Goto Kazuya, Imamura Keiko, Komatsu Kenichi, Mitani Kohnosuke, Aiba Kazuhiro, Nakatsuji Norio, Inoue Makoto, Kawata Akihiro, Yamashita Hirofumi, Takahashi Ryosuke, Inoue Haruhisa
Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto 6068507, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 6068507, Japan.
Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 6068507, Japan.
Mol Ther Methods Clin Dev. 2017 Jan 10;4:115-125. doi: 10.1016/j.omtm.2016.12.007. eCollection 2017 Mar 17.
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal degenerative disorder of motor neurons (MNs). Embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) now help us to understand the pathomechanisms of ALS via disease modeling. Various methods to differentiate ESCs/iPSCs into MNs by the addition of signaling molecules have been reported. However, classical methods require multiple steps, and newer simple methods using the transduction of transcription factors run the risk of genomic integration of the vector genes. Heterogeneity of the expression levels of the transcription factors also remains an issue. Here we describe a novel approach for differentiating human and mouse ESCs/iPSCs into MNs using a single Sendai virus vector encoding three transcription factors, LIM/homeobox protein 3, neurogenin 2, and islet-1, which are integration free. This single-vector method, generating HB9-positive cells on day 2 from human iPSCs, increases the ratio of MNs to neurons compared to the use of three separate Sendai virus vectors. In addition, the MNs derived via this method from iPSCs of ALS patients and model mice display disease phenotypes. This simple approach significantly reduces the efforts required to generate MNs, and it provides a useful tool for disease modeling.
肌萎缩侧索硬化症（ALS）是一种运动神经元（MNs）的进行性致命性退行性疾病。胚胎干细胞（ESCs）/诱导多能干细胞（iPSCs）现在通过疾病建模帮助我们了解ALS的发病机制。已经报道了通过添加信号分子将ESCs/iPSCs分化为MNs的各种方法。然而，传统方法需要多个步骤，而使用转录因子转导的更新的简单方法存在载体基因基因组整合的风险。转录因子表达水平的异质性仍然是一个问题。在这里，我们描述了一种使用编码三种转录因子（LIM/同源盒蛋白3、神经生成素2和胰岛-1）的单个仙台病毒载体将人和小鼠ESCs/iPSCs分化为MNs的新方法，该方法无整合。这种单载体方法从人iPSCs在第2天产生HB9阳性细胞，与使用三种单独的仙台病毒载体相比，增加了MNs与神经元的比例。此外，通过这种方法从ALS患者和模型小鼠的iPSCs衍生的MNs表现出疾病表型。这种简单的方法显著减少了生成MNs所需的工作量，并为疾病建模提供了一个有用的工具。
