molecular cloning of self-renewal factor for hematopoietic stem cells and its clinical application

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Recent studies on the developing mouse embryo have shown that although primitive erythropoiesis is detectable in the yolk sac (YS) at 7.5 days post coitum (dpc), long-term repopulating hematopoietic stem cells (LTR-HSC) are first identified in the aorta-gonad-mesonephros (AGM) region at 10 dpc, prior to such activity being observed in YS and fetal liver, and expand in 11 dpc AGM region, suggesting that the AGM region at 10 to 11 dpc provides a microenvironment suitable for the development of LTR-HSC.These observations prompted us to establish stromal cell lines from the AGM region at 10 to 11 dpc, which would support ex vivo expansion of hematopoietic stem cells. We succeeded the establishment of a novel stromal cell line (AGMA9) from the AGM region of 10.5 dpc mouse embryo. When co-cultured with the AGMA9, lin-Sca-1+c-Kit+ cells isolated form adult mouse bone marrow and human cord blood CD34+ cells, significantly proliferated without additional cytokines. Expanded cocultured human CD34+ cells with AGMA9 for 4 weeks could reconstitute bone marrow of NOD/SCID mice suggesting that the cell line express some novel molecules which affect proliferation of not only murine but also human hematopoietic stem cells. This cell line can now be used to elucidate the molecular mechanisms regulating early hematopoiesis, and provide strategies for manipulation of primitive hematopoietic progenitor/stem cells. In RT-PCR analysis, AGMA9 produced detectable levels of SCF, SDF-1, OSM, IL-6, HGF, MIP-1 γ, Gas6, MCP-3, but no detectable levels of M-CSF EPO, TPO, Flk2/Flt3 ligand, MIP-1 α. We started molecular cloning of a novel self-renewal factor of hematopoietic stem cells using cDNA libraries of AGMA9 and AGMA7 which established from the AGM region at 10.5 dpc, and could not support proliferation of hematopoietic stem cells. We succeeded the cloning of more than ten novel genes and started the analysis of their functions.