NANOG在人原始态多能干细胞诱导和维持中的作用机制研究

Embryonic stem cells (ESCs), a type of pluripotent stem cells (PSCs), have the ability to self-renew retaining pluripotency or differentiate into any cell type that compose a body, thus providing a remarkable model to investigate the mechanisms of development and cell fate commitment. PSCs also hold great promise for regenerative medicine. However, human ESCs/PSCs are considered to be in a “primed” state, which corresponds to a later embryonic stage than their “naïve” mouse counterparts. Thus, human ESCs/PSCs have disadvantages on single cell passage, efficiency of genetic engineering, differentiation potential in vitro and the ability to contribute to chimeras. To overcome these issues, scientists all over the world have tried to establish naïve human ESCs/PSCs and got some encouraging results. However, the published achievements have many problems, such as heterogenecity of cells produced by different labs, low or no interspecies chimera efficiency and so on. Thus, demonstrating the mechanism of naïve human ESC/PSC induction and maintenance will be very important to optimize the existing conditions or set up new conditions for optimal derivation... NANOG is an important transcription factor that over-expressed can contribute to induce both mouse and human naïve ESCs. However, paradoxically NANOG is also highly expressed in mouse epiblast stem cells and in human primed ESCs/PSCs. Here, we want to systematically study the role of NANOG in naïve human ESC/PSC induction and maintenance. For this purpose, we will first study the need for endogenous NANOG to either sustain or induce human naïve pluripotency achieved by overexpresion of NANOG and KLF2 or with chemical cocktails. We will also analyze NANOG genome-wide binding pattern in human primed and naïve ESCs (produced with vectors or chemicals), and study the correlation with published epigenetic marks. Finally, we will perform protein interactome and post-translational modification analysis of NANOG in primed versus naïve human ESCs/PSCs. By doing this, we want to understand differences in the role of NANOG in primed versus naïve human ESCs/PSCs, which will provide clues for further optimization of naïve human ESC/PSC culture condition.

胚胎干细胞（embryonic stem cell，ESC）具有无限自我更新和分化为任何一种细胞类型的能力，被称为多能干细胞。其诞生为研究胚胎发育和细胞分化提供了理想模型，也开启起了再生医学研究的热潮。相比处于原始态的小鼠ESC，人ESC处于始发态，在单细胞传代、分化潜能、基因编辑效率和形成嵌合等方面存在缺陷。因此科学家尝试建立了原始态人ESC。然而，目前建立的原始态人ESC仍存在很多问题。转录因子NANOG是诱导小鼠和人多能干细胞由始发态向原始态转变的重要转录因子。本项目试图通过比较NANOG在始发态和原始态人ESC维持和诱导中的必要性、调控的靶基因、全基因组结合位点及其与关键表观遗传修饰的关联性、相互作用蛋白谱以及翻译后修饰等，深入解析转录因子NANOG发挥作用的分子机理，阐述原始态人ESC建立和维持的机制，为进一步优化培养条件获得能形成高比例嵌合的原始态人多能干细胞奠定基础。

胚胎干细胞（embryonic stem cells，ESCs）具有体外培养无限增殖、自我更新和多向分化的特性，是体外研究发育调控的良好模型，也为再生医学带来了无限可能。然而， 相比于小鼠ESCs，人ESCs在细胞均一性、单细胞传代能力、分化潜能、基因编辑效率和形成嵌合体能力等方面存在显著缺陷。这些缺陷被人为是由于人的ESCs处于始发态所导致的。因此，科学家尝试建立类似于小鼠胚胎干细胞的原始态人ESC。转录因子NANOG是诱导小鼠和人多能干细胞由始发态向原始态转变的重要转录因子。本项目试图系统阐述核心多能性转录因子NANOG在人原始态ESCs诱导和维持中的作用及其调控机制。本项目建立了转录因子（KLF2和NANOG）和小分子化合物诱导的原始态人ESC/iPSC培养平台，在此基础上通过基因编辑构建了特异性标记始发态和原始态人多能干细胞的应该报告细胞系并进行了有效性验证。利用原始态人多能干细胞特异性的荧光报告细胞系，研究了NANOG在原始态人多能干细胞诱导中的作用，并进一步阐述了NANOG相互作用蛋白ALKBH1在人多能干细胞维持和分化中的作用及机制。本项目的顺利进行进一步完善了核心多能性转录因子NANOG在多能干细胞诱导、维持和分化中的作用机制研究。