Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts

Osteoclasts are thought to belong to a macrophage lineage. However, the nat ure of common precursors of osteoclasts and macrophages remains to be inves tigated. We have characterized the differentiation potential of mouse bone marrow macrophages into mature osteoclasts. Monocyte macrophage-colony-stim ulating factor (M-CSF) stimulated the proliferation of bone marrow macropha ges in a dose-dependent manner and these M-CSF-dependent bone marrow macrop hage (MDBM) cells efficiently differentiated into the tartrate-resistant ac id phosphatase (TRAP)-positive osteoclasts in the presence of soluble RANKL (sRANKL) and M-CSF in the in vitro culture. The macrophage-like cell line TMC16 was established from tsA58 (temperature-sensitive SV40 large T-antige n) transgenic mice in the same manner to the preparation of MDBM cells and also differentiated into mature osteoclasts. During this differentiation in vitro, the morphology of the cells changed from spindle to round and small er (termed pOC) on day 2 and to multinuclear (termed multinucleated cells [ MNCs]) on day 4. The surface expression of macrophage marker CD14 was down- regulated and that of CD43 was up-regulated on pOC, analyzed by flow cytome try. RNA analysis revealed that osteoclast marker genes such as calcitonin receptor (CTR), carbonic anhydrase II (CAII), cathepsin K (cath K), MMP9, a nd TRAP were strongly expressed in MNCs and weakly in pOC whereas MDBM cell s did not express these genes. However, the osteopontiu (OPN) gene was stro ngly expressed in MDBM cells and this expression became weakened after diff erentiation into pOC. The TMC16 cell line weakly expressed cath K, TRAP, an d OPN, suggesting that the TMC16 cell line is immortalized at a stage sligh tly differentiated from MDBM cells. Furthermore, cell sorting analysis reve aled that osteoclast early progenitors in bone marrow cells are preferentia lly present in the Mac-1(-) F4/80(dull) population, which differentiated in to MDBM cells (the osteoclast progenitor) expressing Mac-1(+) F4/80(int) su ggesting that M-CSF plays roles of a differentiation factor as well as a gr owth factor for osteoclast early progenitors. These results showed the tran sition of morphology, surface markers, and gene expression from the early t o mature stage in osteoclast differentiation. We propose three differentiat ion stages in the osteoclast lineage: the pro-osteoclast (spindle-shaped ma crophage cells), the pre-osteoclast (small round mononucleated TRAP-positiv e cells), and the mature osteoclast (multinucleated TRAP-positive cells) st age.