Figure 6.

GPIbα‐retaining iPSC platelets generated in the presence of KP‐457 exhibited an improved response to vWF in vitro and hemostatic function in vivo. (A): Transmission electron micrographs of imMKCL platelets produced with or without KP‐457. Scale bar, 2 µm. (B): Percentage of double‐colored events served as an aggregation index in a flow cytometry‐based platelet aggregation assay using peripheral blood‐derived platelets or iPSC‐derived platelets generated with or without 15 µmol/l KP‐457. A mixture of APC‐ and V450‐labeled platelets was stimulated using 2 mg/ml ristocetin to evoke vWF/GPIbα‐dependent platelet aggregation. ∗, p < .05 vs. without KP‐457, n ≥ 4. (C): Representative sequential images of GPIbα⁺ iPSC platelets generated with KP‐457 in mouse thrombus formation model. A combination of FITC‐dextran (green), Hoechst33342 (blue), and equal numbers of tetramethylrhodamine‐labeled iPSC platelets (1 × 10⁷ CD41a⁺ platelets, red) generated with or without KP‐457 were transfused into irradiated NOG mice. Hematoporphyrin was also administered to induce thrombus formation by chemical reaction after laser‐induced injury. Mesenteric capillaries were visualized using a confocal laser scanning microscope. Attachment of platelets including tetramethylrhodamine‐positive iPSC platelets on injured blood vessels and subsequent thrombus formation were observed after the injury. White arrow, direction of blood flow; pink arrow, site of thrombus formation. Scale bar, 10 µm. (D): The number of iPSC platelets in thrombus formation was estimated using the images of 30 vessels from two mice of the same group in two independent experiments. GPIbα‐retained iPSC‐derived platelets with KP‐457 exerted more effective hemostatic function in vivo. ∗, p = .0556 vs. vehicle group, n = 30. Abbreviations: APC, allophycocyanin; GP, glycoprotein; imMKCL, immortalized megakaryocyte progenitor cell line; iPSC, induced pluripotent stem cell; vWF, von Willebrand factor.