Effect of erythropoietin on bone marrow mononuclear cells

Stem/progenitor cells are considered an alternative method of heart failure therapy by promoting regeneration of damaged myocardium in myocardial infarction. Effectiveness of cell therapy depends on the population composition and functional activity of the cell graft, and, in turn, it depends on the conditions of microenvironment. Cultivation of stem/progenitor cells with erythropoietin stimulates proliferative potential causing in vitro resistance to hypoxia, and in vivo stimulation of angiogenesis. We aimed for assessing effects of erythropoietin upon hematopoietic cells. We studied some effects of short-term incubation of bone marrow mononuclear cells (BM-MNCs) in patients with coronary heart disease (CHD) with erythropoietin upon cellular phenotype, cell cycle, apoptosis and their proliferative potential. BM-MNCs were isolated from bone marrow aspirate from patients with CHD in a density gradient, then incubated for 60 minutes with erythropoietin (33.4 IU/ml). Using flow cytometric assay of the total BM-MNCs pool, we have shown there endothelial progenitor cells at different stages of maturation and differentiation, mesenchymal stem cells are. Their total number did not exceed 30%. Short-term incubation of BM-MNCs with erythropoietin reduces expression of CD184 “homing receptor” molecules on CD34+ cells, and causes increase of CD184 on CD31+ cells in the BM-MNCs pool (p < 0.05). In addition, erythropoietin has been shown to cause a delay of CD34+ cells in the resting phase (G0G1), reduce a proportion of cells in the synthetic phase (S) and mitosis (G2/M) (p<0.05), and does not affect apoptosis, as shown by Annexin V-FITC Apoptosis Detection Kit. Erythropoietin had no significant effects on expression on BM-MNCs surface molecules involved in providing adhesion, such as CD18, CD29, CD44, CD49a, CD54, CD62E, CD146, and CD202b. MTT-method has shown that the short-term preincubation of BM-MNCs with erythropoietin contributed to a significant decrease in proliferative activity of BM-MNCs (p < 0.05). However, there was a tendency towards increased resistance of erythropoietin-pretreated BM-MNCs to oxidative stress induced by hydrogen peroxide. We have also revealed a correlation between the numbers of endothelial progenitor cells at different stages of differentiation, and numbers of hematopoietic stem cells in the total BM-MNCs pool. The number of CD34+/CD133+, CD34- / CD31+, CD45+/EpoR+, and CD34+/EpoR+ in BM-MNCs pool are dependent on the age of patients. Hence, a short-term incubation of BM-MNCs with erythropoietin promotes the cells to be retained in resting phase of the cell cycle, thus, in turn, helping to reduce proliferative potential of BM-MNCs.

erythropoietin, bone marrow, mononuclear cells, phenotype, cell cycle, apoptosis, proliferation

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