Liu Shu-Ping, Li Yan-Xin, Xu Jing, Gu Hai-Hui, Zhang Hong-Yan, Liang Hao-Yue, Liu Han-Zhi, Zhang Xiao-Bing, Cheng Tao, Yuan Wei-Ping
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China.
State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2014 Jun;22(3):580-7. doi: 10.7534/j.issn.1009-2137.2014.03.002.
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.
诱导多能干细胞（iPSC）的基因组不稳定性和致瘤性阻碍了其在临床应用中的巨大潜力。使用游离型载体生成iPSC是目前解决安全问题的最佳方法。该方法简单，且外源基因未整合到宿主基因组中。然而，该方法的重编程效率非常低，因此限制了其应用。本研究旨在改进从脐带血单个核细胞（CB MNC）生成诱导多能干细胞的游离型方法，建立无整合的iPSC技术体系，为未来临床使用的个体化iPSC奠定基础。为提高iPSC的重编程效率，采用游离型方法，测试了游离型载体、预刺激培养基和氧气条件的各种组合以优化该方法。结果表明，使用红系培养基培养8天，在低氧条件（3%）下用具有SFFV（脾集落形成病毒）启动子的游离型载体转染，CB MNC可最有效地重编程，效率为0.12%。此外，结果表明，红细胞（CD36(+)CD71(+)CD235a(low)）是培养8天后重编程效率高的细胞。结论是成功建立了一种高效、安全的无整合iPSC生成方法，可用于临床研究。
