Integrin activation and the structure of human iPSC platelets are comparable to those in human PB-derived platelets. (A–C) Integrin activation in fresh human platelets (Fresh-P), aged human platelets (48-h incubation at 37°C; Aged-P), TkDN4-M (three-factor iPSC) platelets (3f-iPSC-P), TkDA3-4 (four-factor iPSC) platelets (4f-iPSC-P), and ESC platelets (ESC-P), with or without c-MYC O/E. The binding of PAC-1 (indicative of platelet activation) to individual platelets was quantified in the absence and presence of 50 µM ADP using flow cytometry. (A) Representative flow cytometry dot plots. Square indicates CD42b⁺ platelets. (B) Mean fluorescence intensity (MFI) of bound PAC-1, obtained from square gate in A. Error bars depict means ± SEM for four independent experiments (duplicate). (C) Representative flow cytometry analysis of PAC-1–bound platelets generated from integration-free SeV-iPSCs subjected to biphasic activation and, thereafter, decline of c-MYC expression as protocol b shown in Fig. 6 C. Square indicates CD42b⁺ platelets. (D) Spreading of iPSC platelets on fibrinogen. Human CD41a (red) and phalloidin (green) were used to identify F-actin fibers. Arrowheads indicate lamellipodia. Arrows indicate actin stress fibers. Bars, 5 µm. (E) Transmission electron micrographs of hiPSC (TkDA3-4) platelets on day 26. Bar, 3 µm.