Optimization of Culture Conditions for Maintaining Porcine Induced Pluripotent Stem Cells
Optimization of Culture Conditions for Maintaining Porcine Induced Pluripotent Stem Cells
复制标题
猪诱导多能干细胞培养条件的优化
DOI：
10.1089/dna.2013.2095
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发表时间：
2014-01-01
影响因子：
3.1
通讯作者：
Wang, Huayan
中科院分区：
文献类型：
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作者：
Gao, Yi;Guo, Yanjie;Wang, Huayan
基态猪诱导多能干细胞(PiPSCs)保留了产生嵌合动物和生殖系传播的潜力，但很难生产出来。本研究对猪体细胞重编程早期的形态和生物学进程进行了研究，并探索了提高猪PSCs诱导效率的适宜条件。确定的转录因子的鸡尾酒被用来产生piPSCs。用携带人OCT4(O)、SOX2(S)、KLF4(K)、C-myc(M)、TERT(T)和绿色荧光蛋白的两性逆转录病毒感染猪胚胎成纤维细胞(PEF)。OSKM(4F)和OSKMT(4F+T)克隆数显著高于SKM(3F)和SKMT(3F+T)，提示OCT4在猪细胞重编程调控中起关键作用。在含有白血病抑制因子(LIF)和碱性成纤维细胞生长因子(BFGF)的培养液(M1培养液)中，碱性磷酸酶阳性克隆数显著高于胰岛素和2i PD0325901/CHIR99021(M2培养液)，表明胰岛素和2i不能有效地维持PIPSC的增殖。在M1培养基中，PIPSC在明胶涂层和Matrigel涂层平板上不能保持典型的自我更新形态。在没有小鼠胚胎成纤维细胞(MEF)饲养层的情况下，piPSCs开始同步分化。基于多能标记的自我更新和激活的潜力，我们发现4F+T+LIF和bFGF+MEF饲养层的培养条件比本试验的其他条件更有效地促进PEF重编程。获得了两个PIPSC系(IB-1和IB-2)，并在体外培养了20代。
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.