HEMATOPOIETIC STEM CELL BIOLOGY

造血干细胞生物学造血干细胞（HSC）是外周血中所有类型细胞的最终祖细胞。除了能够增殖和分化成这些细胞类型外，HSC还具有增殖而不分化的能力。后一种特性允许少量移植的HSC重新填充骨髓移植受体。我们的研究兴趣是如何控制HSC的分化和增殖。我们设计了纯化HSC超过1000倍的方法，允许检测HSC中的基因表达。我们之前的研究表明，逆转录病毒受体mRNA的编码水平在造血干细胞中非常低，逆转录病毒受体mRNA的水平与造血细胞系或造血干细胞的转导频率有直接的相关性。我们已经证明，在IL-3、Il-6和SCF中长期培养小鼠和人干细胞可以增加受体mRNA的水平和逆转录病毒转导的频率。目前，我们正在比较标准Moloney逆转录病毒载体与最近开发的慢病毒系统的转导。我们还表明，在小鼠，狗和猴子中，细胞因子管理动员具有高水平逆转录病毒受体的干细胞进入外周血。我们目前正在筛选猴子和人类的一些细胞因子组合，以找到最佳的细胞因子组合。此外，我们已经证明，锚定蛋白和带3基因的启动子在转基因小鼠中使珠蛋白基因的表达与位置无关，并导致珠蛋白基因在逆转录病毒载体中的稳定传播和表达。我们将继续开发这些载体作为库利氏贫血和镰状细胞病的潜在基因治疗方法。我们纯化干细胞的能力使我们能够研究干细胞的分子生物学。我们已经建立了几个HSC文库，从中我们提取了2个有趣的基因，酪氨酸激酶跨膜受体Ryk和HMG-4，一个未知的基因在成人组织中表达。每种基因在造血干细胞中都有高水平表达，但在更成熟的造血细胞中表达水平较低。针对Ryk的多克隆抗体证实在干细胞上高水平表达。遗传学研究所的合作者正在描述Ryk配体的特征。我们已经在胚胎干细胞中敲除了HMG-4基因，并将对携带被破坏的等位基因的转基因小鼠进行表征。-骨髓，库利贫血，基因治疗，血液学，白血病，镰状细胞病，干细胞研究，移植研究

HEMATOPOIETIC STEM CELL BIOLOGYThe hematopoietic stem cell (HSC) is the ultimate progenitor of all types of cells found in the peripheral blood. In addition to being capable of proliferation and differentiation into these cell types, HSC also have the capacity to proliferate without differentiation. This latter property allows a small number of transplanted HSC to repopulate a bone marrow transplant recipient. Our research interest is in how the differentiation and proliferation of HSC are controlled. We have devised methods to purify HSC over 1000 fold, allowing the examination of gene expression in HSC. We have previously shown that the level of mRNA encoding retrovirus receptors in HSC is very low, and that there is a direct correlation between the level of retrovirus receptor mRNA and the frequency with which hematopoietic cell lines or HSC can be transduced. We have shown that extended culture of mouse and human stem cells in IL-3, Il-6 and SCF increases the level of receptor mRNA and the frequency of retrovirus transduction. We are currently comparing the transduction with the standard Moloney Retrovirus vectors to the lentivirus system recently developed. We have also shown that in mice dogs and monkeys, cytokine administration mobilizes stem cells with high levels of retrovirus receptor into the peripheral blood. We are currently screening a number of cytokine combinations in monkeys and humans to find the optimal cytokine combination. In addition, we have shown that the promoters from the ankyrin and band 3 genes give position independent expression of globin genes in transgenic mice and lead to stable transmission and expression of globin genes in retroviral vectors. We will continue to develop these vectors as a potential gene therapy for Cooleys anemia and Sickle Cell Disease. Our ability to purify stem cells has allowed us to investigate the molecular biology of stem cells. We have generated several HSC libraries from which we have extracted 2 interesting genes, a tyrosine kinase transmembrane receptor, Ryk, and HMG-4, a gene that was not known to be expressed in adult tissues. Each gene is expressed at high levels in HSC, but is present at low levels in more mature hematopoietic cells. Polyclonal antibodies against the Ryk confirm high level expression on stem cells.Collaborators at Genetics Institute are characterizingthe Ryk ligand. We have knocked out the HMG-4 gene in ES cells and will be characterizing transgenic mice carrying the disrupted allele. - Bone Marrow, Cooley's Anemia, Gene Therapy, Hematology, Leukemia, Sickle Cell Disease, Stem cell Research, Transplantation Research