Osteoclast development from hematopoietic stem cells: apparent divergence of the osteoclast lineage prior to macrophage commitment.

To further clarify the progression of osteoclast development, the relationship of clonogenic osteoclast progenitors (CFU-O) to macrophage or more primitive progenitors was examined. Serum-free culture supernatant of a tumor clone (CESJ) was used as a source of an osteoclast colony stimulating factor (O-CSF). CFU-O-derived colonies were identified by their characteristic positive staining for tartrate resistant acid phosphatase (TRAPase). The effect of macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) on osteoclast progenitors was examined by pre-culturing mouse bone marrow (BM) cells in agar medium containing M-CSF or SCF and overlaying CESJ medium 0-7 days later. The number of TRAPase+ colonies decreased while TRAP- macrophage colonies increased in M-CSF pre-cultures as overlays of CESJ medium were delayed. On the other hand, TRAPase+ and mixed colonies persisted in SCF pre-cultures with CESJ medium overlays. Conversely, all colonies were TRAPase+ and no macrophage colonies developed in O-CSF pre-cultures overlaid with M-CSF. CFU-O, but not CFU-M, survived 7 days without exogenous CSFs in agar medium. In fractionated BM, the majority (> 99%) of CFU-O were in the c-kit positive population; however, a specific antibody to SCF did not affect O-CSF-induced TRAPase+ colony formation, suggesting the proliferation and differentiation of osteoclast progenitors are independent of c-kit-SCF interactions. These studies provide further experimental evidence to support the concept that O-CSF acts on progenitors in earlier stages of development, supporting their differentiation into the osteoclast lineage prior to macrophage commitment.