In Vitro Expansion of Megakaryocytes from Peripheral Blood Hematopoietic Progenitors

Recently, there have been several reports describing the in vitro proliferation and differentiation of megakaryocytic progenitor cells, isolated from either bone marrow (BM) or peripheral blood (PB), into relatively pure human mega-karyocytes (1 ,2 ). These culture systems originated from the discovery that Ficoll isolated human mononuclear PB cells, when stimulated with aplastic sera from thrombocytopenic animals, differentiated into megakaryocytes (3 ), and also from the finding that the megakaryocyte progenitors found in PB or BM typically express the CD34 antigen on their cell surface (4 ). Collectively, these two discoveries led to a system whereby PB isolated CD34⁺ cells are cultured with an exogenously derived cytokine soup of growth and differentiation factors. Finally, after a variable expansion period, from 8–14 d, the cells are analyzed for megakaryocytic antigenic markers, such as αIIb/β3 (CD41). The disadvantage of this system is that it is a short-term one, with most of these primary cells being dead 21 d after their initial plating. Other “long-term” systems where the developing CD34⁺ cells are grown in the presence of cytokine expressing human BM microvascular endothelial cells have demonstrated 200-fold expansions over a 2-month growth period (5 ). Both the short- and long-term culture systems have the potential to generate sufficient numbers of megakaryocytes for doing transient or stable expression studies or for other applications, such as providing sufficient megakaryocyte nuclear extracts for electrophoretic mobility shift assays or nuclei for DNase1 hypersensitivity studies.