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 long-term cultures in the presence of various combinations of early acting cytokines such as FLT3-ligand (FLT3-L) and stem cell factor (SCF) and the differentiating growth factors GM-CSF and TNF-α. Two weeks of incubation with GM-CSF and TNF-α generated fully functional DC. However, clonogenic assays demonstrated that CFU-DC did not survive beyond 1 wk in liquid culture 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 acting cytokines allowed a 100-fold expansion of CFU-DC for >1 month. Phenotypic analysis demonstrated the differentiation of CD34⁺DR^- cells into CD34^-CD33⁺DR⁺CD14⁺ cells, which were inter-mediate progenitors capable of differentiating into functionally active DC upon further incubation with GM-CSF and TNF-α. As expected, GM-CSF and TNF-α generated DC from committed CD34⁺DR⁺ cells. However, only SCF, with or without FLT3-L, induced the expansion of DC precursors for >4 wk, as documented by secondary clonogenic assays. This demonstrates that although GM-CSF and TNF-α do not require additional cytokines to generate DC from primitive human CD34⁺DR^- progenitor cells, they do force terminal differentiation of DC precursors. Conversely, FLT3-L and SCF do not directly affect DC differentiation, but instead sustain the long-term expansion of CFU-DC, which can be induced to produce mature DC by GM-CSF and TNF-α.