Differential Effect of Erythropoietin and GM‐CSF on Megakaryocytopoiesis from Primitive Bone Marrow Cells in Serum‐Free Conditions

In this study we have explored the effect of recombinant human erythropoietin (EPO) and recombinant murine GM-CSF on megakaryocyte progenitors (colony forming units-megakaryocyte [CFU-Mk]) using a serum-free fibrin clot assay and enriched primitive hematopoietic progenitors of marrow cells from day 4 post-5-fluorouracil-treated mice. We have monitored the production of high proliferative potential-colony forming cells ([HPP-CFC]; compact colonies, > 0.5 mm) and studied their relationship to CFU-Mk formation. EPO induced the formation of small numbers of megakaryocyte colonies, but acted together with the megakaryocyte-stimulating factors, stem cell factor (SCF) and interleukin (IL-3), to augment the size of CFU-Mk (colonies with > 50 megakaryocytes/colony). A strong correlation between the number of CFU-Mk and HPP-CFC formation from 5-fluorouracil bone marrow cells was observed when these cells were stimulated with EPO in the presence of SCF and IL-3. On the other hand, GM-CSF alone had no effect on megakaryocyte colony formation. Moreover, GM-CSF in the presence of SCF and IL-3 potentiates the HPP-CFC formation (i.e., an increase of 3.1-fold compared to the effect induced by SCF+IL-3) with strong inhibitory effects on the number and size of megakaryocyte colonies. Although several studies suggest that EPO and GM-CSF can stimulate megakaryocytopoiesis, our results indicate that neither EPO nor GM-CSF alone are sufficient to stimulate primitive progenitors committed to the megakaryocyte lineage. The fact that EPO can exert a strong effect on the size of CFU-Mk induced by SCF/IL-3 suggests that only those megakaryocyte progenitors previously stimulated by other megakaryocyte stimulating factors are able to respond to EPO. These findings may explain the physiological and clinical observations in which high levels of EPO are often associated with thrombocytosis.