Efficient reprogramming of naïve-like induced pluripotent stem cells from porcine adipose-derived stem cells with a feeder-independent and serum-free system.
Efficient reprogramming of naïve-like induced pluripotent stem cells from porcine adipose-derived stem cells with a feeder-independent and serum-free system.
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DOI：
10.1371/journal.pone.0085089
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Zhang Y
中科院分区：
文献类型：
--
作者：
Zhang Y;Wei C;Zhang P;Li X;Liu T;Pu Y;Li Y;Cao Z;Cao H;Liu Y;Zhang X;Zhang Y
诱导多能干细胞（induced pluripotent stem cells，iPSC）是通过转录因子的异位表达或小分子处理而重编程的体细胞，类似于胚胎干细胞（embryonic stem cells，ESC）。它们在改善转基因大型动物的生产方面有很大的希望。然而，目前获得的很少有猪iPSC（piPSC）系可以支持克隆胚胎的发育。在这里，我们从猪脂肪来源的干细胞（pADSC）中产生iPSC，使用定义的人类因子（Oct 4，Sox 2，c-Myc和Klf 4）的药物诱导表达。从pADSC重编程iPSC比从成纤维细胞重编程iPSC更有效，无论使用饲养层非依赖性或饲养层依赖性方式。通过添加含有小鼠Lif、CHIR 99021和PD 0325901（Lif-2 i）的Lif-2 i培养基，在饲养层非依赖性和无血清条件下获得幼稚样piPSC。这些成功重编程的piPSC的特征在于短细胞周期间隔、碱性磷酸酶（AP）染色、Oct 4、Sox 2、Nanog、SSEA 3和SSEA 4的表达以及正常核型。piPSC与幼稚ESC的相似性通过它们的密集圆顶形态、单细胞解离后的生长、Life依赖性、Stella和Eras的上调、TRA-1-60、TRA-1-81和MHC I的低表达水平以及两个X染色体的活化来证实。通过Lin 28、Esrrb、Utf 1和Dppa 5的显著上调，评估了幼稚样piPSC的完全重编程，在体外和体内分化成所有三个胚层的细胞类型。此外，来自幼稚样piPSC的核移植胚胎可以发育成质量提高的囊胚。因此，我们提供了一种有效的方案，用于在饲养层非依赖性和无血清系统中从pADSC产生幼稚样piPSC，并控制外源基因的调节，这可能有助于优化培养基和生产转基因猪。
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.
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