Bifurcated dendritic cell differentiation in vitro from murine lineage phenotype-negative c-kit⁺ bone marrow hematopoietic progenitor cells

We have recently established the culture system to generate dendritic cells (DCs) from murine Lin^-c-kit⁺ bone marrow hematopoietic progenitor cells (HPCs) in the presence of granulocyte-macrophage colony-stimulating factor (GMCSF) + stem call factor (SCF) + tumor necrosis factor-α (TNF-α). We present here the identification of two DC precursor subsets originated from HPCs with the phenotype of CD11b(-/dull)CD11c⁺ and CD11b(+hi)D11c⁺ that develop independently at early time points (days 4 to 6) in the same culture conditions. Both of CD11b(-/dull)CD11c⁺ and CD11b(+hi)CD11c⁺ precursors could differentiate at day 10 to 14 into CD11b(-/dull)CD11c⁺ mature DCs with typical morphology, phenotype, and the ability to stimulate allogenic mixed leukocyte reaction (MLR). However, the endocytic capacity of fluorescein isothiocyanate-dextran was markedly reduced during the differentiation. CD11b(-/dull)CD11c⁺ precursors expressed high levels of 1a, CD86, CD40, and E-cadherin molecules, but not c-fms transcript, end mature DCs derived from this precursor subset continue to express abundant E- cadherin antigen, a discernible marker for Langerhans cells. In contrast, CD11b(+hi)D11c⁺ precursors expressed c-fms mRNA, but low levels of 1a, CD86, and E-cadherin, whereas CD40 was undetectable. CD11b(-/dull)CD11c⁺ mature DCs differentiated from these precursors displayed abundant c-fms mRNA end nonspecific esterase activity. Interestingly, CD11b(+hi)D11c⁺ precursors, but not CD11b(-/dull)CD11c⁺ precursors, may be bipotent cells that can be induced by M-CSF to differentiate into macrophages. All of these results suggest that CD11b(-/dull)CD11c⁺ and CD11b(+hi)CD11c⁺ cells are distinct DC precursors derived from Lin^-c-kit⁺ HPCs, which differentiate into mature DCs through bifurcated and independent DC differentiation pathways.