Department of Animal Biotechnology, College of Veterinary Medicine , Northwest A&F University, Yangling, Shaanxi, China .
DNA Cell Biol. 2014 Jan;33(1):1-11. doi: 10.1089/dna.2013.2095. Epub 2013 Nov 20.
Ground state porcine induced pluripotent stem cells (piPSCs), which retain the potential to generate chimeric animal and germline transmission, are difficult to produce. This study investigated morphological and biological progression at the early stage of porcine somatic cell reprogramming, and explored suitable conditions to increase the induction efficiency of piPSCs. A cocktail of defined transcription factors was used to generate piPSCs. The amphotropic retrovirus, which carried human OCT4 (O), SOX2 (S), KLF4 (K), C-MYC (M), TERT (T), and GFP, were used to infect porcine embryonic fibroblasts (PEFs). The number of clones derived from OSKM (4F) and OSKMT (4F+T) was significantly higher than that from SKM (3F) and SKMT (3F+T), suggesting that OCT4 played a critical role in regulating porcine cell reprogramming. The number of alkaline phosphatase-positive clones from a medium with leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) (M1 medium) was significantly higher than that with insulin and 2i PD0325901/CHIR99021 (M2 medium), indicating that insulin and 2i could not effectively maintain piPSC propagation. In the M1 medium, piPSC lines could not maintain the typical self-renewal morphology on gelatin-coated and Matrigel-coated plates. Without the mouse embryonic fibroblast (MEF) feeder, piPSCs started to simultaneously differentiate. Based on the potential for self-renewal and activation of pluripotent markers, we found that the culture condition of 4F+T plus LIF and bFGF plus MEF feeder promoted PEF reprogramming more efficiently than the other conditions tested here. Two piPSC lines (IB-1 and IB-2) were derived and maintained for up to 20 passages in vitro.
猪诱导多能干细胞（piPSC）的基础状态，保留了产生嵌合动物和种系传递的潜力，难以产生。本研究调查了猪体细胞重编程早期的形态和生物学进展，并探索了提高 piPSC 诱导效率的合适条件。使用定义的转录因子鸡尾酒来生成 piPSC。携带人 OCT4（O）、SOX2（S）、KLF4（K）、C-MYC（M）、TERT（T）和 GFP 的双嗜性逆转录病毒用于感染猪胚胎成纤维细胞（PEF）。来自 OSKM（4F）和 OSKMT（4F+T）的克隆数量明显高于来自 SKM（3F）和 SKMT（3F+T）的克隆数量，表明 OCT4 在调节猪细胞重编程中起关键作用。来自白血病抑制因子（LIF）和碱性成纤维细胞生长因子（bFGF）（M1 培养基）的碱性磷酸酶阳性克隆数量明显高于含有胰岛素和 2i PD0325901/CHIR99021（M2 培养基）的克隆数量，表明胰岛素和 2i 不能有效维持 piPSC 增殖。在 M1 培养基中，piPSC 系不能在明胶包被和 Matrigel 包被板上维持典型的自我更新形态。没有小鼠胚胎成纤维细胞（MEF）饲养细胞，piPSC 开始同时分化。基于自我更新的潜力和多能标志物的激活，我们发现 4F+T 加 LIF 和 bFGF 加 MEF 饲养细胞的培养条件比这里测试的其他条件更有效地促进了 PEF 重编程。从猪胚胎成纤维细胞（PEF）中成功诱导出了 2 株 piPSC 系（IB-1 和 IB-2），并在体外维持了多达 20 代。
