MULTINUCLEATED CELLS CAN CONTINUOUSLY GENERATE MONONUCLEATED CELLS INTHE ABSENCE OF MITOSIS - A STUDY OF CELLS OF THE AVIAN OSTEOCLAST LINEAGE

The multinucleated bone-resorbing osteoclast has a hematopoietic origi n. We have demonstrated previously that osteoclasts are derived from t he monocytic lineage by fusion of mononuclear macrophage precursors. U sing an in vitro osteoclast differentiation model derived from pure po pulations of chick macrophage cultures, osteoclast-like multinucleated giant cells (MNGCs) can be formed by fusion following an active proli feration phase. However, after reaching a peak with 70% of the culture being MNGCs, a new round of expansion of the mononuclear cells is obs erved. 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, ini tially 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 confir m that these mononuclear cells were indeed derived from a parent MNGC, single MNGCs were cloned into single wells. Within a week, the MNGC w as surrounded by mononuclear cells, which eventually populated the ent ire well. These mononuclear cells could then give rise to a second gen eration of MNGCs following a three-week period of culture. To determin e whether this process required mitosis, MNGCs were cultured for three days in the presence of the mitotic inhibitor, Ara-C, prior to microi njection with Lucifer Yellow. Fluorescent mononuclear cells were still seen to arise from a single injected MNGC under these conditions. Det ailed observations by scanning electron microscopy and confocal micros copy indicated that these mononuclear cells arise by a budding process from the central zone of the apical cell surface. A continuum of nucl ei was observed to exist in MNGCs, with a distinct and characteristic spatial localization of nuclei. Nuclei located at the basal surface we re uniformly oval and regular in shape, being clustered in a central l ocation in a single plane at the base of the MNGCs. Nuclei at the apic al surface, in contrast, were clustered and irregular, and twisted in shape. Taken together, our data provide the first evidence that mononu clear cells can be generated from a multinucleated 'differentiated' ce ll type, by a budding process that is independent of mitosis.