Differentiation of hematopoietic stem cells isolated from peripheral blood to megakaryocyte

Objective: Thrombocytopenia remains a serious problem in patients treated with highdose chemotherapy and bone marrow transplantation. In recent years, infusion of ex vivo expanded megakaryocytes (Mk) progenitors into patients has been proposed as a strategy for shortening the time of platelet engraftment. The development of in vitro culture methods to obtain sufficient numbers of Mks from haematopoietic stem cells (HSC) is an important target in basic and clinical research projects. The aim of this study was to develop a two-step ex vivo expansion culture system of Mk progenitors from peripheral blood stem cells (PBSC). Methods: PBSC were harvested from three healthy adult donors. CD34+ cells were isolated and cultured in serum free media supplemented with thrombopoietin (TPO) (50 ng/ml), Interleukin 3 (IL-3) (20 ng/ml) and Interleukin 6 (IL-6) (20 ng/ml) for 12 days followed by an incubation with IL-6 (20 ng/ml) and TPO (50 ng/ml) for another 9 days. The differentiation of Mks was monitored by flow cytometry (% of CD34+/41+ cells). The morphology of the cells was studied by light, electron and fluorescence microscopy. Results: Morphological analysis of cells generated after 7 days of culture showed typical aspects of developing Mks. The percentage of CD41+ cells was higher than 70 on day 21. Conclusion: The results obtained in this study demonstrated that this two-step culture system is an effective method to obtain high rates of megakaryocytes. It is obvious that this promising method needs further development for clinical applications.