Esther S. Choi

Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia.

The present study shows that recombinant human megakaryocyte growth and development factor (r-HuMGDF) behaves both as a megakaryocyte colony stimulating factor and as a differentiation factor in human progenitor cell cultures. Megakaryocyte colony formation induced with r-HuMGDF is synergistically affected by stem cell factor but not by interleukin 3. Megakaryocytes stimulated with r-HuMGDF demonstrate progressive cytoplasmic and nuclear maturation. Measurable levels of megakaryocyte growth and development factor in serum from patients undergoing myeloablative therapy and transplantation are shown to be elaborated in response to thrombocytopenic stress. These data support the concept that megakaryocyte growth and development factor is a physiologically regulated cytokine that is capable of supporting several aspects of megakaryopoiesis.

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.