Manipulation of hematopoietic cells for regenerative medicine

再生医学中造血细胞的操作

基本信息

批准号：
12557080
负责人：
NAKANO Toru
金额：
$ 8.58万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557080/
关键词：
Hematopoiesis Embryonic Stem Cell Cell Differentiation Transcription Factor GATA-2 Gene Regulation Stroma Cell 細胞分化 GATA転写因子 Myb転写因子

项目摘要

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.

我们通过在OP9基质细胞系（OP9系统）上共同培养ES细胞，从而从小鼠胚胎（ES）细胞到造血细胞建立了一种体外分化诱导方法。尽管OP9系统可以引起多能造血祖细胞，但具有自我更新活性的造血干细胞永远不会出现。为了改善造血活性，我们将条件基因表达方法引入了OP9系统。四环素基因调节系统（TET-OFF系统）和OP9系统的组合使我们能够通过OP9系统开发的造血祖细胞中所需的基因。系统。当GATA-2过表达时，个别菌落中造血菌落的数量和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抑制了血细胞的末端分化。但是，我们的数据相反。即，增强了对红细胞和巨核细胞谱系的终末分化。我们推测，这种差异应该是由于采用的实验系统，因为先前的研究使用了GATA-2的融合蛋白和雌激素受体（GATA-2/ER）的配体结合结构域，以将转录因子的条件激活改为真实的GATA-2。然后，我们比较了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的结合能力和C-MYB截然不同。 - 研究造血分化的分子机制和未来再生医学的分子机制。