利用iPSCs技术辅助建立猪Naive胚胎干细胞系及Naive与Primed胚胎干细胞转化机制研究

Increasingly pigs are becoming an important model for understanding human medicine and the potential source of organs for xenotransplantation, because their phenotype in many cases is more similar to humans than other animals. However, it is relative hard to get transgenic pig via somatic cell transgene and somatic cell nuclear transfer without pig naive ESC for transfection and long term screening. In this study, the traditional ESC culture method will be combined with iPSCs technique. The pig fetal fibroblasts will be transfected with Tet-inducible reprogramming factors vector followed by somatic cell nuclear transfer for getting blastocysts. The ICM of the blastocyst will be isolated and seeded in medium with LIF and DOX. The pig naive ESC hope to be established via this combination of traditional ESC and iPSCs method. The naive ESC derived from this project and the primed ESC established in our Lab recently will be compared for microRNA and lncRNA expression. According to the expression differences between the naive and primed ESC ,the different expressed microRNA or lncRNA will be selected,and the application of microRNA-mediated or lncRNA-mediated iPSCs on pig naive ESC establishment will be studied. Also,the mechanism of the conversion between the naive and primed ESC will be investigated，the conversion from pig primed ESC to naive ESC will be reached. This research may help to clarify the mechanism why the derivation of naive ESC from pigs and other ungulates has proved so difficult.

由于代谢生理、基因组和个体大小等与人类更为接近，猪已经日益成为人类疾病研究模型和潜在的异种器官移植供体。但由于没有naive胚胎干细胞（ESC）用于基因转染和长期筛选，通过细胞遗传修饰培育转基因猪的研究受到限制。本研究拟在传统胚胎干细胞建系方法中融入iPSCs技术，首先将转录因子表达载体导入猪胎儿成纤维细胞，通过体细胞克隆获得囊胚，囊胚内胚团接种于LIF培养体系，同时启动DOX诱导外源转录因子表达，从而建立猪naive ESC；比较猪naive 和primed ESC与猪体外受精胚胎内细胞团的microRNA和lncRNA表达谱，选择差异表达的microRNA或lncRNA，探讨RNA介导猪naive ESC建系的可行性；研究猪naive和primed ESC的相互转化机制，实现primed ESC 向naive ESC的转化。本研究结果有助于揭示大动物naive ESC建系难的分子机制。

本研究在传统胚胎干细胞(ESC)建系方法中融入iPSCs技术，将鼠源OCT4、Sox2、KLF4和C-Myc（mOSKM）表达载体导入猪胎儿成纤维细胞(PEF)，通过体细胞克隆获得囊胚，内细胞团(ICM)接种的同时启动DOX诱导mOSKM表达，从而建立猪naive ESCs；分析microRNA和lncRNA的差异表达,探讨其介导猪naive ESCs建系的可行性；研究猪naive和primed ESCs的相互转化机制。结果如下：(1)采用LIF+FGF+3i培养体系，成功建立了猪naive-like ESCs。所建细胞系已传至135代，仍然保持旺盛生长状态。所建干细胞系在三维立体形态及信号通路等诸多方面与小鼠naive ESCs类似，体外可分化为神经细胞和肾前体细胞。（2）所建细胞系同时表达猪内源pOSKM和鼠源mOSKM，兼具ESC和iPSC两种特性。naive-like ESCs中内源pOSKM的表达极显著高于相应的iPSCs中pOSKM的表达，而外源mOSKM的表达极显著地低于iPSCs。逐步降低培养液中DOX浓度至完全去除之后，naive-like ESCs中外源mOSKM表达极显著降低，内源pOCT4和pKLF4的表达未获提高，但内源pSOX2和pc-Myc的表达显著提高。所建ESCs在不添加DOX的培养系统中仍然持续生长，可继续传10代以上。（3）Naive-like ESCs 和primed ESCs(本实验室之前建立)的microRNA表达模式接近于猪体外受精胚胎ICMs,其中，ssc-miR-205和 ssc-miR-885-3p的表达在ICMs、naive-like和primed ESCs之间呈递减趋势，ssc-miR-205在ICMs中的表达分别是两种ESCs的285和473倍。构建miR-205表达载体，对mOSKM阳性PEFs再转染，结果显示，miR-205可提高猪iPSCs的建系效率。（4）在DOX培养条件下，naive-like ESCs的全基因组表达谱和Lnc-RNA表达谱接近于猪iPSCs，去除DOX后，其全基因组表达谱和Lnc-RNA表达谱与ICMs更为接近。 (5) Naive-like ESCs可在单纯更换培养条件下,实现与primed ESCs的相互转化。本研究结果有助于揭示大动物naive胚胎干细胞建系难的分子机理。