Kidder Benjamin L
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 7B04, 10 Center Drive, Bethesda, MD, 20892, USA,
Methods Mol Biol. 2014;1150:227-36. doi: 10.1007/978-1-4939-0512-6_15.
Generation of induced pluripotent stem (iPS) cells from differentiated cells has traditionally been performed by overexpressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. However, inclusion of c-Myc in the reprogramming cocktail can lead to expansion of transformed cells that are not fully reprogrammed, and studies have demonstrated that c-Myc reactivation increases tumorigenicity in chimeras and progeny mice. Moreover, chemical inhibition of Wnt signaling has been shown to enhance reprogramming efficiency. Here, we describe a modified protocol for generating iPS cells from murine fibroblasts using chemical inhibition and overexpression of three transcription factors. Using this protocol, we observed robust conversion to iPS cells while maintaining minimal contamination of partially reprogrammed transformed colonies.
传统上，从分化细胞中生成诱导多能干细胞（iPS细胞）是通过过表达四种转录因子来实现的：Oct4、Sox2、Klf4和c-Myc。然而，在重编程混合物中加入c-Myc会导致未完全重编程的转化细胞增殖，并且研究表明，c-Myc的重新激活会增加嵌合体和子代小鼠的致瘤性。此外，Wnt信号通路的化学抑制已被证明可提高重编程效率。在此，我们描述了一种使用化学抑制和三种转录因子过表达从鼠成纤维细胞生成iPS细胞的改良方案。使用该方案，我们观察到向iPS细胞的强劲转化，同时保持部分重编程的转化集落的污染最小。
