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 (GM-CSF) + stem cell factor (SCF) + tumor necrosis factor-alpha (TNF-alpha). We present here the identification of two DC precursor subsets originated from HPCs with the phenotype of CD11b-/dullCD11c+ and CD11b+hiCD11c+ that develop independently at early time points (days 4 to 6) in the same culture conditions. Both of CD11b-/dullCD11c+ and CD11b+hiCD11c+ precursors could differentiate at day 10 to 14 into CD11b-/dullCD11c+ 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-/dullCD11c+ precursors expressed high levels of Ia, CD86, CD40, and E-cadherin molecules, but not c-fms transcript, and mature DCs derived from this precursor subset continue to express abundant E-cadherin antigen, a discernible marker for Langerhans cells. In contrast, CD11b+hiCD11c+ precursors expressed c-fms mRNA, but low levels of Ia, CD86, and E-cadherin, whereas CD40 was undetectable. CD11b-/dullCD11c+ mature DCs differentiated from these precursors displayed abundant c-fms mRNA and nonspecific esterase activity. Interestingly, CD11b+hiCD11c+ precursors, but not CD11b-/dullCD11c+ precursors, may be bipotent cells that can be induced by M-CSF to differentiate into macrophages. All of these results suggest that CD11b-/dullCD11c+ and CD11b+hiCD11c+ cells are distinct DC precursors derived from Lin-c-kit+ HPCs, which differentiate into mature DCs through bifurcated and independent DC differentiation pathways.