title: Development of a liquid culture system for megakaryocyte terminal differentiation: fibrinogen promotes megakaryocytopoiesis but not thrombopoiesis.
abstract: 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.
authors.affiliation: Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sato
authors.forename: Minoru
authors.initials: M
authors.lastname: Ono
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fujita
authors.forename: Nobuhito
authors.initials: N
authors.lastname: Tanaka
authors.forename: Junji
authors.initials: J
authors.lastname: Tomiyama
authors.forename: Yujiro
authors.initials: Y
authors.lastname: Sakamoto
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Takano
authors.forename: Sei-Itsu
authors.initials: SI
authors.lastname: Murota
authors.forename: Ikuo
authors.initials: I
authors.lastname: Morita
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D001792:Blood Platelets / Q000648:ultrastructure*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002478:Cells, Cultured; D003470:Culture Media; D005340:Fibrinogen / Q000494:pharmacology*; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology*; D008854:Microscopy, Electron; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000032:analysis; D040201:Platelet Membrane Glycoprotein IIb / Q000032:analysis; D019089:Stem Cell Factor; D013926:Thrombopoietin
chemical_list: D018952:Antigens, CD34; D015415:Biomarkers; D003470:Culture Media; D019038:Platelet Glycoprotein GPIb-IX Complex; D040201:Platelet Membrane Glycoprotein IIb; D019089:Stem Cell Factor; D005340:Fibrinogen; D013926:Thrombopoietin