Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot, Israel.
Biochem Biophys Res Commun. 2010 Mar 26;394(1):189-93. doi: 10.1016/j.bbrc.2010.02.150. Epub 2010 Feb 25.
Reprogramming of differentiated cells into induced pluripotent cells (iPS) was accomplished in 2006 by expressing four, or less, embryonic stem cell (ESC)-specific transcription factors. Due to the possible danger of DNA damage and the potential tumorigenicity associated with such DNA damage, attempts were made to minimize DNA integration by the vectors involved in this process without complete success. Here we present a method of using RNA transfection as a tool for reprogramming human fibroblasts to iPS. We used RNA synthesized in vitro from cDNA of the same reprogramming four transcription factors. After transfection of the RNA, we show intracellular expression and nuclear localization of the respective proteins in at least 70% of the cells. We used five consecutive transfections to support continuous protein expression resulting in the formation of iPS colonies that express alkaline phosphatase and several ESC markers and that can be expanded. This method completely avoids DNA integration and may be developed to replace the use of DNA vectors in the formation of iPS.
重编程分化细胞为诱导多能干细胞（iPS）是在 2006 年通过表达四个或更少的胚胎干细胞（ESC）特异性转录因子来实现的。由于涉及该过程的载体可能存在 DNA 损伤的危险以及与这种 DNA 损伤相关的潜在致瘤性，人们试图通过最小化 DNA 整合来降低这种危险，但并未完全成功。在这里，我们提出了一种使用 RNA 转染作为重编程人成纤维细胞为 iPS 的工具的方法。我们使用从相同重编程的四个转录因子的 cDNA 体外合成的 RNA。在 RNA 转染后，我们在至少 70%的细胞中显示了各自蛋白质的细胞内表达和核定位。我们使用五次连续转染来支持持续的蛋白质表达，从而形成表达碱性磷酸酶和几种 ESC 标记物的 iPS 集落，并且可以进行扩增。这种方法完全避免了 DNA 整合，并且可以开发出来替代 DNA 载体在 iPS 形成中的应用。
