ZEB2介导的血管内皮生血化在人多能干细胞来源血细胞谱系发育中的作用研究

The process of hematopoietic cells budding from endothelium is called endothelial-to-hematopoietic transition (EHT), which sequentially ensures the circulation formation during embryo development. However, the molecular mechanism regulating EHT remains largely unknown. We previously found that during hematopoietic differentiation, expression of the transcript factor, ZEB2, was upregulated in human pluripotent stem cells (hPSC)-derived CD34+ cells. The budding hematopoietic cells were dramatically decreased in ZEB2-knockout hPSC-derived EHT. In addition, Zeb2-/- mice exhibited symptoms of myeloid over-proliferation, indicating that ZEB2 as a regulator in cellular transformation, plays an important role in determination of EHT-related blood fate. In this study, we will focus on the function of ZEB2 in hPSC's hematopoietic differentiation by using the genome editing technology to generate ZEB2-knockout or ZEB2-reexpressing hPSC sublines. The time-phase of hPSC-EHT, the hematopoietic colony-forming capacity of the budding cells and the difference of hPSC-derived hematopoietic lineage will be investigated in ZEB2-KO or ZEB2-reexpressing hPSCs. The function of ZEB2 on homing capability in regulating hPSC-derived blood cells will be also determined by in vivo image technique. The data of this study will clarify the role of the master transcript, ZEB2, in blood development and will enable to find the molecular target in regulating hPSC's hematopoietic differentiation.

血细胞从血管内皮上以出芽方式释放入血的过程即血管内皮生血化(EHT)，这种时空发育顺序保证了哺乳动物胚胎的血循环建立，但EHT及血细胞发育过程中的分子调控机制尚未明了。我们将人多能干细胞(hPSC)造血分化作为研究人类胚胎期血细胞发生的模型，发现ZEB2在hPSC-CD34+细胞中的表达增加；而下调ZEB2后，hPSC来源内皮细胞上的芽生血细胞明显减少。此外，Zeb2缺失的小鼠有髓系过度增殖的表现，说明这一调控细胞表型转化的转录因子在血细胞谱系发育中有重要作用。本课题以基因编辑技术获取敲除(或再诱导)ZEB2表达的hPSC亚系，观察这些亚系中EHT的时相改变，分析hPSC来源芽生血细胞的集落形成能力及细胞类型的变化，并以活体小动物成像技术探究ZEB2是否影响hPSC造血分化产物在成体造血器官中的归巢定植。对阐明血细胞发育过程中的分子机制、靶向诱导hPSC造血分化具有重要意义。

血细胞从血管内皮上以出芽方式释放入血的过程即血管内皮生血化(EHT)，这种时空发育顺序保证了哺乳动物胚胎的血循环建立。ZEB2是上皮间质转化在启动上皮间质转化过程中不可或缺，在造血系统中具有重要作用。我们建立可诱导型CRISPR/Cas9基因编辑系统，在iPSCs上完全敲除ZEB2，对ZEB2缺失的iPSCs运用spinEB分化方法进行体外造血分化以研究ZEB2在造血分化中的作用。发现ZEB2缺失不仅阻碍了EHT过程及抑制了人多能干细胞分化产生HSPCs，而且体外实验发现ZEB2缺失后分化的HSPCs在造血集落形成中也存在缺陷。同时，我们进一步明确了ZEB2缺失引起的造血分化缺陷发生在内皮-造血转化阶段。ZEB2缺失不影响造血内皮细胞的产生，而是通过影响内皮-造血转化过程阻碍造血干/祖细胞的生成。本研究发现ZEB2对于人多能干细胞向造血系分化的影响，加深了对HSC分化调控机制的了解，有望通过对ZEB2的调控来促进人多能干细胞向造血系分化的成熟度。随后我们的关注点聚焦于骨髓造血衰竭及hPSC造血分化产物在成体移植后的相关研究。发现改善免疫介导骨髓衰竭小鼠模型的骨髓微环境及aGVHD进展的新机制。阐明AA及aGVHD相关机制对造血细胞移植的应用具有重要意义。