猪naive-like胚胎干细胞向神经细胞体外诱导分化

The induced differentiation of naive-like porcine embryonic stem cells into nerve cells in vitro

目的:将猪naive-like胚胎干细胞体外定向诱导分化为神经细胞,为猪多能干细胞诱导神经分化方法的建立提供参考。方法:利用小分子化合物β-巯基乙醇、B-27、肝素等组成的神经诱导培养液,采用直接分化法,分化得到神经样细胞,对其进行神经特异性基因表达鉴定,同时检测分化后的细胞是否已经丧失多能性。结果:分化所得到的神经细胞具有明显的神经细胞结构特征,RT-PCR和免疫荧光结果显示分化后的神经细胞表达多种神经细胞特异性基因,qPCR结果显示分化后多能因子的表达显著降低,神经特异性基因表达显著升高,免疫荧光结果经统计分析后显示,低分子量神经微丝蛋白(light neurofilament protein,NF-L)阳性细胞率可达79%。结论:成功实现了体外直接诱导猪naive-like胚胎干细胞向神经细胞分化,为神经系统疾病修复与治疗研究奠定了基础。

Objective:Porcine naive-like embryonic stem cells were induced to differentiate into neural cells in vitro,which provided basic information for the repair and treatment of the neurological disease.Methods:The neural-inducing culture medium with small molecule compoundsβ-mercaptoethanol,B-27 and heparin was used to differentiate the porcine naive-like embryonic stem cells towards the neural-like cells.We have derived neural-like cells with identification of the neural specific gene expression.We detected whether these cells could show limited pluripotency.Results:The neural-like cells had obvious neuronal cell structure.RT-PCR and immunofluorescence data showed that the differentiated neurons expressed a variety of neural cell-specific genes.QPCR results showed that the expression of pluripotent factors was decreased significantly after differentiation process and the expression of nerve-specific genes was significantly increased.The results of immunofluorescence showed that the rate of light neurofilament protein(NF-L)positive cells reached 79%.Conclusion:Neural cells could be directly induced from porcine naive-like embryonic stem cells in vitro.