Manipulation of hematopoietic cells for regenerative medicine

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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.