Development of a liquid culture system for megakaryocyte terminal differentiation: fibrinogen promotes megakaryocytopoiesis but not thrombopoiesis

Summary. Megakaryocyte differentiation is composed of three distinct stages: formation of erythromegakaryocytic progenitor cells, maturation of megakaryocytes and production of platelets. We have developed a liquid culture system for megakaryocyte terminal differentiation from haematopoietic stem cells into proplatelets. In this system, CD34+ cells isolated from human cord blood, differentiated to CD41+ cells, were classified either as propidium iodide (PI)+ cells (large) or PI– cells (small) by fluorescence‐activated cell sorting analysis on the late‐stage CD41+ cells. Transmission electron microscopy showed that the cultured small cells were morphologically identical to platelets isolated from normal peripheral blood. Moreover, the number of differentiated cells that were CD42b‐positive attained an approximately 60‐fold expansion over that of the primary CD34+ cells in this culture system. Furthermore, gene expression of megakaryocytopoietic transcriptional factors, GATA‐1 and NF‐E2, and several megakaryocytic markers such as glycoprotein (GP)IIb and thromboxane synthase was observed in the individual differentiation stage. Treatment with fibrinogen, a ligand of GPIIb/IIIa, increased the number of CD41+/PI+ cells, but treatment in the late stage suppressed CD41+/PI– cell formation, suggesting that fibrinogen promotes megakaryocytopoiesis, but not thrombopoiesis. We conclude that this liquid culture system using human CD34+ cells may be used to mimic the physiological development from haematopoietic stem cells into megakaryocytes, as well as promote subsequent thrombopoiesis.

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