Effect of c-kit andflt-3 overexpression on primitive hematopoietic cells

Recent studies have demonstrated that hematopoietic stem cell (HSC) self-renewal divisions in vitro are favoured by exposure of the cells to elevated levels of Steel factor (SF) or flt-3 ligand (FL). This suggested that enhanced HSC self-renewal by these cytokines might be limited by the level of expression of the corresponding c-kit or flt-3 receptors on HSCs. In this thesis, I investigated this hypothesis by examining how retroviral-mediated overexpression of either c-kit or flt-3 affects the responsiveness of hematopoietic cells to SF and FL, respectively, first in a cell line model and then in primary mouse bone marrow cells. c-kit and flt3-transduced BaF3 cells were able to proliferate in lower concentrations of SF and FL, respectively, than control-transduced cells, although evidence of high dose inhibition was also noted. In primitive mouse bone marrow cells transduced with the same c-kit or flt-3-encoding vectors, a similar effect on short term total cell expansion in vitro was seen. This included a cytokine-specific dose sensitization of progenitor and HSC expansion without detectable effects on their subsequent commitment or differentiation. Depressed responsiveness to very high cytokine concentrations was also seen in primary transduced cells such that no significant further net amplification of the transduced HSCs could be achieved in vitro. In vivo competitive reconstitution-assays were performed to determine whether the enhanced cytokine sensitivity of receptor-overexpressiog HSCs would give them a growth advantage in vivo. Although continued expression of functional receptors on the in vivo generated progeny of the transduced HSCs could be clearly documented and their self-renewal shown to be intact, their behaviour in vivo could not be distinguished from control-transduced cells, even when the corresponding ligand was administered exogenously or induced endogenously. It thus appears that neither c-kit nor fit-3 receptor levels on HSCs are limiting to their capacity to self-amplify in vivo. These results suggest that other molecular interactions of HSCs with their environment may be more potent regulators of HSC self-renewal responses in vivo and need to be identified to improve HSC amplification in vitro.