Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency
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DOI：
10.1038/nmeth.1325
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发表时间：
2009-05-01
期刊：
影响因子：
48
通讯作者：
Ellis, James
中科院分区：
文献类型：
--
作者：
Hotta, Akitsu;Cheung, Aaron Y. L.;Ellis, James
诱导多能干细胞（iPS）可能用于再生医学。然而，重编程的低效率是产生患者特异性iPS细胞系的主要障碍。在这里，我们报告了用于分离人iPS细胞的第一选择系统。我们开发了EOS（早期转座子启动子和Oct-4（Pou 5 f1）和Sox 2增强子）慢病毒载体，在小鼠和人胚胎干细胞中特异性表达，但不在原代成纤维细胞中表达。双顺反子EOS载体用EGFP标记新出现的小鼠和人iPS细胞集落，并且我们使用嘌呤霉素选择来帮助分离表达内源性多能性标记物的iPS细胞系。这些细胞系分化成来自所有三个胚层的细胞类型。报告基因表达在分化后消失，因此监测形成畸胎瘤的残留多能细胞。最后，我们使用EOS选择来建立在MECP 2中具有已知突变的Rett综合征特异性小鼠和人iPS细胞系。
Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome-specific mouse and human iPS cell lines with known mutations in MECP2.