DNA甲基化和去甲基化对猴Naive（全能）干细胞重编程的作用机制研究

The key mechanisms that reprogramming technology changes cell fate and rebuilds new functions are to complete epigenetic reprogramming, including DNA methylation and demethylation. Compared to mice and rat pluripotent stem cells (PSCs), we could not prove whether human PSCs are naive ground with development totipotency due to lack of function tests, such as chimera assays. Thus, it is unclear that the functions and mechanisms of DNA methylation modification for human naive stem cell formation and maintenance. Monkey PSCs are an ideal model to study primate stem cell pluripotency due to their close morphologies, growth characteristics, and gene expression profiles similarity to those of human PSCs. . In our previous studies, we successfully reprogrammed cynomolgus monkey primed embryonic stem cells (ESCs) into naive-like ESCs in adjusted culture conditions. These naive-like ESCs are able to incorporate into host embryos and develop into chimeras with contribution in all three germ layers and in germ cell progenitors once reintroduced into host monkey embryos. Furthermore, these naive-like ESCs lowly expressed de novo methyltransferase DNMT3A and DNMT3B, suggesting that there may be differential patterns in DNA methylation modification between naive-like and primed ESCs. . Following the previous findings, the project objective is to figure out the DNA methylation regions specific for naive stem cells and understand how DNA methylation modification together with exogenous environment promotes primate naive stem cell reprogramming. To answer the two questions, we plan to perform the five following experiments. First, we shall investigate the functions of DNA methylation modification for reprogramming primed ESCs into naive ESCs by establishing primed DNMT3A-/- and DNMT3B-/- DNMT3A-/-DNMT3B-/-, DNMT3A+/-, DNMT3B+/- and DNMT3A+/-DNMT3B+/- ESC lines. Second, we try to draw the change dynamic curves of DNA methylation and demthylation during the course of primed ESCs into naive ESCs. Third, we shall want to figure out the differential DNA methylation regions (DMRs) specific for naive stem cells by comparing the genome-wide DNA methylation and demthylation among chimera competent naive ESCs, primed ESCs and non-chimera competent ESCs grown in naive culture media. Forth, we plan to analyze the regulatory relationship between these DMRs and gene expression profiles of naive ESCs. Finally, we set out to understand which signaling pathways, such as LIF- Stat3, TGF-β1, ERK1/2i, Wnt, P38-MAPK and JNK, in naive culture media are involved in the contributions of DMRs specific for naive stem cells by dropout experiments. We hope to reveal the mechanisms of DNA methylation and demethylation in regulating primate pluripotent stem cell fates and functions by these serial experiments.

重编程技术改变细胞的命运和功能的关键在于实现细胞表观遗传的重编程，包括DNA甲基化修饰。同大小鼠相比，人多能干细胞无法通过嵌合体等功能实验证明发育全能性，即细胞的Naive（全能）特性，因此甲基化修饰对全能干细胞的作用机制，仍然是一个世界性难题。猴多能干细胞与人高度相似，是研究灵长类全能干细胞的最佳模型。在前期研究中，我们首次改变培养体系，将猴Primed胚胎干细胞重编程为能产生嵌合体猴的Naive干细胞，并发现后者低水平表达甲基化酶。本项目在此基础上，拟通过研究猴Primed到Naive干细胞重编程过程中甲基化修饰的功能和动态变化，并与Naive培养条件下无嵌合能力的胚胎干细胞进行比较分析，筛选出Naive干细胞特异性的甲基化和去甲基化区域，分析它们的生物学功能以及与Naive培养条件下信号因子的作用关系，揭示培养环境与DNA甲基化修饰相结合促进灵长类Naive干细胞重编程的作用机制。

本项目在2015年我们发表在《Cell Stem Cell》的文章证明Naive-like多能干细胞具有发育的全能性，能够产生嵌合体猴的基础上，希望通过对不同嵌合能力细胞系的DNA甲基化分析，筛选出维持Naive-like多能干细胞特定的甲基化区域。针对项目目标，我们在过去一年里开展了系统地研究，完成了项目的预订目标，并新增加了项目里面没有涉及的研究内容。完成项目中的研究内容如下：1）DNA甲基化对维持灵长类多能干细胞Naive状态的作用，获得了DNMT3A 和DNMT3B敲除的稳定细胞系；2）灵长类Primed 多能干细胞重编程到Naive 多能干细胞过程中的DNA甲基化动态变化的研究，目前正在进行数据的整理以及相应的文章发表；3-4）Naive 干细胞特异甲基化和去甲基化差异性区域（DMRs）的筛选和研究Naive 多能干细胞特异性区域与基因表达之间的相关性，目前已经筛选到一些特定的区域，由于项目只有一年时间，正在进行功能分析；5）研究Naive 培养条件下信号通路与Naive 多能干细胞关系，发现Wnt信号通路具有重要的关系。项目完成了申请书中没有而新增的三个研究内容：1）首次证明传统培养的猴ESCs、诱导多能干细胞（iPSCs）以及核移植来源的ESCs (nt-ESCs)具有产生嵌合猴的能力。我们的研究结果首次通过体内实验证明了传统的猴多能干细胞具有发育全能性，对传统灵长类PSCs的观点提出了巨大的挑战，为干细胞的转化奠定了重要的基础；2）证明了猴多能干细胞的嵌合能力与细胞状态之间的关系，并揭示了传统培养的ESCs，iPSCs和nt-ESCs嵌合以及它们之间嵌合能力差异的分子机制；3）建立了新的Naive干细胞培养体系的建立，并采用该体系直接从人和猴囊胚中获得了15株Naive胚胎干细胞系。这些研究成果促进我们灵长类干细胞多能性的理解，促进细胞的标准化培养。目前在该项目支持下发表了1篇SCI论文，有1篇文章正在国际一流杂志审稿。培养3名硕士研究生和1名博士生。