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Derivation of induced pluripotent stem cells from pig somatic cells

机译：猪体细胞诱导多能干细胞的衍生

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For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after 22 days, providing an overall reprogramming efficiency of 0.1 %. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of 17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and tera-tomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age.
机译：由于不清楚的原因，从有蹄类动物产生胚胎干细胞已被证明是难以捉摸的。在这里，我们描述了通过慢病毒转导4个人类（h）基因hOCT4，hSOX2，hKLF4和hc-MYC的慢病毒转导从猪胎儿成纤维细胞衍生的诱导多能干细胞（iPSC），这种组合通常用于在小鼠和人类中创建iPSC。在辐射的小鼠胚胎成纤维细胞（MEF）和补充有基因敲除血清替代品和FGF2的培养基中培养细胞。 22天后出现碱性磷酸酶阳性细胞的紧凑菌落，总体重编程效率为0.1％。该细胞表达猪OCT4，NANOG和SOX2，具有高端粒酶活性，但仍继续表达4种人类转基因。与人ESC不同，猪iPSC（piPSC）对SSEA-1呈阳性，但对SSEA-3和-4呈阴性。在Affymetrix（猪）微阵列上的转录谱分析和实时RT-PCR支持重新编程为多能性的结论。一种细胞系ID6具有正常的核型，细胞倍增时间为17 h，并已通过> 220倍倍数维持。 ID6系形成胚状体，在分化条件下培养时表达代表所有3个胚层的基因，并在裸鼠中形成包含外胚层，中胚层和内胚层来源组织的畸胎瘤。我们得出结论，猪体细胞可以重新编程以形成piPSC。从个体动物中分离出来的这种细胞系可以提供一种方法，用于在晚年返回同一只猪时测试源自干细胞的组织移植物的安全性和有效性。

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