Pamela Hunt

Osteoclast markers accumulate on cells developing from human peripheral blood mononuclear precursors.

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 × 106 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‐ α‐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. J. Cell. Biochem. 72:67–80, 1999.

The role of megakaryocyte growth and development factor in terminal stages of thrombopoiesis.

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 μg/kg/d to mice revealed a biphasic response in platelet production. Administration of the drug at concentrations from 6 to 60 μg/kg/d resulted in platelet counts 5‐fold above normal. However, doses >60 μg/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.

Osteoblast Precursor Cells are Found in CD34+ Cells from Human Bone Marrow

It is known that osteoblast precursor cells are found in the low‐density mononuclear (LDMN) fraction of human bone marrow (BM) aspirates. The purpose of this study was to investigate whether CD34, a hematopoietic progenitor cell marker, is present on osteoblast progenitor cells. LDMN, CD34+, and CD34− cells were cultured under conditions that promote growth and differentiation of mineral‐secreting osteoblasts in a limiting dilution manner. With LDMN cells, osteoblast progenitor cells were found at an average frequency of 1/36,000 cells. With CD34− cells, osteoblast progenitor frequency remained at an average of 1/33,000, similar to LDMN cells. With CD34+ selected cells, osteoblast progenitor frequency increased to an average of 1/5,000. This osteoblast progenitor frequency is maintained in sorted CD34+/CD38+ cells. The osteoblasts generated from CD34+ cells were morphologically normal, and expression of skeletal‐specific alkaline phosphatase and osteonectin increased upon differentiation induced by dexamethasone (DEX) treatment. Ultrastructurally, these CD34+ cell‐derived osteoblasts displayed osteoblast‐specific features. Functionally, these CD34+ cell‐derived osteoblasts differentiated with DEX treatment, increased the level of cyclic adenosine monophosphate in response to parathyroid hormone stimulation, increased the level of alkaline phosphatase activity, and increased mineral secretion. These results demonstrate that osteoblast progenitor cells are enriched in the CD34+ cell population from BM and that these progenitor cells can differentiate into functional osteoblasts in culture.

Recombinant Human Ligand for MPL, Megakaryocyte Growth and Development Factor (MGDF), Stimulates Thrombopoiesis in Vivo in Normal and Myelosuppressed Baboons

Megakaryocyte growth and development factor (MGDF) is a ligand for c‐mpl and a member of the hematopoietic growth factor superfamily. Recombinant murine MGDF specifically stimulates thrombopoiesis in mice. Recombinant human (rHu) MGDF stimulates megakaryocytic differentiation of baboon CD 34+ marrow cells in vitro. Therefore, we determined the in vivo biological effects of rHuMGDF administered to normal baboons in the absence and presence of myelosuppression with 5‐fluorouracil (5‐FU). rHuMGDF was administered to normal baboons as single s.c. injection at doses of 1, 10, 25 and 50 μg/kg/day for 10 days and, as a control, heat‐inactivated MGDF was administered at a dose of 10 μg/kg/day. Platelet counts were markedly increased in all animals administered native rHuMGDF but not in animals given heat‐inactivated rHuMGDF. Platelet counts began to increase between three and six days after starting rHuMGDF administration and the maximum average increases were 1.7‐, 3.4‐, 5.1‐ and 4.0‐fold above baseline in animals administered 1, 10, 25 and 50 μg/kg/day, respectively. Maximum platelet counts were reached between 7 and 10 days after starting rHuMGDF and maintained for four days after the last dose. Thereafter, platelet counts decreased, reaching stable pretreatment values between 11 and 14 days after the last dose of rHuMGDF. No changes in red cell mass, peripheral blood white blood cell counts or differentials were observed during rHuMGDF treatment. For animals administered 10, 25 and 50 μg/kg/day of rHuMGDF, megakaryocytes increased more than threefold in marrow, were markedly enlarged, and had increased numbers of lobes. Overall marrow cellularity remained unchanged, as did red cell and white cell morphology. No marrow fibrosis was detected. Progenitor cells were not increased in marrow but did increase modestly in the peripheral blood, associated with increased numbers of CD34+ cells in circulation.

Multiple myeloma cells and cells of the human osteoclast lineage share morphological and cell surface markers

This study demonstrates that the multiple myeloma cell (MMC) in its plasma cell form is morphologically indistinguishable from human osteoclast‐like cells that form in culture when peripheral blood mononuclear cells (PBMCs) are plated at high density in serum containing medium. MM has been described as a disease of B‐cell lineage, monoclonal immunoglobulin (Ig) producing cells with unique properties: MM precursor cells lodge in bone, where they proliferate and differentiate into plasma cell tumors. Then, by some mechanism, presumably involving cytokines, these cells mediate an increase in neighboring osteoclast numbers and activity, leading to excessive bone erosion and hypercalcemia. Three days after plating PBMCs, tartrate resistant acid phosphatase‐ (TRAP‐) blasts as well as TRAP+ cells, each with an eccentric nucleus, appear in culture. By day 10, TRAP+, vitronectin+ (VR+) cells, appear to be morphologically indistinguishable from multiple myeloma plasma cells (MMPCs) on cytocentrifuge preparations. These cells are CD19‐ and CD38++, as are MMCs reported by others. Other surface markers are also shared. Furthermore, Ig mRNA is demonstrated in the cytoplasm of cells at 8 days by in situ hybridization with the IgG FcA3 sequence. This novel finding is not unusual, in light of reports, demonstrating non‐B‐lineage Ig‐producing cells. Thus, this study raises some serious questions about the true nature of MMCs. J. Cell. Biochem. 71:559–568, 1998.

The new generation of recombinant human hematopoietic cytokines

In the past year, the most exciting development in the field of hematopoietic growth factors has been the identification of the platelet-inducing factor Mpl ligand. Administration of recombinant Mpl ligand may alleviate the potential for hemorrhagic complications following cancer therapies. Stem cell factor continues to be studied clinically in the mobilization of peripheral blood cells for transplantation.