In vitro expansion of human megakaryocytes as a tool for studying megakaryocytic development and function

Research on normal human megakaryopoiesis has been limited by technical pro blems in obtaining megakaryocytic cells in sufficient quantities for experi mental purposes. We describe here an ex vivo serum-free liquid culture syst em to expand normal human megakaryoblasts from purified bone marrow-, cord blood- or peripheral blood- derived CD34(+) cells. The early megakaryocytic cells are expanded in the presence of recombinant thrombopoietin (TpO) and interleukin-3 (IL-3), and if necessary further purified by employing anti- CD61 immunomagnetic beads. Our expansion system generates normal human mega karyoblasts in quantities sufficient to perform various functional studies on these cells as well as to isolate from them proteins and mRNA for molecu lar analysis. Megakaryocytic cells isolated from these cultures (i) express several markers characteristic of this lineage (CD41, CD61, CD62 P, CXCR-4 , PAR-1, etc.), (ii) respond by calcium flux and phosphorylation of various intracellular proteins to stimulation by thrombin and (iii) adhere to fibr inogen and vitronectin. However, human megakaryoblasts derived from the cul tures supplemented with TpO + IL-3, in contrast to murine megakaryocytic ce lls cultured under similar conditions, display poor polyploidization and do not release platelets. Since IL-3 has been reported to inhibit final matur ation of megakaryocytic cells, we recently modified our expansion strategy. In this new approach CD34(+) cells are first expanded for 11 days in the p resence of TpO + IL-3. Then megakaryoblasts derived are expanded for an add itional 7 days supplemented with TpO only. We found that megakaryocytic cel ls expanded in this 'two step culture' model are more differentiated, are p olyploid and release platelets. The model described here provides normal hu man megakaryoblasts in adequate numbers, to study megakaryopoiesis and mega karyocyte function.