Effects of recombinant erythropoietin on murine megakaryocytic colony formation in vitro.

: The availability of pure recombinant erythropoietin permits the study of its effects on hematopoietic progenitors free from those of other factors that may copurify with it. Mouse bone marrow cells were cultured in plasma clots in the presence of recombinant human erythropoietin. This factor supported megakaryocytic colony formation in a dose-dependent fashion, with plateau growth at 1 U erythropoietin per milliliter of culture medium. Erythropoietin did not increase the number of granulocyte-macrophage colonies. Plasma clots per se were not essential for megakaryocytic colony formation, because recombinant erythropoietin also supported colony growth in soft agar containing 25% serum. However, little colony formation was observed in serum-free soft agar cultures containing erythropoietin. Colony formation supported by suboptimal concentrations of erythropoietin was additive to that supported by suboptimal amounts of medium conditioned by pokeweed mitogen-stimulated spleen cells or by WEHI-3 cells. Delayed addition of conditioned medium to cultures resulted in a 50% to 100% decline in the number of megakaryocytic colonies by 12 to 48 hours, which was abolished by the inclusion of erythropoietin in the original culture medium. Delayed addition of erythropoietin by 24 hours to cultures resulted in loss of its effect on colony formation. These results indicate that erythropoietin has an effect on murine megakaryocytic colony formation in vitro and show that at least a portion of this effect is exerted during the early stages of colony development.