MK maturation derived from H9 hES cell. (A) CD41 and CD42 flow cytometry analysis. Expression of CD41 and CD42 MK differentiation markers, shown, respectively on the y-axis and x-axis of the dot plot, were analyzed in embryonic (left) and adult (right) megakaryocytic cells. As previously described in Figure 1A, the cells were incubated with isotype-matched irrelevant antibodies as negative controls, and dead cells were excluded from the analyses. Representative histograms and values in each quadrant are the mean ± SD of 3 independent experiments. (B) May-Grünwald-Giemsa-stained cytospins of CD41⁺CD42⁺ subsets isolated from a hES/OP9 coculture from day 14 to day 16. Various levels of maturation were observed in immature MK (first on the left) compared with mature MK (last on the right). (C) Light microscopy image of proplatelet formation in serum-free cultures. (D) Immunofluorescent staining of an MK with anti-VWF polyclonal antibody (green) and phalloidin (red) showed a granular pattern of labeling for VWF. MKs were left to adhere on polylysine-coated slides for immunolabeling and detection of cytoskeletal components. The images were acquired using a Zeiss laser scanning microscope (LSM 510; Carl Zeiss) or an inverted Leica DM IFBE microscope (Leica Microsystems) with a 63×/1.0 NA oil objective. (E) Ploidy distribution was measured by flow cytometry after staining hES-derived MKs with propidium iodide from day 14 to 16, CD34⁺ cord blood–derived MKs at day 10, and CD34⁺ leukapheresis-derived MKs at day 10.