原始态与始发态多能性干细胞差异选择性剪接转录本分析

According to the derivation stage and the pluripotency levels, the embryo-derived pluripotent stem cells could be categorized as either naive or primed pluripotent state. The naive pluripotent stem cells, including mouse embryonic stem cells (mESCs), have higher development potential compared to the primed pluripotent stem cells. The mouse epiblast stem cells (EpiSCs) and the human embryonic stem cells (hESCs) are regarded as the primed pluripotent cells due to their capacities of forming chimeric. The recent researches showed the hESCs could be converted to the naive pluripotent state through transgenic reprogramming or modifying culture medium. However, the systematical comparison at transcriptome level between naive and primed state cells among different species has not been carried out. Here we propose to systematically identify the differentially expressed alternative splicing events between distinct pluripotent cells among three species, mouse, crab-eating macaque and human. We attempt to analyze the relationship between the alternative splicing events and the different pluripotent states in different species from evolutionary perspective. At last, we want to identify the pluripotent state specific alternative splicing events as the indicative markers of pluripotent levels and explore the function and mechanism of alternative splicing events in pluripotent regulation.

胚胎来源的多能性干细胞按照其来源及多能性水平，可分为原始态及始发态两类状态。原始态多能性干细胞相对于始发态多能性干细胞具有更高的发育潜能。一般认为小鼠胚胎干细胞属于原始态多能性干细胞；小鼠的上胚层干细胞及人胚胎干细胞属于始发态多能性干细胞。最近的研究通过重编程或改变培养条件的手段可将人胚胎干细胞转变为原始态类似的多能性干细胞。但到目前为止，对于不同物种间的两类多能性状态的细胞缺乏系统的比较分析。本研究拟通过系统比较小鼠、食蟹猴及人三个物种的两类多能性状态的细胞的转录组数据，鉴定在两类多能性状态细胞间的差异的选择性剪接事件；从进化角度解析差异选择性剪接事件在三物种间的保守关系及与多能性状态的关系；识别可作为多能性水平标志的选择性剪接事件作为筛选多能性状态细胞的分子标记并研究重要选择性剪接事件对多能性的调控机制。

选择性剪接作为调控基因功能多样性的重要机制参与多种细胞生物学过程；胚胎来源的多能性干细胞按照其来源及多能性水平，可分为原始态及始发态两类状态。本课题设计通过分析原始态与始发态多能性干细胞选择性剪接事件差异解析选择性剪接事件对多能性水平的调控。首先，通过比较Rbm14敲除与野生型小鼠胚胎干细胞发现其参与调控的选择性剪接事件，进而探索选择性剪接事件在多能性维持过程中的作用。通过该部分工作的开展，优化了选择性剪接事件的分析流程，为后续课题开展奠定基础，该部分工作发表在Cell Proliferation 杂志上。其次，通过与其他课题组合作，申请人进一步优化了人原始态与始发态多能性干细胞的培养体系，解析了原始态多能性维持机制，首次利用原始态与始发态多能性状态转换模拟了X染色体失活机制，为后续解析多能性相关选择性剪接事件提供了细胞资源。该部分工作发表在Cell Stem Cell杂志上。在此基础上，通过比较原始态、始发态及各种类型的体细胞间选择性剪接的差异，识别了一批与多能性水平相关的选择性剪接事件，经过验证及敲除实验发现参与原始态多能性调控的选择性剪接事件，为解析多能性调控提供了全新思路。