Okahara-Narita Junko, Umeda Rieko, Nakamura Shinichiro, Mori Takahide, Noce Toshiaki, Torii Ryuzo
Research Center for Animal Life Science, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga, 520-2192, Japan.
Primates. 2012 Apr;53(2):205-13. doi: 10.1007/s10329-011-0283-1. Epub 2011 Nov 11.
Induced pluripotent stem (iPS) cells have the potential to become a universal resource for cell-based therapies in regenerative medicine; however, prior to the use of such iPS cell-based therapies, preclinical assessment of their safety and efficacy is essential. Non-human primates serve as valuable animal models for human diseases or biomedical research; therefore, in this study, we generated cynomolgus monkey iPS cells from adult skin and fetal fibroblast cells by the retrovirally mediated introduction of four human transcription factors: c-Myc, Klf4, Oct3/4, and Sox2 (the so-called "Yamanaka factors"). Twenty to 30 days after the introduction of these factors, several cynomolgus monkey embryonic stem (ES) cell-like colonies appeared on SNL and mouse embryonic fibroblast (MEF) feeder layers. These colonies were picked and cultivated in primate ES medium. Seven iPS cell lines were established, and we detected the expression of pluripotent markers that are also expressed in ES cells. Reverse transcription polymerase chain reaction (PCR) showed that these iPS cells expressed endogenous c-Myc, Klf4, Oct3/4, and Sox2 genes, whereas several transgenes were silenced. Embryoid body and teratoma formation showed that the cynomolgus iPS cells had the developmental potential to differentiate into cells of all three primary germ layers. In summary, we generated cynomolgus monkey iPS cells by retrovirus-mediated transduction of the human transcription factors, c-Myc, Klf4, Oct3/4, and Sox2 into adult cynomolgus monkey skin cells and fetal fibroblasts. The cynomolgus monkey is the most relevant primate model for human disease, and the highly efficient generation of monkey iPS cells would allow investigation of the treatments of various diseases in this model via therapeutic cloning.
诱导多能干细胞（iPS细胞）有潜力成为再生医学中基于细胞治疗的通用资源；然而，在使用此类基于iPS细胞的治疗方法之前，对其安全性和有效性进行临床前评估至关重要。非人灵长类动物是人类疾病或生物医学研究的宝贵动物模型；因此，在本研究中，我们通过逆转录病毒介导将四种人类转录因子：c-Myc、Klf4、Oct3/4和Sox2（即所谓的“山中因子”）导入成年食蟹猴皮肤和胎儿成纤维细胞中，从而产生了食蟹猴iPS细胞。在导入这些因子后的20至30天，几个食蟹猴胚胎干细胞样集落出现在SNL和小鼠胚胎成纤维细胞（MEF）饲养层上。挑选这些集落并在灵长类胚胎干细胞培养基中培养。建立了7个iPS细胞系，我们检测到了在胚胎干细胞中也表达的多能性标志物的表达。逆转录聚合酶链反应（PCR）表明这些iPS细胞表达内源性c-Myc、Klf4、Oct3/4和Sox2基因，而几个转基因被沉默。胚状体和畸胎瘤形成表明食蟹猴iPS细胞具有分化为所有三个原始胚层细胞的发育潜力。总之，我们通过逆转录病毒介导将人类转录因子c-Myc、Klf4、Oct3/4和Sox2转导入成年食蟹猴皮肤细胞和胎儿成纤维细胞中，从而产生了食蟹猴iPS细胞。食蟹猴是与人类疾病最相关的灵长类动物模型，高效产生猴iPS细胞将有助于通过治疗性克隆在该模型中研究各种疾病的治疗方法。
