High-throughput screening-compatible single-step protocol to differentiate embryonic stem cells in neurons
High-throughput screening-compatible single-step protocol to differentiate embryonic stem cells in neurons
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DOI：
10.1089/scd.2007.0130
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发表时间：
2008-06-01
影响因子：
4
通讯作者：
Filosa, Stefania
中科院分区：
文献类型：
--
作者：
Fico, Annalisa;Manganelli, Genesia;Filosa, Stefania
高通量筛选（HTS）等生物技术能够评估大型化合物文库的生物活性和毒性。在药物开发领域，胚胎干细胞已成为HTS测试新化合物效果的重要工具。我们报告了一种一步分化胚胎干细胞在神经元和神经胶质细胞中的创新方法。四种不同的神经元亚型，γ -氨基丁酸（GABA）能，多巴胺能，血清素能和运动神经元，在培养中形成。从z因子值可以看出，该方案适用于小井，重现性高。此外，通过在我们的培养条件下使用白血病抑制因子（LIF）或重组Cripto蛋白，我们提供了证据，证明该方案适用于测试不同分子对ES细胞神经元分化的影响。最后，由于实验简单，该方法提供了通过延时视频显微镜跟踪体外分化神经元细胞形态演变的可能性。我们的实验系统为测试不同物质对神经元和胶质细胞存活和/或分化的影响提供了一个强大的工具。此外，利用基因修饰的胚胎干细胞，将有可能筛选对特定神经病变有治疗作用的药物，或者通过延时视频显微镜观察它们在体外分化的能力。
Biotechnologies such as high-throughput screening (HTS) enable evaluation of large compound libraries for their biological activity and toxic properties. In the field of drug development, embryonic stem (ES) cells have been instrumental in HTS for testing the effect of new compounds. We report an innovative method in one step to differentiate ES cells in neurons and glial cells. The four different neuronal subtypes, gamma-aminobutyric acid (GABA)-ergic, dopaminergic, serotonergic, and motor neurons, are formed in culture. This protocol is adaptable to small wells and is highly reproducible, as indicated by the Z-factor value. Moreover, by using either leukemia inhibitory factor (LIF) or recombinant Cripto protein in our culture conditions, we provide evidence that this protocol is suitable for testing the effect of different molecules on neuronal differentiation of ES cells. Finally, thanks to the simplicity in carrying out the experiment, this method provides the possibility of following the morphological evolution of the in vitro differentiating neuronal cells by time-lapse videomicroscopy. Our experimental system provides a powerful tool for testing the effect of different substances on survival and/or differentiation of neuronal and glial cells in an HTS-based approach. Furthermore, using genetically modified ES cells, it would be possible to screen for drugs that have a therapeutic effect on specific neuronal pathologies or to follow, by time-lapse videomicroscopy, their ability to in vitro differentiate.