Figure 6: Functional assessment of fopMK in vitro platelets.

(a) Adhesion to fibrinogen of fopMK and cbMK in vitro platelets upon combined TRAP+ADP stimulation compared to blood platelets using a flow cytometry bead based assay. Percentages of adhesion on BSA or fibrinogen-coated beads are shown (mean±s.e.m., n=4, 4, 2 for blood, fopMK and cbMK respectively; **P<0.01 versus blood by two-tail t-test). (b) Representative pictures from in vitro spreading assay. Washed platelets were sown on fibrinogen-coated slides, incubated for 45 min at 37 °C and immunostained for alpha-tubulin (TUBA) and F-actin (scale bars, 10 μm). (c) Aggregation of fopMK platelets upon agonists stimulation was tested both with and compared with blood platelets using a flow cytometry-based assay: representative dot plots are shown. (d) Percentages of aggregation from Calcein-AM+ live platelets upon stimulation are shown for blood and fopMK platelets reactions (2 × 10⁷ platelets per ml; mean±s.e.m., n=7 for each reaction group; no statistical difference versus blood at P<0.01 by two-tail t-test). (e) Associated delta-aggregation defined as ((% ADP+TRAP aggregation)—(% no agonist aggregation)) for the different reaction groups (mean ±s.e.m., n=7; P values by two-tail t-test versus blood indicated). (f) Thrombus formation in vitro under arterial shear stress. The participation of Calcein-AM live platelets spiked into human blood (at 1 × 10⁷ per ml) is shown per 100 μm² thrombus area. Normal or thrombocytopenic blood (>150 × 10⁹ and <50 × 10⁹ l⁻¹ respectively) was used as recipient. Spiked platelets were sourced from day-8 concentrate unit (blood) or from fopMK platelets (iPSC#1 and #5; n>30 analysed thrombi per group; P values by two-tail t-test versus blood indicated). (g) Representative pictures from in vitro thrombus formation assays. Thrombi identified using bright field images are delineated and Calcein-AM platelets fluorescing in green; in vitro platelets Calcein-AM labelling is intrinsically dimmer than donor-derived platelets (Supplementary Fig. 5b). Scale bar, 50 μm. (h) Thrombus formation in vivo by laser injury of an arteriole in the cremaster muscle of NSG mice and intravital confocal microscopy. The incorporation of human Calcein-AM-labelled platelets (50 million transfused per mouse) to mouse thrombi is shown per 100 μm² thrombus area at T_max (thrombus maximum size). Mean values±s.e.m. and P values by two-tail t-test versus donor platelets are shown (n=16/4/8 thrombi analysed for blood, fopMK#1 and #5 platelets respectively). (i) Representative snapshots of Calcein-AM+ human platelets incorporated to mouse thrombus (scale bar 10 μm).