Abstract
We have recently described an assay system for human peripheral blood megakaryocyte colony-forming unit cells (CFU-M) using an anti-platelet glycoprotein antiserum probe to define megakaryocyte colonies grown in vitro. This system was applied to study the nature and regulation of human bone marrow CFU-M. In the absence of a specific megakaryocyte growth-promoting factor, 12.4 +/- 3.0 (means +/- SEM) megakaryocyte colonies were cloned per 5 X 10(5) cells cultured. Colonies were present after 6 d of incubation reaching peak numbers between days 10 and 14 and slowly decreasing thereafter. Erythropoietin in concentrations of up to 4 U/ml failed to augment colony numbers. Also failing to enhance megakaryocyte colony plating efficiency were media containing burst-promoting activity and colony-stimulating activity. A medium conditioned by human embryonic kidney cells, which has been previously demonstrated to contain thrombopoietin, also had no effect on megakaryocyte colony numbers. In contrast, sera from three patients with severe aplastic anemia produced significant enhancement of CFU-M-derived colony formation in vitro. Both the number of megakaryocyte colonies present and the number of megakaryocytes per colony were increased in proportion to the final concentration of aplastic anemia serum. In the presence of 10% aplastic anemia serum, cultured megakaryocyte colony numbers were linear with respect to the number of bone marrow mononuclear cells plated suggesting a clonal origin of each of the colonies. This in vitro assay for bone marrow CFU-M is a reliable means by which to study the regulation of human megakaryocytopoiesis. Initial data suggest that megakaryocyte production is stimulated by a factor detectable in aplastic anemia serum that may be distinct from other known hematopoietic stem cell regulators.