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

最近对发育中的小鼠胚胎的研究表明，尽管在妊娠后7.5天（dpc）在卵黄囊（YS）中可检测到原始红细胞生成，但在10 dpc时在卵黄囊-性腺-中肾（AGM）区域中首先鉴定出长期再生造血干细胞（LTR-HSC），然后在YS和胎肝中观察到这种活性，并在11 dpc AGM区域中扩增，提示10 - 11 dpc的AGM区为LTR-HSC的发育提供了适宜的微环境。这些观察促使我们从10 - 11 dpc的AGM区建立基质细胞系，这将支持造血干细胞的体外扩增。我们成功地从10.5dpc小鼠胚胎AGM区建立了一个新的基质细胞系（AGMA 9）。当与AGMA 9共培养时，从成年小鼠骨髓和人脐带血CD 34+细胞分离的lin-Sca-1+c-Kit+细胞在没有额外细胞因子的情况下显著增殖。扩增后的人CD 34+细胞与AGMA 9共培养4周后可重建NOD/SCID小鼠的骨髓，提示该细胞系表达一些新的分子，不仅影响小鼠造血干细胞的增殖，而且也影响人造血干细胞的增殖。该细胞系可用于阐明调控早期造血的分子机制，并为操纵原始造血祖细胞/干细胞提供策略。RT-PCR检测结果显示，AGMA 9可表达SCF、SDF-1、OSM、IL-6、HGF、MIP-1 γ、Gas 6、MCP-3，但不能表达M-CSF、EPO、TPO、Flk 2/Flt 3配体、MIP-1 α。我们利用在10.5dpc时从AGM区域建立的AGMA 9和AGMA 7的cDNA文库开始了一种新的造血干细胞自我更新因子的分子克隆，并且不能支持造血干细胞的增殖。我们成功地克隆了十多个新基因，并开始了它们的功能分析。

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.