Stem cell factor and FLT3-ligand are strictly required to sustain the long-term expansion of primitive CD34(+)DR(-) dendritic cell precursors

We studied cytokine-driven differentiation of primitive human CD34(+)HLA-DR - cells to myeloid dendritic cells (DC). Hemopoietic cells were grown in lo ng-term cultures in the presence of various combinations of early acting cy tokines such as FLT3-ligand (FLT3-L) and stem cell factor (SCF) and the dif ferentiating growth factors GM-CSF and TNF-alpha. Two weeks of incubation w ith GM-CSF and TNF-alpha generated fully functional DC. However, clonogenic assays demonstrated that CFU-DC did not survive beyond 1 wk in liquid cult ure regardless of whether FLT3-L and/or SCF were added. FLT3-L or SCF alone did not support DC maturation. However, the combination of the two early a cting cytokines allowed a 100-fold expansion of CFU-DC for >1 month. Phenot ypic analysis demonstrated the differentiation of CD34(+)DR(-) cells into C D34(-)CD33(+)DR(+)CD14(+) cells, which were intermediate progenitors capabl e of differentiating into functionally active DC upon further incubation wi th CM-CSF and TNF-alpha. As expected, GM-CSF and TNF-alpha generated DC fro m committed CD34(+)DR(+) cells. However, only SCF, with or without FLT3-L, induced the expansion of DC precursors for >4 wk, as documented by secondar y clonogenic assays. This demonstrates that although GM-CSF and TNF-alpha d o not require additional cytokines to generate DC from primitive human CD34 (+)DR(-) progenitor cells, they do force terminal differentiation of DC pre cursors. Conversely, FLT3-L and SCF do not directly affect DC differentiati on, but instead sustain the long-term expansion of CFU-DC, which can be ind uced to produce mature DC by GM-CSF and TNF-alpha.