EOS lentiviral vector selection system for human induced pluripotent stem cells
EOS lentiviral vector selection system for human induced pluripotent stem cells
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DOI：
10.1038/nprot.2009.201
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发表时间：
2009-01-01
期刊：
影响因子：
14.8
通讯作者：
Ellis, James
中科院分区：
文献类型：
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作者：
Hotta, Akitsu;Cheung, Aaron Y. L.;Ellis, James
从患者体内产生诱导多能干细胞 (iPS) 在研究疾病的分子机制、筛选药物以及最终用于细胞治疗方面具有令人兴奋的应用。然而，重编程的低效率和异质性是产生个性化 iPS 细胞系的主要障碍。我们在 NatureMethods (6, 370-376, 2009) 中报道了第一个富集重编程人类 iPS 细胞的选择系统。使用在多能干细胞中特异性表达增强型绿色荧光蛋白和嘌呤霉素抗性基因的慢病毒载体，现在可以标记和富集表达内源多能性标记的人类 iPS 细胞集落。在这项研究中，我们描述了用于生产多能状态特异性慢病毒载体的详细方案以及用于诱导健康和疾病特异性人类 iPS 细胞的选择系统。总体而言，选择系统的准备需要2周的时间，而人类iPS细胞的生成则需要大约2个月的时间。
Generation of induced pluripotent stem (iPS) cells from patients has exciting applications for studying molecular mechanisms of diseases, screening drugs and ultimately for use in cell therapies. However, the low efficiency and heterogeneous nature of reprogramming is a major impediment to the generation of personalized iPS cell lines. We reported in Nature Methods (6, 370-376, 2009) the first selection system to enrich for reprogrammed human iPS cells. Using a lentiviral vector that specifically expresses the enhanced green fluorescence protein and puromycin resistance genes in pluripotent stem cells, it is now possible to mark and enrich for human iPS cell colonies expressing endogenous pluripotency markers. In this study, we describe a detailed protocol for the production of the pluripotent state-specific lentiviral vector and the selection system for the induction of healthy and disease-specific human iPS cells. Overall, preparation of the selection system takes 2 weeks, and the generation of human iPS cells takes similar to 2 months.