BMP4促进诱导多能干细胞向感觉神经元分化

BMP4 promotes differentiation of induced pluripotent stem cells into sensory neurons

目的探讨骨形成蛋白-4(BMP4)在诱导多能干细胞(iPSCs)向感觉神经元分化中的作用。方法采用碱性磷酸酶染色(AP)和拟胚体(EB)形成实验对iPSCs进行鉴定。采用神经营养因子诱导法将iPSCs向感觉神经元样细胞分化两周,对照组诱导方案为DMEM/12中加入2%B27、20 ng/mL碱性成纤维因子(bFGF)、200 ng/mL音猬因子(SHH)及10μmol/L全反式维甲酸(ATRA),BMP4组为对照组基础上添加10 ng/mL BMP4。通过细胞免疫荧光染色,检测诱导后细胞神经元活性,通过Realtime-PCR比较两组间细胞内感觉元相关基因mRNA相对表达量。结果iPSCs呈现出典型的类似胚胎干细胞样克隆细胞团块,AP为阳性,EB随机分化实验中能表达三个胚层相关基因,符合iPSCs生物学特性。BMP4组两周的神经元定向诱导后,iPSCs展现出双极或多极形态,神经元相关标志蛋白β-Tublin-Ⅲ表达阳性,表明诱导分化后的细胞具有神经元功能活性。Realtime-PCR结果显示在对照组vs BMP4组中,BRN3A相对表达量为(0.11±0.03)vs(0.25±0.04)、Neurog1为(1.02±0.32)vs(1.93±0.52)、GFAP为(0.21±0.04)vs(0.65±0.07),差异均具有显著统计学意义(P<0.01),NeuroD为(0.45±0.05)vs(0.52±0.04),差异无统计学意义(P>0.05),表明BMP4能显著提升诱导后细胞BRN3A、Neurog1、GFAP相对表达量。结论BMP4能显著提升iPSCs向感觉神经元样细胞诱导分化的效率,为后续实验研究提供了有力的保障。

Objective To investigate the role of bone morphogenetic protein-4(BMP4)in the induction of pluripotent stem cells(iPSCs)into sensory neurons.Methods iPSCs were identified by alkaline phosphatase staining and embryoid body formation experiments.Using neurotrophic factor induction,iPSCs were differentiated into sensory neuron-like cells for 2 weeks.The control group was induced with DMEM/12 with 2%B27,20 ng/mL basic fibroblast factor(bFGF),200 ng/mL sonic hedgehog homolog(SHH),and 10μmol/L all-trans retinoic acid(ATRA).The BMP4 group added 10 ng/mL BMP4 based on the treatment of the control group.The neuronal activity of the cells was detected by immunofluorescence staining,and the relative expression levels of sensory neuron-related genes were compared by realtime-PCR.Results iPSCs exhibited the morphological characteristics of embryonic stem cell,which were positive for alkaline phosphatase staining.Three embryonic layer-related genes could be expressed in the embryonic body random differentiation experiment.After 2 weeks of neuronal induction in the BMP4 group,iPSCs exhibited bipolar or multipolar morphology,and the sensory neuron-associated marker proteinβ-Tublin-Ⅲwas positive.Realtime-PCR results showed that the relative expression of BRN3A,Neurog1,and GFAP in the control group vs BMP4 group were(0.11±0.03)vs(0.25±0.04),(1.02±0.32)vs(1.93±0.52),(0.21±0.04)vs(0.65±0.07),and the differences were statistically significant(P<0.01).The relative expression of NeuroD was(0.45±0.05)vs(0.52±0.04),and the difference was not significant(P>0.05).Conclusion BMP4 can significantly improve the efficiency of iPSCs differentiation into sensory neuron-like cells,which provides a powerful guarantee for subsequent experimental research.