Fig. 4.

More functional platelets generated from TPO knock-in cells. a Representative morphology of proplatelets derived from megakaryocytes in Ctr or TPO-KI-1/-2 H1 hESC groups. Scale bar = 40 μm. b, c Flow cytometry analysis showing percentage of CD41a⁺ (b) or CD41a⁺CD42b⁺ (c) platelets in Ctr or TPO-KI-1/-2 H1 hESC groups, respectively. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. d Fold change of CD41a⁺CD42b⁺ platelets in Ctr or TPO-KI-1/-2 H1 hESC groups, respectively. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. e Comparative analyses of CD41a⁺CD42b⁺ PLTs per MKs or seeded H1 hESCs, MKs per HPCs. Data shown as mean ± SEM (n = 3). *P < 0.05; **P < 0.01. f Phase-contrast images (left panel) and fluorescence images (right panel) of PB-PLTs or cultured platelets in Ctr or TPO-KI-1/-2 H1 hESC groups. Platelets stained for β-tubulin (microtubule cytoskeleton, orange). Representative line functions for single platelet shown by distance (μm) and intensity (inset). Scale bar = 2 μm. g Distribution of maximal particle diameter of PB-PLT or cultured platelets, quantified by immunofluorescence micrographs. Sizes of platelets shown as mean ± SEM (n = 3). **P < 0.01; NS, not significant. h Immunofluorescence images of platelets bound to immobilized fibrinogen with F-actin filament in absence (left panel) or presence (right panel) of 1 U/ml thrombin. Scale bar = 5 μm. i Aggregates of a mixture of 2 × 10⁵ Calcein-AM (red)-labeled blood or cultured platelets and 2 × 10⁷ blood platelets. Red, β-tubulin staining of both populations. Scale bar = 5 μm. j Representative flow cytometry analysis showing percentage of P-selectin (CD62P)-positive events in gated CD41a⁺ platelets in indicated groups. Blood platelets (right panel) used as positive controls. Ctr control, hESC human embryonic stem cell, HPC hematopoietic progenitor cell, MK megakaryocyte, PB-PLT platelet from peripheral blood, PLT platelet, TPO thrombopoietin, PLP platelet-like particle