The aim of this proposal is to determine whether basic fibroblast growth factor enables human hematopoietic stem cells to be expanded in vitro with the maintenance of a primitive phenotype. Basic fibroblast growth factor is a mitogen for stem cells of neuronal and embryonal origin and promotes both their proliferation and the maintenance of their primitive undifferentiated phenotype. Basic fibroblast growth factor has recently been shown to be a hemopoietic cytokine. It is found in hemopoietic cells in vivo and it is produced by bone marrow stromal cells in vitro. It directly stimulates the proliferation of hematopoietic progenitor cells in concert with other cytokines. In this respect it is analogous to stem cell factor that also synergises with other cytokines. Basic fibroblast growth factor and stem cell factor also synergise with each other and their is evidence that they are both critical cytokines that act on the most primitive stem cells. This proposal aims to characterize the biologic effects of basic fibroblast growth factor on early stem cells and define its relationship to stem cell factor and other cytokines. In order to define the stem cells, in vitro assays for the high proliferative potential colony forming cells and other progenitors will be used. RT-PCR and in situ hybridization techniques will be used to identify fibroblast growth factor receptor mRNA's present on CD34+Lin- hematopoietic stem cells that are further separated according to their expression of c-kit and their rhodamine staining characteristics. The fibroblast growth factor receptor expressing cells will be isolated using biotinylated fibroblast growth factor and/or antibodies to the fibroblast growth factor receptors. Using purified stem cells and isolated fibroblast growth factor receptor expressing cells, we shall attempt to define culture conditions that will be able to maintain or expand primitive stem cells without differentiation. This could result in new strategies for the culture of primitive stem cells for gene therapy and/or bone marrow transplantation studies.
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