Abstract
Osteoclasts (OCs) are large, multinucleated bone resorbing cells originating from the bone marrow myeloid lineage, and share a common progenitor with macrophages and dendritic cells. Bone marrow cells (BMCs) are a common source for in vitro osteoclastogenesis assays but are a highly heterogeneous mixture of cells. Protocols for in vitro osteoclastogenesis vary considerably thus hindering interpretation and comparison of results between studies. Macrophage colony-stimulating factor (M-CSF) pretreatment is commonly used to expand OC progenitors (OCPs) in BMC cultures before in vitro differentiation. However, the failure of osteoclastogenesis of M-CSF primed bone marrow myeloid blasts has been reported. In this study, we used a simple method of differential adherence to plastic to enrich OCP from mouse BMCs. We found that M-CSF pretreatment of plastic-adherent BMCs (adBMCs) increased the number of CD11b-F4/80+ macrophages and decreased the number of CD11b+ monocytes resulting in decreased OC formation. M-CSF pretreatment of purified c-Kit+ progenitors weakly inhibited OC formation, whereas M-CSF pretreatment of purified c-Kit-CD11b+ progenitors promoted the formation of large OC. M-CSF pretreatment increased the proliferation of both purified c-Kit+ and c-Kit-CD11b+ cells and increased the percentage of CD11b-F4/80+ cells from c-Kit+ progenitors. In addition, M-CSF pretreatment increased the percentage of CD11b+ F4/80- cells from purified c-Kit-CD11b+ cells. M-CSF pretreatment increased the percentage of CD14 + CD16 + intermediate monocytes and subsequent OC formation from human 2adBMCs, and increased OC formation of purified CD14 + cells. Together, these results indicate that in vitro OCP expansion in the presence of M-CSF and bone marrow stromal cells is dependent upon the developmental stage of myeloid cells, in which M-CSF favors macrophage differentiation of multipotent progenitors, promotes monocyte maturation and supports differentiation of late-stage OCP cells.