定向诱导人尿液细胞源性多能干细胞向多巴胺能神经元的分化

Directed differentiation of human urine cell-derived pluripotent stem cells into dopaminergic neurons

目的探讨定向诱导人尿液来源诱导性多能干细胞(U-iPS)向多巴胺(DA)能神经元分化。方法单层贴壁法体外培养U-iPS,通过时向性添加TGF-β及BMP信号通路抑制剂,SHH细胞因子,GSK3β抑制剂Chir99021将U-iPS诱导为DA能神经前体细胞(NPCs),通过添加BDNF/GDNF等细胞因子将其进一步分化为DA能神经元。在诱导分化的4个时间点(第0天、7天、14天及25天)采用RT-qPCR和免疫荧光检测多能干细胞标志物Oct4与Nanog的表达;NPCs标志物Nestin、Pax6和Foxa2的表达;DA神经前体细胞标志物Lmx1a和En1的表达;神经元标志物Tuj1的表达以及DA能神经元标志物TH的表达情况。以人皮肤成纤维细胞来源的iPS(F-iPS)向DA能神经元的诱导分化作为对照进行分化效率的比较。结果U-iPS在第0天表达多能干标志物Oct4和Nanog;诱导第7天能转化为Nestin、Pax6及Foxa2阳性的NPCs;第14天转化为Lmx1a和En1阳性的DA能神经前体细胞;第25天分化得到的神经元样细胞表达神经元标志物Tuj1,其中部分细胞表达DA能神经元标志物TH。各时间点神经类细胞标志物的表达与F-iPS诱导分化得到的神经类细胞标志物的表达差异无统计学意义(P>0.05)。结论U-iPS细胞具有向DA能神经元分化的潜力,能在体外经过诱导分化得到TH阳性的DA能神经元,且与F-iPS向DA能神经元分化的效率无明显差异。

Objective To explore directed differentiation of human urine cell-derived pluripotent stem cells(U-iPS)into dopaminergic neurons.Methods U-iPS was cultured in vitro by single-layer adherence method,and U-iPS was induced into dopaminergic neural precursor cells by adding TGF-βand BMP signaling pathway inhibitors,SHH cytokine and GSK3βinhibitor Chir99021.Cytokines such as BDNF/GDNF are added to further differentiation into dopaminergic neurons.RTqPCR and immunofluorescence were used to detect the expression of pluripotent stem cell markers Oct4 and Nanog,the expression of neural precursor cell marker Nestin,Pax6 and Foxa2,the expression of the dopaminergic neuron precursor cell markers Lmxla/Enl,the expression of the neuronal marker Tuj1 and the expression of the dopaminergic neuron marker TH at four time points(days 0,7,14 and 25 days)during the induction and differentiation period.The differentiation efficiency of iPS derived from human skin fibroblasts(F-iPS)to dopaminergic neurons was used here as a control.Results U-iPS expressed pluripotent markers Oct4 and Nanog on day 0,it was then transformed into Nestin,Pax6 and Foxa2-positive neural progenitor cells on i nduction day 7 and converted into Lmx1 a and Enl positive dopaminergic progenitor cells on day 14.Neuron-like cells differentiated on day 25 express the neuronal marker Tuj1,some of which express the dopaminergic neuron marker TH.There is no significant difference in the expression of neuronal cell markers at each time point and the expression of neuronal cell markers obtained by F-iPS-induced differentiation(P>0.05).Conclusion U-iPS cells have the potential to differentiate into dopaminergic neurons.They can be induced into TH-positive DA neurons in vitro,and there is no significant difference in the dopaminergic neuron differentiation efficiency between F-iPS and U-iPS.