Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Mariensee, Germany.
Stem Cells Dev. 2013 Jan 1;22(1):124-35. doi: 10.1089/scd.2012.0382. Epub 2012 Nov 2.
The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (iPS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives of the 3 germ layers. Upon injection of putative iPS cells under the skin of immunodeficient mice, we observed teratomas in 3 of 6 cases. These results form the basis for in-depth studies toward the derivation of porcine iPS cells, which hold great promise for preclinical testing of novel cell therapies in the pig model.
家猪是临床前新型细胞疗法测试的重要大型动物模型。最近，我们生产了多能性报告猪，其中 Oct4 启动子驱动增强型绿色荧光蛋白（EGFP）的表达。在这里，我们使用非病毒 Sleeping Beauty 转座子系统对 Oct4-EGFP 成纤维细胞进行重编程，以传递重编程因子 Oct4、Sox2、Klf4 和 cMyc。细胞形态的变化和 Oct4-EGFP 报告基因的重新激活表明成功地重编程为多能状态。转座子重编程的诱导多能干细胞（iPS）在体外培养超过 40 代时表现出长期增殖，表达胚胎干细胞的典型转录因子，包括 OCT4、NANOG、SOX2、REX1、ESRRB、DPPA5 和 UTF1 以及多能性的表面标记物，包括 SSEA-1 和 TRA-1-60。体外分化导致 3 个胚层的衍生物。在将推定的 iPS 细胞注射到免疫缺陷小鼠的皮下后，我们在 6 例中有 3 例观察到畸胎瘤。这些结果为深入研究猪 iPS 细胞的衍生奠定了基础，这为在猪模型中进行新型细胞疗法的临床前测试提供了巨大的潜力。
