ISOLATION AND CHARACTERIZATION OF OSTEOCLAST PRECURSORS THAT DIFFERENTIATE INTO OSTEOCLASTS ON CALVARIAL CELLS WITHIN A SHORT-PERIOD OF TIME

Osteoclasts are formed in cocultures of mouse calvarial cells and hema topoietic cells in the presence of osteotropic factors such as 1 alpha ,25-dihydroxyvitamin D-3 [1 alpha,25(OH)(2)D-3], parathyroid hormone ( PTH) and prostaglandin E-2 (PGE(2)). We isolated osteoclast precursors (OCPs) from the coculture and examined their characteristics. After c oculture for 7 days of mouse calvarial cells and bone marrow cells in the absence of osteotropic factors, hematopoietic cells were recovered and applied to a Sephadex C-10 column. Cells which passed through the column were collected as OCPs. When OCPs were cultured on calvarial c ell layers in the presence of 1 alpha,25(OH)(2)D-3, tartrate-resistant acid phosphatase (TRAP)-positive cells first appeared within 24 h, an d their number increased thereafter. OCPs also differentiated into TRA P-positive cells within 48 h on the calvarial cell layer which had bee n pretreated with either 1 alpha,25(OH)(2)D-3, PTH, or PGE(2). Autorad iography using [I-125]-labeled calcitonin showed that TRAP-positive ce lls formed on the calvarial cell layer expressed calcitonin receptors. Direct contact between OCPs and calvarial cells was required for the differentiation of OCPs into TRAP-positive cells. Flow cytometric anal ysis revealed that OCPs were positive for Mac-1, Mac-2, and Gr-1 but n egative for F4/80, B220 and CD3e. Calvarial cells obtained from macrop hage-colony stimulating factor (M-CSF)deficient osteopetrotic (op/op) mice did not support OCP formation. A cell preparation disaggregated f rom long bones of newborn mice contained OCPs that differentiated into TRAP-positive cells on calvarial cells within 48 h, but cell preparat ions of freshly isolated bone marrow cells and alveolar macrophages di d not. These results suggest that OCPs are specific cells which are fo rmed only in the bone microenvironment and that OCPs recognize a signa l(s) expressed by stromal cells in response to osteotropic factors and differentiate into osteoclasts. J. Cell. Physiol. 177:26-35, 1998. (C ) 1998 Wiley-Liss, Inc.