Enrichment of generated murine osteoclasts

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Abstract
Biochemical and molecular studies of osteoclasts generally require cells in a reasonable degree of purity. The chicken has been extremely useful 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 efficients, 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 explore the mechanisms by which stromal cells promote murine osteoclast generation and survival. We find that 106 fractionated murine marrow cells enriched, for marrow-residing colony-forming units (CFU-cs), yield 3000–4000 tartrate-resistant acid phosphatase (TRAP)-expressing multinucleated giant cells when cultured for 12 days with ST-2 stromal cells. 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 osteoclast cultures with bacterial collagenase for 2 hours at 37° selectively removes virtually all ST-2 cells, yielding a >60% pure population of TRAP and calcitonin receptor-expressing cells, 90% of which are viable. These cells continue to respond to calcitonin and survive for 24 hours in the absence of ST-2 cells. We also found that murine osteoclast generation depends upon contact of osteoclast precursors with viable ST-2 cells. Furthermore, the stromal cells secrete macrophage colony-stimulating factor (CSF-1), and the anti-CSF-1 antibody 5A1 inhibits murine osteoclastogenesis. Exogenous CSF-1, in turn, partially overrides the anti-osteoclastogenic effect of 5A1. We conclude that (1) the purity of murine osteoclasts generated from bone marrow cells enriched for CFU-cs can be greatly enhanced by selective removal of associated stromal cells, and (2) both soluble and membraneresiding stromal cell factors are necessary for generation of murine osteoclasts.

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