The identification and cloning of thrombopoietin was certainly a defining moment for the study of megakaryopoiesis and thrombopoiesis ex vivo. This and other progresses made in the development of culture processes for hematopoietic stem cells have paved the way for ongoing clinical trials and, in the future, for the potential therapeutic use of ex vivo produced blood substitutes such as platelets. This chapter describes a 14-day culture protocol for the production of megakaryocytes (MK) and platelets from human cord blood stem cells. The CD34+ cells are grown in a serum-free medium supplemented with a newly developed cytokine cocktail optimizing MK differentiation, expansion, and maturation. A detailed methodology for flow cytometry analysis of the cells and platelets is also presented together with supporting figures. A brief review on megakaryocytic differentiation and ex vivo MK cultures is first presented.
