Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector.
Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector.
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重编程转基因的切除可改善用单个可置矢量产生的IPS细胞的分化潜力。
DOI：
10.1002/stem.255
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发表时间：
2010-01
期刊：
影响因子：
--
通讯作者：
Mostoslavsky G
中科院分区：
文献类型：
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作者：
Sommer CA;Sommer AG;Longmire TA;Christodoulou C;Thomas DD;Gostissa M;Alt FW;Murphy GJ;Kotton DN;Mostoslavsky G
诱导多能干细胞(IPS)分化后残留整合的转基因是非常不可取的。在这里，我们展示了使用可切除的多顺反子慢病毒载体有效地获得没有外源重编程转基因的iPS细胞。该载体的一个新版本包含一个报告荧光色素，可以实时直接显示活的iPS细胞中的载体切除。我们发现，去除重编程载体显著提高了iPS细胞的发育潜力，并显著增强了其在体外进行定向分化的能力。我们进一步提出，有效地切除重编程转基因的方法，以及最少的培养传代，是至关重要的，因为我们发现iPS细胞在培养扩增后可能获得类似于ESC的染色体异常，如8号染色体的三体。我们的发现说明了一种产生无转基因iPS细胞的有效方法，并强调了消除整合的重新编程因子可能产生的潜在益处。此外，我们的结果强调了iPS细胞临床应用需要考虑的长期培养的后果。
The residual presence of integrated transgenes following the derivation of induced pluripotent stem (iPS) cells is highly undesirable. Here we demonstrate efficient derivation of iPS cells free of exogenous reprogramming transgenes using an excisable polycistronic lentiviral vector. A novel version of this vector containing a reporter fluorochrome allows direct visualization of vector excision in living iPS cells in real time. We find that removal of the reprogramming vector markedly improves the developmental potential of iPS cells and significantly augments their capacity to undergo directed differentiation in vitro. We further propose that methods to efficiently excise reprogramming transgenes with minimal culture passaging, such as those demonstrated here, are critical since we find that iPS cells may acquire chromosomal abnormalities, such as trisomy of chromosome 8, similar to ESC after expansion in culture. Our findings illustrate an efficient method for the generation of transgene-free iPS cells and emphasize the potential beneficial effects that may result from elimination of integrated reprogramming factors. In addition, our results underscore the consequences of long-term culture that will need to be taken into account for the clinical application of iPS cells.