Tashiro Katsuhisa, Kawabata Kenji, Inamura Mitsuru, Takayama Kazuo, Furukawa Norihisa, Sakurai Fuminori, Katayama Kazufumi, Hayakawa Takao, Furue Miho Kusuda, Mizuguchi Hiroyuki
National Institute of Biomedical Innovation, Osaka, Japan.
Cell Reprogram. 2010 Oct;12(5):501-7. doi: 10.1089/cell.2010.0023.
We examined the transduction efficiency in human embryonic stem (ES) and induced pluripotent stem (iPS) cells using an adenovirus (Ad) vector. RT-PCR analysis revealed the expression of the coxsackievirus and adenovirus receptor, a receptor for Ad, in these cells. However, gene expression after the transduction with an Ad vector was observed only in the periphery of ES and iPS cell colonies, when human ES and iPS cells were passaged as small colonies. This suggests that the Ad vector could not enter inside the ES and iPS cell colonies by their tight connection. We thus attempted to transduce foreign genes into the dissociated form of human ES and iPS cells, which were passaged using Rho-associated kinase inhibitor. In this condition, transduction efficiency in human ES and iPS cells was markedly increased and transgene expression was observed even inside the colonies by using Ad vectors. Furthermore, Ad vector-mediated transduction did not alter the expression of undifferentiated markers such as Oct-3/4, Nanog, and SSEA-4. Our results indicate that Ad vectors are effective tools for transduction into human ES and iPS cells.
我们使用腺病毒（Ad）载体检测了其在人胚胎干细胞（ES）和诱导多能干细胞（iPS）中的转导效率。逆转录聚合酶链反应（RT-PCR）分析显示，这些细胞中存在腺病毒的受体——柯萨奇病毒和腺病毒受体。然而，当人ES细胞和iPS细胞以小克隆形式传代时，用Ad载体转导后仅在ES和iPS细胞克隆的周边观察到基因表达。这表明Ad载体无法通过它们紧密的连接进入ES和iPS细胞克隆内部。因此，我们尝试将外源基因导入解离形式的人ES细胞和iPS细胞，这些细胞是使用Rho相关激酶抑制剂传代的。在这种情况下，人ES细胞和iPS细胞的转导效率显著提高，并且通过使用Ad载体，即使在克隆内部也观察到了转基因表达。此外，Ad载体介导的转导并未改变未分化标志物如Oct-3/4、Nanog和SSEA-4的表达。我们的结果表明，Ad载体是用于转导人ES细胞和iPS细胞的有效工具。
