人原始态多能性特异表面标志物ALPPL2的鉴定、作用机制及应用研究

Human naive pluripotency, established in the epiblast cells of the preimplantation blastocysts and captured in vitro, represents the research hotspot in the study fields of pluripotency and stem cells. During the past 10 years, important breakthroughs have been made in optimization of induction and culture systems and evaluation of molecular criteria for naive pluripotency. However, the studies involved in the identification of naive state-specific surface markers and the related molecular mechanisms remain poorly understood..By systematically comparing the membrane proteins between naive and primed pluripotent stem cells, this project is to identify naive specific cell surface marker candidates. Using multiple cell lines including secondary naive/primed reprogramming system, naive-primed transition system, and naive/primed pluripotent stem cells, combined with iTRAQ-MASS and bioinformatics analysis, we recently identify ALPPL2 as the prominent surface marker which can specifically indicate naive pluripotency. Using monoclonal antibody production as well as inducible CRISPR/Cas9 genome editing system, this project tries to illustrate ALPPL2’s function and the involved mechanisms during naive pluripotency establishment and maintenance. Moreover, we also try to perform sgRNA libraries screening using ALPPL2 as the naive pluripotency indicator. .Taken together, our study can identify the first functional surface marker for naive pluripotency. By functional analysis, our study will shed light on the molecular network of naive pluripotency, which can further provide the mechanical basis for optimization of naive induction/culture system.

人原始态多能性研究是当前多能性及干细胞领域的关注热点。在针对于这一捕获于植入前囊胚晚期细胞的多能性状态10年来的研究中，人们在诱导系统的优化及其分子特征的鉴定标准等方向上取得了一系列重要突破，然而原始态特异表面分子标志物的鉴定及分子机理研究仍处于起步阶段。.本项目拟以系统比较原始态和始发态多能干细胞的表面膜蛋白组差异入手，运用包括2°原始态及始发态体细胞重编程系统、原始态-始发态转换系统及原始态和始发态多能干细胞在内的多种细胞系，结合iTRAQ-MASS定量、组学分析、单克隆抗体制备及可诱导的CRISPR/Cas9基因编辑等技术，鉴定能特异性指征原始态多能性的首个功能性表面分子标志物ALPPL2。并通过功能分析，深化其在原始态多能性建立及维持过程中的分子作用机制的理解；最后，通过该表面标志物的应用，为原始态多能性建立过程中细胞命运决定机制及诱导培养体系的优化等研究提供理论基础。

人原始态多能性研究是当前多能性及干细胞领域的关注热点。在针对于这一捕获于植入前 囊胚晚期细胞的多能性状态10年来的研究中，人们在诱导系统的优化及其分子特征的鉴定标准 等方向上取得了一系列重要突破，然而原始态特异表面分子标志物的鉴定及分子机理研究仍处 于起步阶段。 .本项目以系统比较原始态和始发态多能干细胞的表面膜蛋白组差异入手，运用包括2° 原始态及始发态体细胞重编程系统、原始态-始发态转换系统及原始态和始发态多能干细胞在内的多种细胞系，结合iTRAQ-MASS定量、组学分析、单克隆抗体制备及可诱导的CRISPR/Cas9 基因编辑等技术，鉴定出能特异性指征原始态多能性的首个功能性表面分子标志物ALPPL2。并阐明其在原始态多能性建立及维持过程中的分子作用机制；在此基础上，建立了能精准指征naïve态多能性的ALPG/OCT4-ΔPE的双荧光报告系统，描绘了naïve多能性建立过程中标志分子表达动态图谱和细胞命运转变动态图谱，并实现了基于人原态诱导体系的新型类囊胚模型构建和原肠胚前发育模拟；开展了基于ALPG/OCT4-ΔPE双荧光报告系统的naïve态新型培养体系筛选，并获得相应的候选小分子。此外，本项目也系统解析了全能性关键转录因子Dux在体内早期胚胎发育系统中、胚胎基因组激活过程中的作用，并揭示锌指蛋白靶向逆转录引物结合位点序列抑制逆转录病毒的机制。项目发表Cell Research、Nature Communications、Cell Reports、Protein & Cell、PNAS等论文多篇，申请国家发明专利4项，其中两项获得授权。