重组蛋白诱导猪iPSCs及其表观遗传重编程变化规律研究

Pig is generally regarded as an economically important domestic animal and an ideal animal model for human medical research, and it's induced pluripotent stem cells (iPSCs) hold great promise for disease diagnosis and drug screening, animal breeding and conservation, biology basic research. However, innative risks of virus-mediated iPSCs generation method, limited source cell type, sup-optimal culture conditions, and unideal iPSCs quality limit the wide application of iPSCs. Based on our previous work, non-virus-mediated and non-genome- integration strategy, this study aims to establish and improve the generation of iPSCs by using recombinant proteins, in order to efficiently generating high-quality, safe iPSCs lines, and to reveal epigenetic dynamic changes during the reprogramming. We will combine the recombinant protein, the source cell type, and small molecule intervention to optimise iPSCs induction program. iPSCs generated by virus-mediated methods serve as control, we then will examine the phenotype, biology properties and genome characteristics of iPSCs induced by the recombinant proteins. Dynamic changes of DNA methylation, histone modifications and microRNAs during reprogramming will also be explored. We will monitor the in vivo and in vitro developmental fate of the iPSCs cloned embryo to assess stringently the quality and developmental pluripotency of iPSCs. Besides, we will enrich criteria for iPSCs quality evaluation by exploring specific microRNAs for maintenance of iPSCs pluripotent state. The study will be of great help to deepen our understanding of cell reprogramming, promote continual improvement of iPSC technology, and lay the foundation for advancing the iPSCs to serve human medicine and animal husbandry.

猪作为重要经济动物和人类医学研究的动物模型，其iPSCs在疾病诊治、动物育种保种、生物学基础研究等方面具有重要价值。病毒介导获取的iPSCs存在安全隐患，iPSCs质量不理想等都限制了iPSCs的应用。本研究拟在前期工作基础上，建立并完善重组蛋白诱导iPSCs的技术方案，创建高质量、安全的iPSCs，并揭示iPSCs产生期间表观遗传重编程动态变化规律。重点从重组蛋白、源头细胞等环节优化iPSCs诱导方案；以病毒介导的iPSCs为对照，检测iPSCs的细胞表型、基因组特征及iPSCs产生期间的DNA甲基化、组蛋白修饰和microRNA变化规律，监测iPSCs克隆胚体内外发育，深入评估iPSCs质量和发育多能性，发掘iPSCs特异的microRNAs，丰富iPSCs质量评价标准。研究有助于加深我们对细胞重编程的认识、促进iPSC技术的不断完善和造福人类医学及畜牧业等。

猪作为重要经济动物和理想人类医学动物模型，其iPSCs在畜牧、医学和基础研究上具有重要学术价值和应用前景。目前，质量不佳限制了iPSCs发展，因而获得猪高质量iPSCs是当前的热点和难点。为创建猪高品质iPSCs，揭示iPSCs重要事件，项目开展了猪iPSCs重组蛋白、药物调节的慢病毒系统诱导及培养体系优化，从形态、细胞分子等方面对所猪 iPSCs 特性进行鉴定，对可能参与 iPSCs 多能性建立/维持的特异表观修饰、miRNAs 等进行发掘，评估iPSCs克隆胚发育潜能。结果如下：（1）原核表达出Sox2-11R 、Lin28-11R 、Oct4-11R等，真核表达出Oct4-9R、Sox2-9R、Klf4-9R、c-Myc-9R、Lin28-9R 等重组蛋白，真核蛋白粗提物可诱导猪体细胞发生 MET 现象，意味着重编程程序启动，但是无法进一步获得可传代的 iPSCs。（2）以 pADSCs 为源头细胞，在无饲养层、无血清条件下诱导出猪高质量的naïve like iPSCs，获得了外源因子随时可被调控表达的 iPSCs 系，为深入探讨间充质来源的猪iPSCs性状、表观修饰特征及应用提供了极好的素材。（3）在无饲养层条件下从pADSCs诱导出一种新型的部分重编程pPiPSCs，揭示了pPiPSCs的生物学特性，为重编程机制研究和克隆猪的生产提供了新材料。（4）Vc 、CHIR-99021 显著提高猪体细胞重编程，不同年龄、类型猪体细胞对猪体细胞重编程的影响不同，为高效获得猪 iPSCs奠定基础；揭示了分化基因、多能性基因在pADSCs重编程中表达规律，发现在iPSCs获取效率上， pADSCs 显著高于 PEFs 和 APEFs。（5）揭示了naïve样 iPSCs的 miRNAs 表达谱。预测 miR-7138-3p、miR-432-5p 和 miR-31 可能是潜在的猪iPSCs多能性建立/维持调控因子。首次揭示了猪 iPSCs 的lncRNA/mRNA 表达谱，在差异表达的201 种已知 lncRNA 、 5 种新 lncRNA发现IL-6 及 LOC102166377、LOC102159188两种 lncRNA 可能负调控猪 iPSCs 多能性基因。总之，本研究结果将有助于丰富对猪干细胞多能性调控的认识，为猪iPSCs、ESCs研究提供宝贵的参考依据。