人体细胞重编程为naïve多能干细胞的分子机制研究

Naïve pluripotency which is established in the epiblast of the pre-implantation blastocyst during development and can be captured in vitro, represents the research trend in the fields of stem cells and reprogramming. Compared with the primed pluripotent state in post-implantation embryos, naïve state represents higher pluripotency and more plasticity; therefore, offers more potential use for early embryo development studies and therapeutic manipulations. However, the current studies mainly focus on derivation of human naïve pluripotent cell lines and modification of naïve culture conditions, lacking in-depth mechanical studies in naïve pluripotentcy establishment and maintenance during reprogramming processes..Using human inducible secondary naïve reprograming system together with the primary reprogramming system, this project is to systematically study the biological changes along the timeline from human somatic cells to naïve pluripotent stem cells. Combined with high-throughput sequencing and bioinformatics analysis, this project also tries to illustrate important molecular mechanisms involved in this reprogramming process. Moreover, using iTRAQ (isobaric tags for relative and absolute quantification)-MASS spectrometry analysis as well as CRISPR/Cas9 genome editing system, we try to identify the specific surface antigen markers for naïve pluripotency, and study their kinetics during naïve pluripotency establishment and stabilization by reporter tracing system. .In summary, our study can deepen the understating of molecular network in naïve pluripotency. Based on the mechanism study of naïve reprograming process, our study can further provide theoretical basis for improvement of the existing naïve reprogramming system, modification of naïve cell differentiation system, and further clinical applications of naïve pluripotent stem cells.

Naïve态多能性作为当今干细胞及重编程领域研究的热点，在体内发育中建立于植入前成熟囊胚的上胚层细胞中。与植入后胚胎所处的primed态相比，具有更高的多能性及可塑性，在早期胚胎发育研究及未来临床应用中具有更广阔的前景。目前的研究主要集中在人naïve态多能干细胞的建立与培养条件上，对人类naïve多能性建立及维持过程中分子机制未有深入探索。本项目以可诱导的人二代（2°）重编程系统为研究对象，结合一代（1°）重编程系统，通过生物信息学分析，运用膜蛋白标定－质谱定量分析及CRISPR/Cas9技术，系统研究人类体细胞重编程至naïve iPSCs过程中所发生的关键生物学事件及特异的表面抗原动态，并对naïve多能性建立及稳定的分子机理进行深入探索。这项研究将深入理解naïve状态多能性建立及维持的分子网络机制，为现有naïve重编程系统优化及其在未来临床治疗上的应用提供理论基础。

原态多能性作为当今干细胞及重编程领域研究的热点，与植入后胚胎所处的始发态相比，具有更高的多能性及可塑性，在早期胚胎发育研究及未来临床应用中具有更广阔的前景。目前的研究主要集中在人原态多能干细胞的建立与培养条件上，对人类原多能性建立及维持过程中分子机制未有深入探索。本项目在成功建立人二代（2°）重编程系统的基础上，结合高通量组学分析，描绘了人体细胞重编程至naïve态多能性过程中转录组及表观修饰组的动态图谱；在此基础上，运用膜蛋白标定－质谱定量分析及CRISPR/Cas9基因技术，系统研究了人类原态多能性特异的表面标志物动态，并鉴定出特异指征人naïve态多能性的功能性表面分子标志物ALPPL2，发现其对于人类naïve态多能性的建立及稳定维持具有重要作用。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。上述研究对原态多能性建立及稳定的分子机理进行了深入探索。