基于GATA2敲除及GATA2-GFP标记的hES细胞系研究内皮-造血转变的调控机制

The inability of obtaining hematopoietic stem cell (HSC) capable of long-term reconstitution and full lineage specification is partially due to lack of understanding about hematopoiesis during early embryo development. Among the early events of early hematopoiesis, endothelial-to-hematopoietic-transition (EHT) is a key time point. Data from Gata2 knock-out mouse model suggest that GATA2 plays an important regulatory role during EHT. However, parallel study in human hematopoiesis is still missing. Due to the difference between human and mouse hematopoiesis, we chose GATA2 as point of entry to study mechanism of EHT in human system. We have recently established both GATA2 knock-out and GATA2-GFP knock-in hES cell line and plan to compare transcriptome of HE and HSPC obtained from in vitro hematopoietic differentiation of these cell lines. We aim to reveal the regulatory mechanism of EHT by discovering GATA2 effectors and transcription factors cooperating with GATA2. Functional tests such as function rescue or shRNA knockdown were also planned to validate the regulatory role of these effectors and transcription factors. Dissection of mechanism of EHT regulation will pave our way to fully understand hematopoiesis regulation during early human embryo development and eventually lead to high efficient method for generation of transplantable HSC through in vitro differentiation of pluripotent stem cells.

明确人早期造血发育的调控机制，对通过体外定向分化途径获得人的功能性造血干细胞(HSC)至关重要。早期造血发育中，内皮-造血转变(EHT)过程是重要节点之一。基于小鼠模型研究发现，GATA2在EHT过程中起重要的调控作用，但迄今未有GATA2在人的造血发育EHT的平行研究报导。鉴于人鼠造血发育的差异性，本课题组直接选择以人的GATA2敲除和内源GATA2-GFP标记的ES细胞系体外造血分化模型来研究EHT调控。本课题拟通过比较GATA2敲除、GFP标记敲入情况下，体外造血分化获得的生血内皮、造血干祖细胞的转录组，寻找GATA2在EHT调控过程中的效应因子和共同作用因子，并通过功能补偿和shRNA敲除等手段加以验证，来揭示GATA2相关的EHT调控机制，完善对人类早期造血发育分化调控的认识，从而为多能干细胞定向造血分化获得HSC奠定基础。

明确人早期造血发育的调控机制，对通过体外定向分化途径获得人的功能性造血干细胞至关重要。早期造血发育中，内皮-造血转变(EHT)过程是重要节点之一。本项目构建了GATA2敲除和内源GATA2-GFP标记的ES细胞系体外造血分化模型来研究EHT调控。结果表明GATA2敲除的hES内皮-造血转变过程受到影响，产生CD34+CD43+造血干祖细胞的能力下降。进一步机制研究发现，SPI1是GATA2调控此过程的效应因子。此外，本项目构建了GATA2/eGFP基因敲入的报告细胞系，证明在造血分化过程中GATA2/eGFP+的内皮细胞为生血内皮(HECs)；在此基础上，找到HECs表面特异标记分子CD61，证明该分子能在人多能干细胞造血分化和小鼠胚胎发育过程中示踪HECs。本项目的成果完善了对人类早期造血发育阶段中EHT过程调控的认识，从而为多能干细胞定向造血分化获得HSC奠定基础。