Characterization of osteoclast precursor cells.

(A) Bone marrow mononuclear cells were subdivided into 3 fractions: c-Fms⁺RANK⁻, c-Fms⁺RANK⁺, and c-Fms⁻RANK⁺ cells. Isolated osteoclast precursor cells were cultured with M-CSF alone or M-CSF and sRANKL for 6 days and subjected to TRAP-solution assay (B) or TRAP staining (C). Note that the TRAP activity was greatest in c-Fms⁺RANK⁻ cells cultured with M-CSF and sRANKL (panel B). Large numbers of multinuclear TRAP⁺ cells were observed among c-Fms⁺RANK⁻ cells (i), but only mononuclear TRAP⁺ cells developed from c-Fms⁺RANK⁺ cells (ii), and c-Fms⁻RANK⁺ cells did not proliferate in this culture (iii). (iv) Many multinuclear TRAP⁺ cells were also observed when 10 times more c-Fms⁺RANK⁺ cells were cultured. (v) c-Fms⁺RANK⁻ cells cultured with M-CSF alone were TRAP⁻. (vi) A significant reduction in the frequency of TRAP⁺ cells and multinuclear cell formation was observed in methylcellulose medium compared to liquid culture in the presence of M-CSF and sRANKL. The bar indicates 100 μm.