Center for iPS Cell Research and Application (CiRA), Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan.
Nat Protoc. 2010 Mar;5(3):418-28. doi: 10.1038/nprot.2009.231. Epub 2010 Feb 11.
Reprogramming of somatic cells into pluripotent stem cells has been reported by introducing a combination of several transcription factors (Oct3/4, Sox2, Klf4 and c-Myc). The induced pluripotent stem (iPS) cells from patient's somatic cells could be a useful source for drug discovery and cell transplantation therapies. However, to date, most iPS cells were made using viral vectors, such as retroviruses and lentiviruses. Here we describe an alternative method to generate iPS cells from mouse embryonic fibroblasts (MEFs) by continual transfection of plasmid vectors. This protocol takes around 2 months to complete, from MEF isolation to iPS cell establishment. Although the reprogramming efficiency of this protocol is still low, the established iPS cells are most likely free from plasmid integration. This virus-free technique reduces the safety concern for iPS cell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency.
已报道通过引入几种转录因子（Oct3/4、Sox2、Klf4 和 c-Myc）将体细胞重编程为多能干细胞。来自患者体细胞的诱导多能干细胞（iPS）可以成为药物发现和细胞移植治疗的有用来源。然而，迄今为止，大多数 iPS 细胞都是使用病毒载体（如逆转录病毒和慢病毒）制成的。在这里，我们描述了一种通过连续转染质粒载体从小鼠胚胎成纤维细胞（MEF）生成 iPS 细胞的替代方法。该方案大约需要 2 个月的时间才能完成，从 MEF 分离到 iPS 细胞的建立。尽管该方案的重编程效率仍然较低，但所建立的 iPS 细胞很可能没有质粒整合。这种无病毒技术降低了 iPS 细胞生成和应用的安全性问题，并为研究重编程和多能性的机制提供了细胞来源。
