Manipulation of hematopoietic cells for regenerative medicine

我们建立了小鼠胚胎干细胞（ES）在OP9基质细胞系（OP9系统）上共培养向造血细胞分化的体外诱导方法。虽然OP9系统可以产生多能造血祖细胞，但无法产生具有自我更新能力的造血干细胞。为了提高造血活性，我们将条件基因表达法引入OP9系统。四环素基因调控系统（Tet-Off系统）与OP9系统的结合，使我们能够在OP9系统发育的造血祖细胞中表达所需的基因。该系统检测了锌指型转录因子GATA-2的功能。当GATA-2过表达时，造血集落的数量和单个集落中未成熟造血细胞的百分比增加到10倍。先前的报道表明GATA-2抑制血细胞的终末分化。然而，我们的数据是相反的；即，红细胞和巨核细胞系的终末分化增强。我们推测这种差异可能与实验系统的不同有关，因为以往的研究使用的是GATA-2的融合蛋白与雌激素受体的配体结合域（GATA-2/ER）来条件激活转录因子，而不是真正的GATA-2。然后比较GATA-2和GATA-2/ER的生物学功能。令人惊讶的是，GATA-2的功能与GATA-2/ER的功能相反。GATA-2与其他造血转录因子PU.1和c-Myb的结合能力和抑制转录相互作用与GATA-2/ER有很大不同。本研究揭示了OP9系统与et- off系统的结合在研究造血分化分子机制和未来再生医学中的应用价值。

We had established an in vitro differentiation induction method from mouse embryonic stem (ES) cells to hematopoietic cells by co-culturing the ES cells on OP9 stroma cell line (OP9 system). Although OP9 system can give rise to multipotential hematopoietic progenitors, hematopoietic stem cells with self-renewing activity never emerge. To improve the hematopoietic activity, we introduced conditional gene expression method to OP9 system. Combination of tetracycline gene regulating system (Tet-Off system) and OP9 system enabled us to express the desirable genes in the hematopoietic progenitor cells which are developed by OP9 system.Function of a zinc-finger type transcription factor GATA-2 was examined by the system. When GATA-2 was over-expressed, the numbers of hematopoietic colonies and the percentages of immature hematopoietic cells in individual colonies increased to 10 fold. Previous reports had suggested that GATA-2 inhibited the terminal differentiation of blood cells. However, our data was opposite ; i.e., terminal differentiation into erythroid and megakaryocyte lineages were enhanced. We speculated that this discrepancy should have been due to the adopted experimental system, since the previous studies used the fusion protein of GATA-2 and the ligand binding domain of estrogen receptor (GATA-2/ER) for conditional activation of the transcription factor instead of the authentic GATA-2. Then, we compared the biological functions of GATA-2 and GATA-2/ER. Surprisingly, the function of GATA-2 was opposite to that of GATA-2/ER. Binding capacity and inhibitory transcriptional interaction of GATA-2 to the other hematopoietic transcription factors, PU.1 and c-Myb, were quite different from those of GATA-2/ER.This study reveals the utility of the combination of OP9 system and Tet-Off system to investigate the molecular mechanisms of hematopoietic differentiation and for the future regenerative medicine.