RESUMO
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.
