ENRICHMENT OF GENERATED MURINE OSTEOCLASTS

Biochemical and molecular studies of osteoclasts generally require cel ls in a reasonable degree of purity. The chicken has been extremely us eful in this regard, as abundant avian osteoclasts can be generated in vitro entirely from pure populations of marrow macrophage precursors, Propagation of murine osteoclasts is, in contrast, far less efficient , demanding the presence of stromal cells. The aims of this study were to develop a method by which murine osteoclasts generated in culture, can be effectively enriched while maintaining viability and, to explo re the mechanisms by which stromal cells promote murine osteoclast gen eration and survival, We find that 10(6) fractionated murine marrow ce lls enriched, for marrow-residing colony-forming units (CFU-cs), yield 3000-4000 tartrate-resistant acid phosphatase (TRAP)-expressing multi nucleated giant cells when cultured for 12 days with ST-2 stromal cell s. These cells are osteoclasts as evidenced by their ability to ''pit' ' bone slices, resorb radiolabeled bone particles, and generate cyclic AMP in response to calcitonin. Treatment of these generated osteoclas t cultures with bacterial collagenase for 2 hours at 37 degrees select ively removes virtually all ST-2 cells, yielding a >60% pure populatio n of TRAP and calcitonin receptor-expressing cells, 90% of which are v iable. These cells continue to respond to calcitonin and survive for 2 4 hours in the absence of ST-2 cells. We also found that murine osteoc last generation depends upon contact of osteoclast precursors with via ble ST-2 cells. Furthermore, the stromal cells secrete macrophage colo ny-stimulating factor (CSF-1), and the anti-CSF-1 antibody 5A1 inhibit s murine osteoclastogenesis. Exogenous CSF-1, in turn, partially overr ides the anti-osteoclastogenic effect of 5A1. We conclude that (1) the purity of murine osteoclasts generated from bone marrow cells enriche d for CFU-cs can be greatly enhanced by selective removal of associate d stromal cells, and (2) both soluble and membrane-residing stromal ce ll factors are necessary for generation of murine osteoclasts.