and inhibit the migration ability of cells at the later stage of differentiation.