The multinucleated bone-resorbing osteoclast has a hematopoietic origin. We have demonstrated previously that osteoclasts are derived from the monocytic lineage by fusion of mononuclear macrophage precursors. Using an in vitro osteoclast differentiation model derived from pure populations of chick macrophage cultures, osteoclast-like multinucleated giant cells (MNGCs) can be formed by fusion following an active proliferation phase. However, after reaching a peak with 70% of the culture being MNGCs, a new round of expansion of the mononuclear cells is observed. The following experiments suggest that these mononuclear cells were derived directly from the MNGCs by a budding process, selectively from the central zone of the apical surface. After microinjection of the membrane-impermeable probe, Lucifer Yellow, into single MNGCs, initially only diffuse fluorescence, limited to the whole MNGC injected, was observed. However, after 24-48 hours fluorescent mononuclear cells were observed adjacent but distinct from the injected MNGC. To confirm that these mononuclear cells were indeed derived from a parent MNGC, single MNGCs were cloned into single wells. Within a week, the MNGC was surrounded by mononuclear cells, which eventually populated the entire well. These mononuclear cells could then give rise to a second generation of MNGCs following a three-week period of culture. To determine whether this process required mitosis, MNGCs were cultured for three days in the presence of the mitotic inhibitor, Ara-C, prior to microinjection with Lucifer Yellow. Fluorescent mononuclear cells were still seen to arise from a single injected MNGC under these conditions. Detailed observations by scanning electron microscopy and confocal microscopy indicated that these mononuclear cells arise by a budding process from the central zone of the apical cell surface. A continuum of nuclei was observed to exist in MNGCs, with a distinct and characteristic spatial localization of nuclei. Nuclei located at the basal surface were uniformly oval and regular in shape, being clustered in a central location in a single plane at the base of the MNGCs. Nuclei at the apical surface, in contrast, were clustered and irregular, and twisted in shape. Taken together, our data provide the first evidence that mononuclear cells can be generated from a multinucleated ‘differentiated’ cell type, by a budding process that is independent of mitosis.

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