EFFECT OF ERYTHROPOIETIN (EPO) ON GRANULOCYTE-MACROPHAGE COLONY-FORMING UNITS (CFU-GM) IN HUMAN CELL CULTURES

Erythropoietin (EPO) is a hematopoietic cytokine primarily recognized for its role in erythropoiesis. However, recent studies suggest that EPO may also influence other hematopoietic lineages, including granulocytes and macrophages. This study aimed to evaluate the effect of EPO on CFU-GM formation in human bone marrow and peripheral blood samples by comparing two commercial culture media, with and without EPO supplementation. To quantitatively compare CFU-GM formation in cell cultures supplemented with or without EPO, in the context of a medium transition due to discontinuation of the EPO-free formulation. Samples were obtained from one healthy donor and four patients undergoing bone marrow transplantation (three with multiple myeloma and one with non-Hodgkin lymphoma). After four days of granulokine stimulation, peripheral blood samples were collected and analyzed by flow cytometry for CD34⁺/CD45^low cells using the ISHAGE protocol on a BD FACSCalibur cytometer. Clonogenic assays were performed using MACS™ HSC-CFU methylcellulose media (Miltenyi Biotec), either with or without EPO (3 U/mL). Cultures were incubated for 14 days at 37°C. CFU-GM colonies were counted under an inverted microscope by two blinded evaluators. Statistical analysis was conducted using the paired Wilcoxon test. In cultures supplemented with EPO, fresh samples yielded an average of 21.63 CFU-GM colonies, while thawed samples yielded 15.75 colonies (p = 0.813). In cultures without EPO, fresh samples yielded 19.47 colonies, and thawed samples yielded 12.67 colonies (p = 0.813). No statistically significant difference in CFU-GM formation was observed with the addition of EPO. These findings suggest that EPO does not significantly affect CFU-GM formation in vitro. Therefore, transitioning to a medium containing EPO does not appear to alter the performance of CFU-GM assays in the laboratory setting. The addition of EPO to hematopoietic culture media does not influence CFU-GM formation, supporting the equivalence of both media and validating the continued reliability of CFU-GM testing following the media transition.