Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation
Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation
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DOI：
10.1073/pnas.0904825106
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发表时间：
2009-08-04
影响因子：
11.1
通讯作者：
Sadelain, Michel
中科院分区：
文献类型：
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作者：
Papapetrou, Eirini P.;Tomishima, Mark J.;Sadelain, Michel
人诱导多能干细胞 (hiPSC) 是通过 4 种重编程因子 (RF) Oct-4、Sox2、Klf4 和 c-Myc 的异位表达从体细胞产生的。为了更好地定义成功 hiPSC 诱导所需的化学计量要求和动态表达模式，我们生成了 4 个双顺反子慢病毒载体，编码与可辨别荧光蛋白共表达的 4 个 RF。使用该系统，我们定义了对 Oct4 剂量高度敏感的 RF 表达的最佳化学计量，并且我们证明了 RF 表达相对比率的变化对 hiPSC 诱导效率的影响。在重编程过程中对单个细胞中每个单独 RF 表达的监测表明，在获得多能细胞标记后载体沉默。明显的慢病毒载体沉默是成功重编程 hiPSC 克隆的一个特征，但缺乏完全沉默并不妨碍 hiPSC 诱导、维持或定向分化。这里描述的载体系统为重编程的机制研究和 hiPSC 生成的优化提供了一个强大的工具。
Human-induced pluripotent stem cells (hiPSCs) are generated from somatic cells by ectopic expression of the 4 reprogramming factors (RFs) Oct-4, Sox2, Klf4, and c-Myc. To better define the stoichiometric requirements and dynamic expression patterns required for successful hiPSC induction, we generated 4 bicistronic lentiviral vectors encoding the 4 RFs co-expressed with discernable fluorescent proteins. Using this system, we define the optimal stoichiometry of RF expression to be highly sensitive to Oct4 dosage, and we demonstrate the impact that variations in the relative ratios of RF expression exert on the efficiency of hiPSC induction. Monitoring of expression of each individual RF in single cells during the course of reprogramming revealed that vector silencing follows acquisition of pluripotent cell markers. Pronounced lentiviral vector silencing was a characteristic of successfully reprogrammed hiPSC clones, but lack of complete silencing did not hinder hiPSC induction, maintenance, or directed differentiation. The vector system described here presents a powerful tool for mechanistic studies of reprogramming and the optimization of hiPSC generation.