Establishment and characterization of bone marrow stromal cell lines that support osteoclastogenesis

We established bone marrow stromal cell lines that support tartrate-resistant acid phosphatase-positive multinucleated cell [TRAcP(+)MNC] formation by using transgenic mice harboring simian virus 40 large T antigen gene. The morphology of these TM cell lines (large T-immortalized marrow cells) was spindle-like at sparse cell density, whereas it became smaller and cuboidal at confluence. The TM cell lines showed diverse ranges of activity in supporting TRAcP(+)MNC formation when they were examined in the cocultures with spleen cells in the presence of 1 alpha,25-dihydroxyvitamin D3. Among these cell lines, TM8 supported the TRAcP(+)MNC formation most efficiently (from 400-1500 cells/well) when cocultured with spleen cells. Another bone marrow-derived cell line, TM5, supported TRAcP(+)MNC formation in the coculture assay, whereas the efficiency was approximately one fifth that of TM8. Interestingly, TM8 cells also supported TRAcP(+)MNC formation even in the cocultures at low serum concentration (0.5% fetal bovine serum) with an efficiency yielding over 200 TRAcP(+)MNCs/well. TM8 cells expressed certain levels of macrophage colony-stimulating factor and stem cell factor messenger RNAs (mRNAs), but low levels of c-fms mRNA. Expression of c-kit mRNA in TM5 and TM8 cells was undetectable. 1 alpha,25-Dihydroxyvitamin D3 treatment enhanced the expression of osteopontin mRNA more than 10-fold in these cells, indicating the presence of the receptor for this steroid. These TRAcP(+)MNCs, which developed in the cocultures of the TM8 and spleen cells, formed pits when cultured on bone slices, indicating that they were capable of resorbing bone. The various levels of expression of these genes and the difference in the supporting activities for the TRAcP(+)MNC development in the diverse TM cell lines suggest the heterogeneity in the marrow cell populations in vivo regarding their activity in supporting osteoclastogenesis.