Two-wave platelet production from ES cells. (A) Cells released to the culture medium on day 15 were analyzed by flow cytometry. A platelet gate was fixed in the forward- and side-scatter profiles of peripheral blood platelets from adult mice (left). Most cells in the culture medium of ES cells were within the gate (right). (B) Cells in culture supernatant were labeled with megakaryocyte- and platelet-specific monoclonal antibodies. Almost all cells were positive for CD41 (left) and GPV (right), as shown by the gray histogram. The open histogram represents cells stained with control antibody. (C) Cells were collected from medium of the same well of a 6-well culture plate every day (days 7 to 16), and the numbers of platelets were counted as platelet-sized and CD41⁺ cells by flow cytometry. Two waves of platelet production were observed. The values shown are the mean ± SD from 5 independent experiments.