Nishimura Ken, Ohtaka Manami, Takada Hitomi, Kurisaki Akira, Tran Nhi Vo Kieu, Tran Yen Thi Hai, Hisatake Koji, Sano Masayuki, Nakanishi Mahito
Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan.
Stem Cell Res. 2017 Aug;23:13-19. doi: 10.1016/j.scr.2017.06.011. Epub 2017 Jun 20.
Transgene-free induced pluripotent stem cells (iPSCs) are valuable for both basic research and potential clinical applications. We previously reported that a replication-defective and persistent Sendai virus (SeVdp) vector harboring four reprogramming factors (SeVdp-iPS) can efficiently induce generation of transgene-free iPSCs. This vector can express all four factors stably and simultaneously without chromosomal integration and can be eliminated completely from reprogrammed cells by suppressing vector-derived RNA-dependent RNA polymerase. Here, we describe an improved SeVdp-iPS vector (SeVdp(KOSM)302L) that is automatically erased in response to microRNA-302 (miR-302), uniquely expressed in pluripotent stem cells (PSCs). Gene expression and genome replication of the SeVdp-302L vector, which contains miRNA-302a target sequences at the 3' untranslated region of L mRNA, are strongly suppressed in PSCs. Consequently, SeVdp(KOSM)302L induces expression of reprogramming factors in somatic cells, while it is automatically erased from cells successfully reprogrammed to express miR-302. As this vector can reprogram somatic cells into transgene-free iPSCs without the aid of exogenous short interfering RNA (siRNA), the results we present here demonstrate that this vector may become an invaluable tool for the generation of human iPSCs for future clinical applications.
无转基因诱导多能干细胞（iPSC）对于基础研究和潜在的临床应用都具有重要价值。我们之前报道过，携带四种重编程因子的复制缺陷型持久性仙台病毒（SeVdp）载体（SeVdp-iPS）能够高效诱导产生无转基因的iPSC。该载体可以在不发生染色体整合的情况下稳定且同时表达所有四种因子，并且通过抑制载体衍生的RNA依赖性RNA聚合酶，可以从重编程细胞中完全消除。在此，我们描述了一种改进的SeVdp-iPS载体（SeVdp(KOSM)302L），它可响应多能干细胞（PSC）中独特表达的微小RNA-302（miR-302）而自动消除。在L mRNA的3'非翻译区含有miRNA-302a靶序列的SeVdp-302L载体的基因表达和基因组复制在PSC中受到强烈抑制。因此，SeVdp(KOSM)302L在体细胞中诱导重编程因子的表达，而在成功重编程以表达miR-302的细胞中它会自动消除。由于该载体无需外源性小干扰RNA（siRNA）的帮助就能将体细胞重编程为无转基因的iPSC，我们在此展示的结果表明，这种载体可能成为未来临床应用中生成人类iPSC的宝贵工具。
