No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells.
No evidence for clonal selection due to lentiviral integration sites in human induced pluripotent stem cells.
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由于人类诱导的多能干细胞中的慢病毒整合位点而导致克隆选择的证据。
DOI：
10.1002/stem.322
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发表时间：
2010-04
期刊：
影响因子：
5.2
通讯作者：
Dunbar, Cynthia E.
中科院分区：
文献类型：
--
作者：
Winkler, Thomas;Cantilena, Amy;Metais, Jean-Yves;Xu, Xiuli;Nguyen, Anh-Dao;Borate, Bhavesh;Antosiewicz-Bourget, Jessica E.;Wolfsberg, Tyra G.;Thomson, James A.;Dunbar, Cynthia E.
关键词：
诱导多能干细胞(IPS)的来源需要在靶细胞中表达确定的转录因子(Oct3/4、Sox2、Klf4、c-Myc、Nanog和Lin28)。慢病毒或标准逆转录病毒基因转移仍然是最可靠和最常用的方法。与最近的非病毒方法相比，低重编程频率，以及利用整合载体进行衍生的效率更高，表明通过前病毒整合位点(IS)的基因激活或破坏可能在获得多能性表型中发挥作用。我们首次对慢病毒在人iPS细胞中的整合情况进行了广泛的分析。我们在8个最近建立的iPS细胞系中鉴定了78个独立的整合位点(IS)，这些细胞系来自于慢病毒基因转移Oct4、Sox2、Nanog和Lin28后的人胎儿成纤维细胞或新生儿包皮成纤维细胞。每个iPS克隆的IS数目从5个到15个不等，75个IS可以被分配到一个独特的染色体位置。不同的iPS克隆没有共同之处。表达分析和广泛的生物信息学分析没有显示不同克隆之间慢病毒靶向基因的功能一致性。有趣的是，在8个iPS克隆中的6个中，发现了一些IS成对存在，整合到彼此6个碱基对内的相同染色体位置上，或者非常接近。我们的研究支持最近的报道，即人类体细胞的有效重新编程不依赖于特定基因或基因类别的插入激活或失活。
Derivation of induced pluripotent stem (iPS) cells requires the expression of defined transcription factors (among Oct3/4, Sox2, Klf4, c-Myc, Nanog and Lin28) in the targeted cells. Lentiviral or standard retroviral gene transfer remains the most robust and commonly used approach. Low reprogramming frequency overall, and the higher efficiency of derivation utilizing integrating vectors compared to more recent non-viral approaches suggests that gene activation or disruption via proviral integration sites (IS) may play a role in obtaining the pluripotent phenotype. We provide for the first time an extensive analysis of the lentiviral integration profile in human iPS cells. We identified a total of 78 independent integration sites (IS) in 8 recently established iPS cell lines derived from either human fetal fibroblasts or newborn foreskin fibroblasts after lentiviral gene transfer of Oct4, Sox2, Nanog, and Lin28. The number of IS ranged from 5 to15 IS per individual iPS clone and 75 IS could be assigned to a unique chromosomal location. The different iPS clones had no IS in common. Expression analysis as well as extensive bioinformatic analysis did not reveal functional concordance of the lentiviral targeted genes between the different clones. Interestingly, in 6 of the 8 iPS clones some of the IS were found in pairs, integrated into the same chromosomal location within six base pairs of each other or in very close proximity. Our study supports recent reports that efficient reprogramming of human somatic cells is not dependent on insertional activation or deactivation of specific genes or gene classes.
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通讯作者：
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