Generation of Footprint-Free Canine Induced Pluripotent Stem Cells Using Auto-Erasable Sendai Virus Vector
Generation of Footprint-Free Canine Induced Pluripotent Stem Cells Using Auto-Erasable Sendai Virus Vector
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使用自动可擦除仙台病毒载体生成无足迹犬诱导多能干细胞
DOI：
10.1089/scd.2018.0084
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发表时间：
2018
影响因子：
4
通讯作者：
Hatoya Shingo
中科院分区：
文献类型：
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作者：
Tsukamoto Masaya;Nishimura Toshiya;Yodoe Kyohei;Kanegi Ryoji;Tsujimoto Yasunori;Alam Md Emtiaj;Kuramochi Mizuki;Kuwamura Mitsuru;Ohtaka Manami;Nishimura Ken;Nakanishi Mahito;Inaba Toshio;Sugiura Kikuya;Hatoya Shingo
犬诱导的多能干细胞（ciPSC）可用于再生医学。然而，没有关于产生耐受酶促单细胞传代的无基因组整合和完全外源基因沉默（无足迹）的ciPSC的报道。在这项研究中，我们使用自可擦除复制缺陷型和持续性仙台病毒载体SeVdp（KOSM）302 L重编程犬胚胎成纤维细胞，并产生两个ciPSC系。即使在SeVdp（KOSM）302 L去除后，ciPSC的多能标记物也呈阳性，包括碱性磷酸酶活性以及OCT 3/4、SOX 2和NANOG转录物，以及NANOG、阶段特异性胚胎抗原-1和部分TRA-1-60蛋白表达。诱导ciPSC分化成所有三个胚层，在体外作为胚状体，在体内作为畸胎瘤。此外，不含SeVdp（KOSM）302 L的ciPSC即使在重复的酶促单细胞传代后也保持正常的核型。因此，据我们所知，我们首次证明了可以使用酶促方法在单细胞阶段传代的无足迹和高质量ciPSC的产生。我们用于产生ciPSC的方法是朝向ciPSC的临床应用的发展的良好步骤。
Canine induced pluripotent stem cells (ciPSCs) can be used in regenerative medicine. However, there are no reports on the generation of genome integration-free and completely exogenous gene-silenced (footprint free) ciPSCs that are tolerant to enzymatic single-cell passage. In this study, we reprogrammed canine embryonic fibroblasts using the auto-erasable replication-defective and persistent Sendai virus vector, SeVdp(KOSM)302L, and generated two ciPSC lines. The ciPSCs were positive for pluripotent markers, including alkaline phosphatase activity as well asOCT3/4,SOX2, andNANOGtranscripts, and NANOG, stage-specific embryonic antigen-1, and partial TRA-1-60 protein expression, even after SeVdp(KOSM)302L removal. The ciPSCs were induced to differentiate into all the three germ layers as embryoid bodies in vitro and as teratomas in vivo. Furthermore, SeVdp(KOSM)302L-free ciPSCs maintained a normal karyotype even after repeated enzymatic single-cell passaging. Therefore, to our knowledge, for the first time, we demonstrated the generation of footprint-free and high-quality ciPSCs that can be passaged at the single-cell stage using enzymatic methods. Our method for generation of ciPSCs is a good step toward the development of clinical application of ciPSCs.