可调控模式GATA-1和GATA-2基因沉默hES/hiPS细胞系的建立及其向初期造血诱导分化的分子调控机制研究

The knowledge about the molecular ragulation of human early hematopoiesis is largely unknow, mostly because we lack a proper in vitro system to mimic the natural progressive course during the development from pluripotent stem cells to functionally mature blood cells. The esbablishment of human embryonic stem cells, and lately, induced pluripotent stem cells (hESC, hiPSC, respectively) greatly expanded our view to subtly analyze the cellular and molecular happenings during the early develoment of various tissues, including blood. Accumulated data, including ours, has achieved various functionally matured blood cells derived from hESC/hiPSCs, highlighting their ideal utilazation both as the research models and clinical applications. However, the hESC/hiPSC-derived hematipoietic cells were phenotpically and functionally immature, and their melacular controlling towards maturation being unclear. We here design a GATA-1/GATA-2 down-regulation system by using molecular targeting manipulation (shRNA)to transfect and establish hESC/hiPSC lines. Using this system, we can further analyze the early hematopoiesis in a subtle and timed way during the early differentiation of hematopoietic stem/progenitor cells from hESC/hiPSC. Our study may dedicate critical information about the molecular controls in human early hematopoiesis, especially the generation of definitive hematopoietic stem cells and their functionally matured progenies.

由于对人类早期造血发生的分子调控机制尚缺乏了解，急需建立一种理想的实验体系。我们报道了体外大量扩增hESC/hiPSC来源的造血干／祖细胞的方法，并证明了hESC可以诱导产生成体造血来源的功能成熟红细胞，为进一步探寻调控hESCs向成体造血分化的关键因子奠定了基础。本研究选择调控造血至关重要转录因子GATA-1和GATA-2，通过构建慢病毒介导的条件诱导式GATA-1和GATA-2基因沉默的hES/hiPS细胞系，探索调控GATA-1和GATA-2基因表达对hESC/hiPSC向成体造血分化的影响。这种条件诱导模式可在多西环素（Doxcycline，Dox）诱导下在特定分化阶段和分化谱系调控GATA-1/GATA-2表达（shRNA）。我们的研究可以精密地检测GATA-1/GATA-2对hESC/hiPSC向造血细胞定向分化的影响，从而为揭开人类初期造血的分子调控机制奠定基础。

GATA转录因子家族的GATA1和 GATA2基因时序性调控对于造血细胞的分化成熟至关重要，一系列基因敲除和获得实验证明GATA1基因是小鼠胚胎造血和成体造血所必需的，GATA2促进成体造血AGM区域生血内皮细胞产生HSC，GATA1和GATA2对人类早期造血发生的影响尚待深入了解。人类多能干细胞作为全能性的人类干细胞在向造血细胞诱导分化的过程中可以很大程度上重现人类初期造血的发生，为探索GATA1和GATA2如何调控人类初期造血的分子调控机制奠定基础。我们建立一套用慢病毒感染或PiggyBac转染获得可诱导调控GATA1和GATA2基因过表达的系统，建立的细胞系在多西环素（Doxcycline，Dox）诱导下稳定表达GATA1和GATA2基因和蛋白，具有多能性和向三胚层分化的能力。在EB和hESC/mAGM-3共培养两种造血分化体系中，探究时序性调控GATA1和GATA2对早期造血发生的影响。结合细胞生物学方法（多色流式分析技术、流式分选、免疫荧光染色、瑞姬氏（May-Grunwald-Giemsa，MGG）染色）和分子生物学方法（荧光定量PCR 技术）探究时序性调控GATA1和GATA2基因对HSPCs来源造血细胞产生的影响。GATA2过表达促进hPSCs向中胚层、生血内皮细胞、造血干祖前体细胞和造血干祖细胞的逐级分化。我们对CD34+CD45+造血干祖细胞（hematopoietic stem progenitor cells, HSPCs）的形态、功能特性、转录表达谱进行分析。GATA2过表达CD34+CD45+细胞处于细胞周期G0/G1期，抑制其向下游血液细胞分化。GATA2过表达CD34+CD45+细胞与细胞周期负调控相关基因CCND3、CDK6、MCM5和CEBPA 表达降低，调控细胞周期的AHR信号通路相关基因AHRR表达降低。我们在人多能干细胞中时序性过表达GATA1和GATA2的研究为获得大量成熟造血细胞（尤其是HSCs的产生）提供可能。过表达GATA1和GATA2对早期内皮细胞造血转换、pre-HSPCs向HSPCs转换的影响有助为我们探索正常和疾病状态下早期造血发生的分子机制。