hiPSC多能标志物的表达水平与谱系特异性分化潜能的相关性研究

Correlation between expression levels of pluripotent markers and lineage specific differentiation potential of hiPSC

目的研究人诱导型多能干细胞(hiPSC)不同多能标志物的相对表达水平与其心肌、神经分化潜能的相关性,为hiPSC作为种子细胞在心脏、神经再生医学研究中的应用提供实验指导。方法以人类胚胎干细胞(hESC)系h1ESC作为对照,通过实时定量聚合酶链反应(RT-qPCR)检测2株hiPSC细胞系多能性基因的表达水平;随后,分别利用心肌分化培养基与神经分化培养基对其进行诱导分化。在心肌分化中,通过比较搏动区域数量与面积以及心肌标志物的表达水平,鉴定不同hiPSC的心肌分化潜能;在神经分化中,通过比较早期花结样细胞团的数量与神经标志物的表达水平,鉴定不同hiPSC的神经分化潜能。最后,通过比对hiPSC多能标志物的表达水平高低与谱系分化潜能高低的对应关系,揭示两者的潜在相关性。结果RT-qPCR结果显示,两株hiPSC相比,hiPSC-0100细胞系中SOX2表达显著较高、NANOG显著较低,hiPSC-WTB细胞系SOX2表达显著较低、NANOG显著较高(P<0.05)。在分化潜能上,形态学与RT-qPCR检测共同显示,hiPSC-0100表现相对较低的心肌分化潜能、较高的神经分化潜能,hiPSC-WTB表现较高的心肌分化潜能、较低的神经分化潜能(P<0.05)。结论高表达SOX2、低表达NANOG的hiPSC细胞系倾向于具有较高的神经分化潜能;高表达NANOG、低表达SOX2的hiPSC细胞系倾向于具有较高的心肌分化潜能。

Objective To study the correlation between the relative expression levels of different pluripotent markers of human induced pluripotent stem cells(hiPSCs)and their myocardial and neural differentiation potential in order to guide the application of hiPSCs as seed cells in the research of heart and nerve regeneration medicine. Methods Human embryonic stem cells(hESCs)line h1ESC was used as control. Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expressions of pluripotent genes in two hiPSC cell lines. Then,they were induced to differentiate by cardiac differentiation medium and neural differentiation medium respectively.In the process of myocardial differentiation,the myocardial differentiation potential of different hiPSCs was identified by comparing the number and size of the beating areas and the expression levels of myocardial markers. In the course of neural differentiation,the number of early rosettelike cell clusters and the expression levels of neural markers were compared to identify the neural differentiation potential of different hiPSCs. Finally,by comparing the levels of expression of hiPSC pluripotent markers with the level of lineage differentiation potential,the potential correlation between the two was revealed. Results RT-qPCR results showed that the expression of SOX2 in the hiPSC-0100 cell line was significantly higher while NANOG was significantly lower,and that the expression of SOX2 in the hiPSC-WTB cell line was significantly lower while NANOG was significantly higher(P<0.05). In terms of differentiation potential,both morphology and RT-qPCR detection showed that hiPSC-0100 showed relatively low myocardial differentiation potential and higher neural differentiation potential,which was the opposite of hiPSC-WTB. Conclusion hiPSC cell lines with high expression of SOX2 and low expression of NANOG tend to have higher neural differentiation potential,while hiPSC cell lines with high expression of NANOG and low expression of SOX2 tend to have higher myocardial differentiation potential.