[An improved method for generating integration-free human induced pluripotent stem cells].
[An improved method for generating integration-free human induced pluripotent stem cells].
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DOI：
10.7534/j.issn.1009-2137.2014.03.002
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发表时间：
2014-06-01
期刊：
影响因子：
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通讯作者：
Yuan, Wei-Ping
中科院分区：
文献类型：
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作者：
Liu, Shu-Ping;Li, Yan-Xin;Yuan, Wei-Ping
诱导性多能干细胞（induced pluripotent stem cells，iPSC）基因组的不稳定性和致瘤性阻碍了其巨大的临床应用潜力。利用附加型载体诱导iPSC是目前解决安全性问题的最佳途径。该方法操作简单，外源基因不会整合到宿主基因组中。然而，这种方法的重编程效率非常低，从而限制了其使用。本研究旨在改进脐血单个核细胞诱导多能干细胞（induced pluripotent stem cells，CB MNC）的游离型方法，建立免整合iPSC技术体系，为将来iPSC的个体化临床应用奠定基础。为了提高iPSC的重编程效率，采用附加体法，对不同附加体载体组合、预刺激培养基和有氧条件进行了优化。结果表明，用红系培养液培养8d，在低氧（3%）条件下，用SFFV（spleen focus forming virus）启动子的附加型载体转染CB MNC，可获得最有效的重编程，重编程效率为0.12%。此外，结果显示成红细胞（CD36（+）CD71（+）CD235a（低））是培养8天后高效重编程的细胞。结论：成功建立了一种高效、安全的无整合iPSC的制备方法，可用于临床研究。
The genome instability and tumorigenicity of induced pluripotent stem cells (iPSC) hinder their great potentials for clinical application. Using episomal vectors to generate iPSC is the best way to solve safety issues at present. This method is simple and the exogenous gene was not integrated into the host genome. However, the reprogramming efficiency for this method is very low and thus limits its usage. This study was purposed to improve episomal method for generating induced pluripotent stem cells from cord blood mononuclear cells (CB MNC), to establish integration-free iPSC technology system, and to lay the foundation for individualized iPSC for future clinical uses. To improve the reprogramming efficiency for iPSC, episomal method was used at various combinations of episomal vectors, pre-stimulating culture mediums and oxygen condition were tested to optimize the method. The results showed that using erythroid culture medium for culturing 8 days, transfecting with episomal vectors with SFFV (spleen focus forming virus) promoter under the hypoxic condition (3%), CB MNC could be mostly efficiently reprogrammed with the efficiency 0.12%. Furthermore, the results showed that erythroblasts (CD36(+)CD71(+)CD235a(low)) were the cells that are reprogrammed with high efficiency after culture for 8 days. It is concluded that a highly efficient and safe method for generation of integration-free iPSC is successfully established, which is useable in clinical study.