一种用于药物神经毒性体外评价的外周神经元分化方法的建立

Establishment of a Peripheral Neuron Differentiation Method for in vitro Drug-induced Neurotoxicity Evaluation

本试验探索了一种人诱导多能干细胞(hiPSC)向外周神经元定向诱导分化的方案,并用以评价药物神经毒性。采用小分子抑制剂及神经营养因子将hiPSC进行定向诱导分化,成功建立hiPSC分化的外周神经元(hiPSC-PNs)模型,并通过免疫荧光法和实时荧光定量逆转录聚合酶链式反应(RT-qPCR)对其相关标志物进行鉴定,结合高内涵成像技术评价长春新碱对所得hiPSC-PNs的神经毒性。结果显示,所分化的hiPSC-PNs能表达特异性标志物,并且长春新碱显著降低了hiPSC-PNs的神经突长度,具有外周神经毒性风险。本试验提供了一种新的hiPSC向外周神经元的分化方法,提高分化效率的同时也改善了分化时程,并且hiPSC-PNs模型结合高内涵成像技术在药物神经毒性评价方面具有广阔的发展前景。

The directed differentiation of human induced pluripotent stem cells(hiPSCs) into peripheral neurons was explored in this paper,and hiPSCs was used to evaluate drug-induced neurotoxicity.The directed differentiation of hiPSCs was induced by a combination of selected small molecule inhibitors and neurotrophins,and the hiPSC-derived peripheral neurons(hiPSC-PNs) model was established successfully.After the identification of peripheral neurons relevant markers using immunofluorescence and real time quantitative polymerase chain reaction(RT-qPCR),vincristine-induced neurotoxicity of hiPSC-PNs was evaluated by combining the high-content imaging technique.The results showed that hiPSC-PNs expressed the specific markers.In addition,vincristine significantly reduced the neurite length of hiPSC-PNs and had the risk of peripheral neurotoxicity.This study provided a new differentiation method in which hiPSCs could be directed to differentiate into peripheral neurons.Moreover,the new method improved the differentiation efficiency and time course.The hiPSC-PNs model combined with a high-content imaging technique could have broad prospects in drug-induced neurotoxicity evaluation.