Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Centre, University of Helsinki, Helsinki, Finland.
J Virol. 2012 Apr;86(8):4463-7. doi: 10.1128/JVI.06302-11. Epub 2012 Feb 1.
Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSC) by ectopic expression of key transcription factors. Current methods for the generation of integration-free iPSC are limited by the low efficiency of iPSC generation and by challenges in reprogramming methodology. Recombinant adeno-associated virus (rAAV) is a potent gene delivery vehicle capable of efficient transduction of transgenic DNA into cells. rAAV stays mainly as an episome in nondividing cells, and the extent of integration is still poorly defined for various replicating cells. In this study, we aimed to induce iPSC from mouse and human fibroblasts by using rAAV vector-mediated transient delivery of reprogramming factors. We succeeded in deriving induced pluripotent stem cells from mouse but not human fibroblasts. Unexpectedly, the rAAV vector-mediated reprogramming led to frequent genomic integration of vector sequences during the reprogramming process, independent of the amount of virus used, and to persistent expression of reprogramming factors in generated iPSC clones. It thus appears that rAAV vectors are not compatible with the derivation of integration-free iPSC.
成纤维细胞可以通过异位表达关键转录因子重编程为诱导多能干细胞（iPSC）。目前生成无整合 iPSC 的方法受到 iPSC 生成效率低和重编程方法学挑战的限制。重组腺相关病毒（rAAV）是一种有效的基因传递载体，能够将转基因 DNA 高效转导到细胞中。rAAV 在非分裂细胞中主要作为附加体存在，并且对于各种复制细胞，整合的程度仍然定义不明确。在这项研究中，我们旨在通过使用 rAAV 载体介导的重编程因子瞬时传递来诱导来自小鼠和人成纤维细胞的 iPSC。我们成功地从小鼠而非人成纤维细胞中诱导出了诱导多能干细胞。出乎意料的是，rAAV 载体介导的重编程导致在重编程过程中载体序列频繁地基因组整合，这与使用的病毒量无关，并导致生成的 iPSC 克隆中重编程因子的持续表达。因此，rAAV 载体似乎与无整合 iPSC 的衍生不兼容。
