Production of functional platelets by differentiated embryonic stem (ES) cells in vitro.
Megakaryocytes and functional platelets were generated in vitro from murine embryonic stem (ES) cells with the use of a coculture system with stromal cells. Two morphologically distinctive megakaryocytes were observed sequentially. Small megakaryocytes rapidly produced proplatelets on day 8 of the differentiation, and large hyperploid megakaryocytes developed after day 12, suggesting primitive and definitive megakaryopoiesis. Two waves of platelet production were consistently observed in the culture medium. A larger number of platelets was produced in the second wave; 104 ES cells produced up to 108 platelets. By transmission electron microscopy, platelets from the first wave were relatively rounder with a limited number of granules, but platelets from the second wave were discoid shaped with well-developed granules that were indistinguishable from peripheral blood platelets. ES-derived platelets were functional since they bound fibrinogen, formed aggregates, expressed P-selectin upon stimulation, and fully spread on immobilized fibrinogen. These results show the potential utility of ES-derived platelets for clinical applications. Furthermore, production of gene-transferred platelets was achieved by differentiating ES cells that were transfected with genes of interest. Overexpression of the cytoplasmic domain of integrin beta3 in the ES-derived platelets prevented the activation of alphaIIbbeta3, demonstrating that this system will facilitate functional platelet studies.
Fujimoto
Tetsuro-Takahiro
TT
Department of Hematology and Oncology, Division of Clinical Pharmacotherapeutics, Program for Applied Biomedicine, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-Ku, Hiroshima, 734-8551, Japan. fujimot@hiroshima-u.ac.jp
Kohata
Satoshi
S
Suzuki
Hidenori
H
Miyazaki
Hiroshi
H
Fujimura
Kingo
K
D000818:Animals; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D048429:Cell Size; D018920:Coculture Techniques; D008533:Megakaryocytes / Q000166:cytology; D051379:Mice; D008854:Microscopy, Electron; D010979:Platelet Function Tests; D013234:Stem Cells / Q000166:cytology*; D017154:Stromal Cells / Q000166:cytology; D034061:Thrombopoiesis; D013997:Time Factors; D014162:Transfection