The hematopoietic stem cell is the ultimate precursor of all types of cells found in the peripheral blood. In addition to the proliferation and differentiation into these cell types, the hematopoietic stem cell retains the capacity to self renew without differentiation, allowing for bone marrow transplantation. Our research interest is in how the proliferation/differentiation and/or self renewal of hematopoietic stem cells can be manipulated. We have previously shown that treating mice with the cytokines Granulocyte Colony Stimulating Factor (G-CSF) and Stem cell Factor (SCF) caused a mobilization of hematopoietic stem cells into the peripheral blood (Bodine et al. Blood 84:1482,1994). We have recently discovered that 14 days after the administration of G-CSF and SCF, the repopulating ability of the bone marrow of treated mice is increased by over 10 fold (Bodine et al. Blood 88: 89, 1996). This increase in repopulating ability is accompanied by a 10 fold increase in the number of cells expressing the highest levels of c-kit, a phenotype we have shown to be descriptive of the repopulating hematopoietic stem cell. Similar studies performed in Rhesus monkeys demonstrated an increase in marrow cellularity and a 5-10 fold increase in the number of CD34+/CD38lo cells in marrow collected 14 days after treatment with G-CSF and SCF. We hypothesize that """"""""cytokine priming"""""""" will be a useful in generating increased numbers of hematopoietic stem cells for transplantation and gene therapy protocols. Our ability to separate hematopoietic stem cells from progenitors and other more mature hematopoietic cells has allowed us to examine gene expression in stem cells and committed hematopoietic cells. We have completed a survey of cytokine receptor expression in stem cells and in developing T- and B- cells. We observed that the mRNAs encoding the common chain of the IL-2 receptor (gc) as well as the IL-4 receptor (IL-4R), IL-2Rb, and IL-9R were expressed in stem cells and at all stages of T- and B-cell maturation. In contrast, L-2Ra mRNA was expressed at low levels in stem cells and reached its peak level in the most mature lymphoid cells. Finally IL-7R mRNA was not expressed in stem cells but was expressed at high levels in the earliest T- and B- cells . From these results we concluded that precise regulation of the gc gene is NOT required for gene therapy of X-linked SCID (which is cause by mutations in the gc gene), and that the expression of the IL-7R is an important early step in the differentiation of T- and B- lymphocytes. In addition to known genes, we are interested in isolating novel genes expressed in stem cells. We have used Differential Display PCR to compare the expression of genes in mRNA purified from hematopoietic stem cells to mRNA purified from unfractionated bone marrow cells. Twenty -five sequences were obtained with this method. These clones were sequenced and screened by a combination of slot blot hybridization and or RT-PCR to determine whether they were expressed preferentially in stem cells. To date 3 distinct clones have been isolated. The sequence of these sequences demonstrates that they are not related to each other, and that they have no homology to any previously characterized genes. We are optimistic that one or more of these genes will be involved in one or more of the unique hematopoietic stem cell functions.
Showing the most recent 10 out of 28 publications
