Osteoclast differentiation in cocultures of a clonal chondrogenic cell line and mouse bone marrow cells

Previous reports have demonstrated that hemopoietic progenitor cells derived from mouse bone marrow can form osteoclast-like cells when cultured in the presence of stromal cells and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We show here that in cocultures of mouse bone marrow cells and a clonal chondrogenic cell line (C5.18), a stimulation of the number of tartrate-resistant acid phosphatase-positive (TRAP+) colonies is seen with or without the addition of 1,25-(OH)2D3 to the cultures. A large proportion of the TRAP+ cells had calcitonin receptors. In addition resorption lacunae were observed on bone slices on which cocultures were maintained, thus these cells had the characteristics of osteoclasts. The number of osteoclast-containing colonies that formed in cocultures varied with the plating density of the C5.18 cells and the length of time the C5.18 cells were cultured before adding mouse bone marrow. These results suggested that osteoclast differentiation decreased with increasing cartilage differentiation. C5.18 cells treated with 1,25-(OH)2D3 before coculture stimulated TRAP+ osteoclast colony formation to a greater extent than untreated C5.18 cells, whereas C5.18 cells cultured in the presence of dexamethasone before coculture inhibited TRAP+ osteoclast colony formation relative to untreated C5.18 cells. Since 1,25-(OH)2D3 inhibits and dexamethasone stimulates cartilage differentiation in C5.18 cells, these results agree with the view that chondroprogenitor cells stimulate osteoclast colony formation, whereas cultures containing predominantly mature chondrocytes do not. Osteoclast-containing colonies were frequently associated with colonies of alkaline phosphatase-positive (AP+) cells. This raised the possibility that C5.18 cells stimulated osteoclast differentiation indirectly by increasing the numbers of AP+ stromal cells from the marrow population, which in turn could stimulate osteoclast differentiation from marrow hemopoietic progenitors. In cocultures in which the C5.18 cells were physically separated from the marrow cells, we also observed increased numbers of TRAP+ colonies growing in association with large colonies of AP+ cells, suggesting that C5.18 cells release a soluble factor that mediates these effects.