Myosin-IIA is not required for MK maturation in vitro. (A-C) MKs differentiated in vitro from Myh9^−/− ES cells. (A) A representative MK colony derived from Myh9^−/− ES cells in a day 8 differentiation culture. (B) A representative GFP-positive Myh9^−/− MK reveals abundant proplatelets (arrowheads); the inset at top right shows a released proplatelet filament. (C) Phase-contrast image of a representative proplatelet (arrowheads)–elaborating MK from similar cultures. Bars represent 15 μm. (D) Flow cytometry analysis of platelets released by MKs differentiated from ES cells. GFP (X-axis) and CD61 (Y-axis) double-positive particles are shown in the red scattergram bounded by the blue polygon. (E) Comparison of platelet numbers released from Myh9^+/− and Myh9^−/− ES cell–derived MKs. Number of particles with platelet properties detected by flow cytometry within 100-μL culture supernatants are expressed per 100 PPF⁺ MKs scored visually within the same cultures. (F) Number of MKs derived from GFP-expressing Myh9^+/− and Myh9^−/− ES cells on differentiation day 9. Cell numbers represent MKs derived from 2 × 10⁵ progenitors collected from day 5 differentiation culture, except that Myh9^+/− MKs (*) are plotted at one-tenth of the original count. The pie charts above the graph represent the fraction of MKs forming proplatelets. PPF in all cultures and MKs in Myh9^−/− samples were counted over full wells in 6-well culture dishes; Myh9^+/− MKs were counted in 6 separate microscope fields and extrapolated to the total surface area. (G) PPF efficiency of cultured primary wild-type MKs after blockade of myosin-IIA ATPase activity. Blebbistatin (100 μM, prepared from a 20 mg/mL stock in dimethyl sulfoxide [DMSO]) or DMSO was added to fetal liver cell cultures after enrichment for MKs over a BSA step-gradient on culture day 3. PPF was assessed on day 4 as described for panel F.