Osteoclastic Potential of Human CFU-GM: Biphasic Effect of GM-CSF

Abstract

Human osteoclasts can be efficiently generated in vitro from cord blood mononuclear cells and derived CFU-GM colonies. However, CFU-M colonies are poorly osteoclastogenic. Short-term (2-48 h) treatment with GM-CSF stimulates osteoclast formation by proliferating precursors, whereas longer exposure favors dendritic cell formation. Osteoclasts (OC) differentiate from cells of the myelomonocytic lineage under the influence of macrophage-colony stimulating factor (M-CSF) and RANKL. However, cells of this lineage can also differentiate to macrophages and dendritic cells (DC) depending on the cytokine environment. The aims of this study were to develop an efficient human osteoclastogenesis model and to investigate the roles of granulocyte macrophage-colony stimulating factor (GM-CSF) and M-CSF in human OC differentiation. A human osteoclastogenesis model, using as precursors colony forming unit-granulocyte macrophage (CFU-GM) colonies generated from umbilical cord mononuclear cells cultured in methylcellulose with GM-CSF, interleukin (IL)-3 and stem cell factor (SCF), has been developed. CFU-GM, colony forming unit-macrophage (CFU-M), or mixed colonies were cultured on dentine with soluble RANKL (sRANKL) and human M-CSF with and without GM-CSF. Major endpoints were OC number, dentine resorption, and CD1a+ DC clusters. Osteoclast generation from CFU-GM and mixed colonies treated with M-CSF and sRANKL for 7-14 days was highly efficient, but CFU-M colonies were poorly osteoclastogenic under these conditions. Pretreatment of precursors with M-CSF for 7 or 14 days maintained the precursor pool, but OCs were smaller and resorption was reduced. The effect of GM-CSF treatment was biphasic, depending on the timing and duration of exposure. Short-term treatment (2-48 h) at the beginning of the culture stimulated cell proliferation and enhanced OC formation up to 100%, independent of sRANKL. Longer-term GM-CSF treatment in the presence of sRANKL, however, inhibited OC generation with the formation of extensive CD1a+ DC clusters, accompanied by downregulation of c-Fos mRNA. Delaying the addition of GM-CSF resulted in progressively less inhibition of osteoclastogenesis. Human CFU-GM, but not CFU-M, progenitors have high osteoclastogenic potential. GM-CSF plays an important role in osteoclastogenesis and has a biphasic effect: Short-term treatment potentiates OC differentiation by proliferating precursors, but persistent exposure favors DC formation.