Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins
Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins
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DOI：
10.1016/j.stem.2009.04.005
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发表时间：
2009-05-08
期刊：
影响因子：
23.9
通讯作者：
Ding, Sheng
中科院分区：
文献类型：
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作者：
Zhou, Hongyan;Wu, Shili;Ding, Sheng
开创性的工作表明，四种转录因子Oct 4、Klf 4、Sox 2和c-Myc的异位表达可以将鼠体细胞重编程为诱导多能干细胞（iPSC）（Takahashi和Yamanaka，2006），并且随后使用类似的遗传操作产生人iPSC（Takahashi et al.，2007年; Yu等人，2007年）。为了解决由在靶细胞基因组中携带整合的外源序列引起的安全性问题，已经开发了许多经修饰的遗传方法，并产生了具有潜在降低的风险的iPSC（关于讨论，参见Yamanaka，2009和其中的参考文献）。然而，迄今为止开发的所有方法仍然涉及使用遗传材料，因此存在靶细胞中外源序列进行意外遗传修饰的可能性。在这里，我们报告了使用重组细胞穿透重编程蛋白从鼠胚胎成纤维细胞产生蛋白诱导的多能干细胞（piPSC）。我们证明了这样的piPSC可以长期自我更新，并且在体外和体内都是多能的。避免将外源性遗传修饰引入靶细胞的一种可能方法是将重编程蛋白直接递送到细胞中，而不是依赖于来自递送基因的转录。先前的研究已经证明，各种蛋白质可以通过将它们与介导蛋白质转导的短肽（例如HIV达特和聚精氨酸）缀合而在体外和体内递送到细胞中（Inoue等人，2006年; Mr. Hue等人，2005年; Wadia和Dowdy，2002年）。此外，还介绍了各种可溶性和复性技术对表达在大肠杆菌中的包涵体蛋白的处理。已经开发了将大肠杆菌转化为生物活性蛋白的方法以允许治疗性蛋白的容易和大规模生产（Lafevre-Bernt等人，2008年）。为了产生可以穿透体细胞质膜的重组蛋白，我们设计了聚精氨酸（即11 R）蛋白转导结构域，并将其融合到四种重编程因子的C末端：Oct 4、Sox 2、Klf 4和c-Myc（参见
Groundbreaking work demonstrated that ectopic expression of four transcription factors, Oct4, Klf4, Sox2, and c-Myc, could reprogram murine somatic cells to induced pluripotent stem cells (iPSCs)(Takahashi and Yamanaka, 2006), and human iPSCs were subsequently generated using similar genetic manipulation (Takahashi et al., 2007; Yu et al., 2007). To address the safety issues arose from harboring integrated exogenous sequences in the target cell genome, a number of modified genetic methods have been developed and produced iPSCs with potentially reduced risks (for discussion, see Yamanaka, 2009, and references therein). However, all of the methods developed to date still involve the use of genetic materials and thus the potential for unexpected genetic modifications by the exogenous sequences in the target cells. Here we report generation of protein-induced pluripotent stem cells (piPSCs) from murine embryonic fibroblasts using recombinant cell-penetrating reprogramming proteins. We demonstrated that such piPSCs can long-term self-renew and are pluripotent in vitro and in vivo. One possible way to avoid introducing exogenous genetic modifications to target cells would be to deliver the reprogramming proteins directly into cells, rather than relying on the transcription from delivered genes. Previous studies have demonstrated that various proteins can be delivered into cells in vitro and in vivo by conjugating them with a short peptide that mediates protein transduction, such as HIV tat and poly-arginine (Inoue et al., 2006; Michiue et al., 2005; Wadia and Dowdy, 2002). In addition, various solubilization and refolding techniques for processing inclusion body proteins expressed in E. coli to bioactive proteins have been developed to allow facile and largescale production of therapeutic proteins (Lafevre-Bernt et al., 2008). To generate recombinant proteins that can penetrate across the plasma membrane of somatic cells, we designed and fused a poly-arginine (ie, 11R) protein transduction domain to the C terminus of four reprogramming factors: Oct4, Sox2, Klf4, and c-Myc (see