Recent studies show that human osteoclasts develop in vitro from hematopoietic cells; however, special cultures conditions and/or cytokine mobilized peripheral blood are apparently required. Here, we report that cells expressing osteoclast markers differentiate from precursors present in nonmobilized peripheral blood mononuclear cells (PBMC), without the addition of stromal cells, growth factors, cytokines or steroids; and characterize their phenotype. Three days after establishing high-density PBMC cultures (1.5 x 10(6) cells/cm2), in serum-containing medium, small adherent colonies of tartrate resistant acid phosphatase positive (TRAP+) cells emerge, amidst massive monocyte cell death. These adherent cells have an eccentrically placed, round nucleus, and express low levels of TRAP and sodium fluoride-resistant- alpha-naphthyl-acetate-esterase (NaF-R-NSE). Over the next week, this cell population accumulates phenotypic markers of osteoclasts (vitronectin receptor [VR], calcitonin receptor, TRAP, cathepsin K protein, and mRNA) with increased nuclearity, covering the entire surface by 15 days. When cultured on bone, VR+, TRAP+ cells of low multinuclearity appear and cover up to 50% of the surface. Resorption lacunae can be observed by day 22. Although these pits are not nearly as numerous as the cells of preosteoclast phenotype, they do represent the activity of a subset of osteoclast-like cells that has achieved osteoclastic maturity under these culture conditions. Transcripts for osteoprotegerin ligand (OPGL), an osteoclast differentiation factor (also known as RANKL and TRANCE) are expressed, likely by adherent cells. Thus, an adherent population of cells, with preosteoclast/osteoclast phenotypic properties, arises selectively under simple culture conditions from normal PBMC. Further characterization of these cells should identify factors involved in the growth, terminal differentiation and activation of osteoclasts.

Thrombopoietin (TPO), the ligand for the c-Mpl cytokine receptor, is a recently identified cytokine with potent effects on platelet production. The receptor-binding portion of c-Mpl ligand is encompassed in another molecule known as megakaryocyte growth and development factor, or MGDF. Although it is clear that the administration of TPO or MGDF to animals dramatically increases the platelet count, the specific stage(s) of thrombopoiesis during which these molecules are principally active have not been unambiguously determined. Pharmacology studies administering MGDF at doses ranging from 0.1 to 630 micrograms/kg/d to mice revealed a biphasic response in platelet production. Administration of the drug at concentrations from 6 to 60 micrograms/kg/d resulted in platelet counts 5-fold above normal. However, doses > 60 micrograms/kg/d resulted in less-than-optimal platelet production. This phenomenon was investigated in vitro. Using an established culture system for the generation of human megakaryocytes and platelets, MGDF was shown to be optimally and equivalently active in the generation of mature megakaryocytes at concentrations from 10 to 1000 ng/ml. However, the cytokine was not required for proplatelet formation and in fact was inhibitory to that process in a dose-dependent manner. When MGDF was added to human megakaryocytes at concentrations of 200 ng/ml or greater, proplatelet formation was inhibited to 30% of control values. MGDF-mediated inhibition was specific, since the addition of the truncated form of the c-Mpl receptor reversed the inhibition in a dose-dependent manner. Other recombinant factors, interleukin-6, interleukin-11 and erythropoietin had no significant positive or negative effects in this human proplatelet assay. Together, these data suggest that although TPO and MGDF promote the full spectrum of megakaryocyte growth and development, they are not necessary for proplatelet formation, and may in part regulate platelet shedding by their absence.

Osteoprotegerin (OPG) and its ligand (OPGL) negatively and positively regulate osteoclastogenesis in the mouse. OPG inhibits osteoclastogenesis by sequestering its ligand, OPGL, the osteoclast differentiation and activation factor. This study demonstrates the effects of soluble muOPGL and huOPG on the developing human osteoclast phenotype, on bone slices, using peripheral blood mononuclear cells (PBMCs), cultured for 2 weeks, without stromal cells. OPGL (2-50 ng/ml), in combination with CSF-1, hydrocortisone (HC), and 1,25(OH)2D3, increases the size of osteoclast-like cells on bone, as defined by the acquisition of osteoclast markers: vitronectin receptor (VR), tartrate-resistant acid phosphatase (TRAP), multinuclearity, and bone resorption. By 14 days, with 20 ng/ml OPGL, the largest cells/10x field have achieved an average diameter of 163+/-38 microm, but only approximately 10-20 microm in its absence and the number of osteoclast-like cells/mm2 bone surface is about 128. By scanning electron microscopy, OPGL-treated (20-ng/ml) cultures contain small osteoclast-like cells on bone with ruffled "apical" surfaces by day 7; by day 15, large osteoclast-like cells are spread over resorption lacunae. At 15 ng/ml OPGL, about 37% of the bone slice area is covered by resorption lacunae. OPG (5-250 ng/ml) antagonizes the effects of OPGL on the morphology of the osteoclast-like cells that form, as well as bone erosion. For cells grown on plastic, Cathepsin K mRNA levels, which are barely detectable at plating, are elevated 7-fold, by 5 days, in the presence, not the absence, of OPGL (20 ng/ml) + CSF-1 (25 ng/ml). Similar findings are observed in experiments performed in the absence of HC and 1,25(OH)2D3, indicating that HC and 1,25(OH)2D3 are not needed for OPGL-induced osteoclast differentiation. In conclusion, this study confirms a pivotal role for OPGL and OPG in the modulation of human osteoclast differentiation and function, suggesting a use for OPG for treating osteoclast-mediated bone disease in humans.

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation. Mortality of this regimen is 94% and is associated with widespread internal bleeding. The daily administration of pegylated recombinant human MGDF (PEG-rMGDF) significantly reduced mortality (to < 15%) and ameliorated the depth and duration of thrombocytopenia. The severity of leucopenia and anemia was also reduced, although it was not clear whether these effects were direct. Platelets generated in response to PEG-rMGDF were morphologically indistinguishable from normal platelets. PEG-rMGDF administered in combination with murine granulocyte colony-stimulating factor completely prevented mortality and further reduced leukopenia and thrombocytopenia. These data support the concept that PEG-rMGDF may be useful to treat iatrogenic thrombocytopenias.

Osteoprotegerin (OPG) and its ligand (OPGL) negatively and positively regulate osteoclastogenesis in the mouse. OPG inhibits osteoclastogenesis by sequestering its ligand, OPGL, the osteoclast differentiation and activation factor. This study demonstrates the effects of soluble muOPGL and huOPG on the developing human osteoclast phenotype, on bone slices, using peripheral blood mononuclear cells (PBMCs), cultured for 2 weeks, without stromal cells. OPGL (2-50 ng/ml), in combination with CSF-1, hydrocortisone (HC), and 1,25(OH)2D3, increases the size of osteoclast-like cells on bone, as defined by the acquisition of osteoclast markers: vitronectin receptor (VR), tartrate-resistant acid phosphatase (TRAP), multinuclearity, and bone resorption. By 14 days, with 20 ng/ml OPGL, the largest cells/10x field have achieved an average diameter of 163+/-38 microm, but only approximately 10-20 microm in its absence and the number of osteoclast-like cells/mm2 bone surface is about 128. By scanning electron microscopy, OPGL-treated (20-ng/ml) cultures contain small osteoclast-like cells on bone with ruffled "apical" surfaces by day 7; by day 15, large osteoclast-like cells are spread over resorption lacunae. At 15 ng/ml OPGL, about 37% of the bone slice area is covered by resorption lacunae. OPG (5-250 ng/ml) antagonizes the effects of OPGL on the morphology of the osteoclast-like cells that form, as well as bone erosion. For cells grown on plastic, Cathepsin K mRNA levels, which are barely detectable at plating, are elevated 7-fold, by 5 days, in the presence, not the absence, of OPGL (20 ng/ml) + CSF-1 (25 ng/ml). Similar findings are observed in experiments performed in the absence of HC and 1,25(OH)2D3, indicating that HC and 1,25(OH)2D3 are not needed for OPGL-induced osteoclast differentiation. In conclusion, this study confirms a pivotal role for OPGL and OPG in the modulation of human osteoclast differentiation and function, suggesting a use for OPG for treating osteoclast-mediated bone disease in humans.