慢病毒直接诱导形成猪iPS细胞无需限定因子

为了证实慢病毒对细胞具有遗传修饰和重编程作用,在本实验中使用慢病毒感染猪胎儿成纤维细胞.结果显示:慢病毒介导的EGFP在猪胎儿成纤维细胞中稳定和高效表达,使用添加LIF和bFGF的细胞培养液,部分猪的胎儿成纤维细胞逐渐改变原有的纤维状形态,形成圆形的细胞,细胞逐步增殖形成细胞集落,细胞集落边界清晰,在饲养层上细胞集落生长迅速,具有稳定的生长性能和正常核型,细胞碱性磷酸酶染色为阳性,表达干细胞特有的标记Oct4、Nanog和SSEA1,在体外能够形成拟胚体,在体内分化形成包含三个生殖层的畸胎瘤.作为核移植的供体细胞,克隆胚的卵裂率为53.33%、桑椹胚率为9.03%、囊胚率为2.07%、孵化囊胚的总细胞数为26.5,在桑椹胚率和囊胚率方面显著低于猪普通胎儿成纤维细胞核移植克隆胚的发育能力(P<0.05).结果证实慢病毒能够直接使猪的胎儿成纤维细胞转变成iPS细胞,因此慢病毒将成为一种理想的材料和工具用于细胞的遗传修饰和细胞重构等方面的研究.

Generation of Porcine iPS Cells From Fetal Fibroblasts by Lentivirus Without Difined Factors

To investigate the effects of lentivirus on the growth and development of porcine fetal fibroblasts (PFBs), in this study PFBs were repeatedly infected by lentivirus. The results showed that lentivirus-mediated enhanced green fluorescent protein (EGFP) had stable and efficient expression in PFBs. Under the condition with leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF), some PFBs gradually changed their fibrous growth pattern into round cell morphology. These round cells proliferated and formed cell clones with clear edge boundary. Clones grew rapidly on feeder layers and passaged stably with normal karyotypes. These cells were positive for alkaline phosphatase (AP) and expressed stem cell markers Oct4, Nanog, and SSEA1. In addition, these cells formed embryoid bodies (EB) in vitro and three germ layers in vivo. After the cells were used as nuclear donors, the cleavage rate of the cloned embryos was 53.33%, the morula rate 9.03%, the blastocyst rate 2.07%, and the total cell number per hatched blastocyst was 26.5. Compared with embryos cloned from non- lentivirus PFBs, the morula rate and blastocyst rate were lower and significantly different (P < 0.05). Lentivirus can result in the generation of porcine iPS cells from PFBs, so it can be used as ideal material and tool for research such as epigenetic modification and cell reprogramming.