In vitro differentiation of osteoclasts - Studying the effect of myeloma derived immunoglobulins on osteoclastogenesis after optimizing the culturing process

Abstract

Multiple myeloma is a hematological cancer caused by accumulation of malignant plasma cells in the bone marrow with extensive bone destruction and heavily decreased bone formation. Osteoclasts are terminally differentiated, large, multinucleated cells formed from fusion of monocytes, and are responsible for the bone resorption in the process of bone remodeling. Monocytes are white blood cells derived from the hematopoietic linage. These cells make up 10-20% of the mononuclear cells of the circulation. Multiple factors that regulate osteoclast development have been identified the most important of which being Macrophage Colony-stimulating factor (M-CSF) and Receptor activator of NFκB ligand (RANKL). In this thesis the optimal method for isolation of monocytes from peripheral blood was by using CD14+ cell isolation. Further an optimal differentiation method for differentiating the monocytes into osteoclasts was established. A hallmark of MM is the overproduction of monoclonal immunoglobulin (M-component) and reduction in numbers of normal plasma cells. The M-component is poorly characterized and little is known about its biological effect. Myeloma cell lines are cell cultures developed from a single cell and therefore consisting of cells with a uniform genetic make-up. An attempt was made to create a model method for stimulation of pre-osteoclasts with M-component isolated from the cell lines available in the lab. Multiple combinations of cell concentrations and medium were tested to elicit immunoglobulin production from the cell lines, but due to little immunoglobulins were secreted a model method was not established. The effect of stimulating pre-osteoclasts with immunoglobulin isolated form bone marrow plasma from patients with multiple myeloma was also studied. The increase in osteoclastogenesis seemed to be mediated through The Fcγ receptor IIa, but this was not conclusively determined. An attempt was also made to determine which signaling pathways were activated upon stimulation with immunoglobulin compared with RANKL controls. Little difference in signaling was detected and not final conclusion could be drawn.