State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China.
Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.
Stem Cell Reports. 2014 May 8;2(5):721-33. doi: 10.1016/j.stemcr.2014.03.013. eCollection 2014 May 6.
Induced pluripotent stem cells (iPSCs) generated from somatic cells by ectopic expression of reprogramming factors, e.g., POU5F1 (OCT4), KLF4, and SOX2, have great potential for regenerative medicine. However, before they can be used in a clinical setting, the mechanism of reprogramming needs to be better understood. Here, by engineering reprogramming factors to a destabilizing protein domain, we achieved inducible generation of mouse and pig iPSCs. Stability of the fusion protein was precisely regulated by the addition of the cell-permeable small molecule trimethoprim (TMP) in a dose-dependent manner. With these tools, we found that during the early and middle stages of reprogramming, exogenous OCT4 or KLF4 could be omitted, whereas exogenous SOX2 expression at early and middle stages was required for successful reprogramming. Our TMP reprogramming system is useful for defining the stoichiometry and temporal requirements of transcription factors for reprogramming.
诱导多能干细胞（iPSCs）是通过异位表达重编程因子，如 POUSF1（OCT4）、KLF4 和 SOX2，从体细胞中产生的，它们在再生医学中有很大的潜力。然而，在它们可以在临床环境中使用之前，需要更好地了解重编程的机制。在这里，通过将重编程因子工程化为不稳定的蛋白质结构域，我们实现了诱导型生成的小鼠和猪 iPSCs。融合蛋白的稳定性可以通过添加细胞通透性的小分子三甲氧苄氨嘧啶（TMP）来精确调节，其剂量呈依赖性。利用这些工具，我们发现，在重编程的早期和中期，外源 OCT4 或 KLF4 可以被省略，而外源 SOX2 在早期和中期的表达是成功重编程所必需的。我们的 TMP 重编程系统有助于确定转录因子在重编程过程中的化学计量和时间要求。
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