Attempt to induce hematoopoietic stem cells from hemangioblasts

造血干细胞已知来源于血管母细胞，血管母细胞是血液和内皮细胞的共同前体细胞。我们发现Podocalyx-like Protein 1(PCLP1)是一个新的血管母细胞标志物。AGM区的PCLP1+CD45-细胞在体外可分化为造血细胞和内皮细胞。当这些细胞被移植到白花丹处理的新生小鼠体内时，它们在肺、小肠和子宫中产生了造血干细胞(HSCs)以及内皮细胞、平滑肌细胞和基质细胞。通过AGM区干细胞的逆转录病毒标记，我们证明了小肠中的血细胞和血管细胞是由单个干细胞来源的。在骨髓CD45阳性侧人群中也发现了类似的体内分化能力，这些侧人群高度浓缩了造血干细胞。因此，AGM区的血管母细胞很可能产生HSCs，而HSCs在活体内转分化为非造血细胞。我们已经证明，两种转录因子，AMI-1和c-myb，对于AGM区HSCs的产生是必不可少的。为了了解这些蛋白的功能，我们从AMI-1或c-myb基因敲除小鼠的agm区域建立了永生化细胞系。然后我们利用可诱导启动子的优势重新导入缺失的基因。基因表达研究表明，AML-1抑制胰岛素样生长因子结合蛋白-3的基因表达。进一步寻找AML-1和c-myb的下游效应分子将阐明AGM区血管母细胞分化为HSC的分子机制。

Hematopoietic stem cells are known to be derived from hemangioblasts, common precursor cells for blood and endothelial cells. We have identified podocalyxin-like protein 1 (PCLP1) as a novel hemangioblast marker. PCLP1+CD45-cells in the AGM region are differentiated to hematopoietic cells and endothelial cells in vitro. When these cells were transplanted into busulfan-treated neonatal mice, they gave rise to hematopoietic stem cells (HSCs) as well as endothelial cells, smooth muscle cells, and stromal cells in lung, small intestine, and uterus. By retrovirus marking of stem cells in the AGM region, we proved that blood cells and vascular cells in small intestine are derived from single stem cells. Similar in vivo differentiation capacity was detected in the bone marrow CD45-positive side population, which are highly enriched for HSCs. Therefore, it is likely that hemangioblasts in the AGM region generated HSCs that are in turn transdifferentiated to non-hematopoietic cells in vivo.We have shown that two transcription factors, AMI-1 and c-myb, are essential for the generation of HSCs in the AGM region. To know functions of these proteins, we newly established immortalized cell lines from the AGM regions of knockout mice ofAMI-1 or c-myb genes. Then we reintroduced missing genes by taking advantage of inducible promoter. Gene expression studies revealed that mRNA for insulin-like growth factor binding protein-3 is suppressed by AML1.Further search of down stream effectors of AML-1 and c-myb would clarify the molecular mechanism of HSC development from hemangioblasts in the AGM region.