Zhang Yu, Wei Chao, Zhang Pengfei, Li Xia, Liu Tong, Pu Yong, Li Yunsheng, Cao Zubing, Cao Hongguo, Liu Ya, Zhang Xiaorong, Zhang Yunhai
Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Bio-breeding, Department of Animal Science, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui, People's Republic of China.
PLoS One. 2014 Jan 20;9(1):e85089. doi: 10.1371/journal.pone.0085089. eCollection 2014.
Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed by ectopic expression of transcription factors or small molecule treatment, which resemble embryonic stem cells (ESCs). They hold great promise for improving the generation of genetically modified large animals. However, few porcine iPSCs (piPSCs) lines obtained currently can support development of cloned embryos. Here, we generated iPSCs from porcine adipose-derived stem cells (pADSCs), using drug-inducible expression of defined human factors (Oct4, Sox2, c-Myc and Klf4). Reprogramming of iPSCs from pADSCs was more efficient than from fibroblasts, regardless of using feeder-independent or feeder-dependent manners. By addition of Lif-2i medium containing mouse Lif, CHIR99021 and PD0325901 (Lif-2i), naïve-like piPSCs were obtained under feeder-independent and serum-free conditions. These successfully reprogrammed piPSCs were characterized by short cell cycle intervals, alkaline phosphatase (AP) staining, expression of Oct4, Sox2, Nanog, SSEA3 and SSEA4, and normal karyotypes. The resemblance of piPSCs to naïve ESCs was confirmed by their packed dome morphology, growth after single-cell dissociation, Lif-dependency, up-regulation of Stella and Eras, low expression levels of TRA-1-60, TRA-1-81 and MHC I and activation of both X chromosomes. Full reprogramming of naïve-like piPSCs was evaluated by the significant up-regulation of Lin28, Esrrb, Utf1 and Dppa5, differentiating into cell types of all three germ layers in vitro and in vivo. Furthermore, nuclear transfer embryos from naïve-like piPSCs could develop to blastocysts with improved quality. Thus, we provided an efficient protocol for generating naïve-like piPSCs from pADSCs in a feeder-independent and serum-free system with controlled regulation of exogenous genes, which may facilitate optimization of culture media and the production of transgenic pigs.
诱导多能干细胞（iPSCs）是通过转录因子的异位表达或小分子处理重编程的体细胞，类似于胚胎干细胞（ESCs）。它们在改善转基因大型动物的培育方面具有巨大潜力。然而，目前获得的猪诱导多能干细胞（piPSCs）系中，很少有能支持克隆胚胎发育的。在此，我们利用特定人类因子（Oct4、Sox2、c-Myc和Klf4）的药物诱导表达，从猪脂肪来源干细胞（pADSCs）中生成了iPSCs。无论采用无饲养层或有饲养层方式，从pADSCs重编程生成iPSCs都比从成纤维细胞更高效。通过添加含有小鼠白血病抑制因子（Lif）、CHIR99021和PD0325901的Lif-2i培养基（Lif-2i），在无饲养层和无血清条件下获得了类似原始态的piPSCs。这些成功重编程的piPSCs具有细胞周期短、碱性磷酸酶（AP）染色阳性、Oct4、Sox2、Nanog、阶段特异性胚胎抗原3（SSEA3）和阶段特异性胚胎抗原4（SSEA4）表达以及正常核型等特征。piPSCs与原始态ESCs的相似性通过其堆积穹顶形态、单细胞解离后的生长、对Lif的依赖性、Stella和Eras的上调、TRA-1-60、TRA-1-81和主要组织相容性复合体I（MHC I）的低表达水平以及两条X染色体的激活得以证实。通过Lin28、雌激素相关受体β（Esrrb）、未受精卵专一转录因子1（Utf1）和发育多能性相关蛋白5（Dppa5）的显著上调、在体外和体内分化为所有三个胚层的细胞类型，评估了类似原始态piPSCs的完全重编程。此外，来自类似原始态piPSCs的核移植胚胎能够发育成质量更好的囊胚。因此，我们提供了一种在无饲养层和无血清系统中通过对外源基因的可控调控从pADSCs生成类似原始态piPSCs的高效方案，这可能有助于优化培养基和转基因猪的生产。
