Identification of Nucleoside Analogs as Inducers of Neuronal Differentiation in a Human Reporter Cell Line and Adult Stem Cells
Identification of Nucleoside Analogs as Inducers of Neuronal Differentiation in a Human Reporter Cell Line and Adult Stem Cells
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DOI：
10.1111/cbdd.12488
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发表时间：
2015-08-01
影响因子：
3
通讯作者：
Brandt, Roland
中科院分区：
文献类型：
--
作者：
Raasch, Katharina;Malecki, Edith;Brandt, Roland
核苷类似物（NSAs）是第一批化疗剂之一，也可用于操纵细胞命运。为了研究NSA诱导神经元分化的潜力，我们开发了一种新的基于人神经元定向畸胎瘤细胞系（NT 2）作为神经元祖细胞模型的表型测定，并构建了一种基于NT 2的报告细胞系，其在神经元特异性启动子的控制下表达eGFP。我们测试了38个结构相关的非结构化氨基酸，并确定其活性诱导神经元分化的神经元标记蛋白，活细胞成像，荧光检测和免疫印迹分析的免疫细胞化学。我们鉴定了12种不同程度诱导神经元分化的神经节苷脂。具有最高活性的NSAs在其嘧啶核碱基处携带卤素取代基，并且未修饰的或2 '-O-甲基取代的2-脱氧-β-D-呋喃核糖基残基作为糖基部分。克拉屈滨是一种具有相似结构特征的嘌呤核苷，用于治疗白血病和多发性硬化症，也诱导成人神经嵴来源的干细胞分化。我们的研究结果表明，NSA可能是有用的细胞替代疗法或治疗神经退行性疾病的神经元细胞命运的操纵。结构和功能关系的数据将有助于设计具有增加的活性和低毒性的化合物。
Nucleoside analogs (NSAs) were among the first chemotherapeutic agents and could also be useful for the manipulation of cell fate. To investigate the potential of NSAs for the induction of neuronal differentiation, we developed a novel phenotypic assay based on a human neuron-committed teratocarcinoma cell line (NT2) as a model for neuronal progenitors and constructed a NT2-based reporter cell line that expressed eGFP under the control of a neuron-specific promoter. We tested 38 structurally related NSAs and determined their activity to induce neuronal differentiation by immunocytochemistry of neuronal marker proteins, live cell imaging, fluorometric detection and immunoblot analysis. We identified twelve NSAs, which induced neuronal differentiation to different extents. NSAs with highest activity carried a halogen substituent at their pyrimidine nucleobase and an unmodified or 2'-O-methyl substituted 2-deoxy-beta-D-ribofuranosyl residue as glyconic moiety. Cladribine, a purine nucleoside with similar structural features and in use to treat leukemia and multiple sclerosis, induced also differentiation of adult human neural crest-derived stem cells. Our results suggest that NSAs could be useful for the manipulation of neuronal cell fate in cell replacement therapy or treatment of neuro-degenerative disorders. The data on the structure and function relationship will help to design compounds with increased activity and low toxicity.