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
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