Figure 5: Platelet release in vitro from fopMKs throughout long-term culture.

(a) Phase contrast picture of spontaneous proplatelet-forming fopMKs (arrowheads) in suspension culture (hiPSC#3, day26; scale bar, 50 μm). (b) Proplatelet-forming fopMK on fibrinogen-coated slide immunostained for alpha-tubulin (TUBA) and P-selectin (SELP). Arrowheads indicate nascent platelet tips containing SELP-positive granules (hiPSC#4, day21; scale bar, 50 μm). (c) Transmission electron microscopy pictures of blood and in vitro-produced platelets showing typical ultrastructure. AG, alpha-granules; MVB, multi-vesicular bodies (scale bars, 1 μm). (d) Representative flow cytometry dot plots of platelet analysis. Platelets are defined within the human platelet size gate as CD41a/CD42a double positive events. (e) cbMK and fopMK from different culture time points were sown on C3H10T1/2 feeder cells for 48 h and the number of platelets released in the supernatant quantified by flow cytometry. Data represent the mean±s.e.m. of platelet number per MK sown (n=7, 4, 10, 6, 2 for cbMK and fopMK (hiPSC#1–4 pool) at day 20–90 respectively; **P<0.01 versus fopMK day 20 by two-tail t-test). (f,g) Mean platelet volume (MPV) ±s.d. and immature platelet fraction (IPF) ±s.d. of washed donor and fopMK platelets as measured on a Sysmex whole-blood analyzer (n=4/2 respectively; hiPSC#1 and #5). (h) Human platelet survival in NSG mice circulation over 24 h measured by flow cytometry following the systemic injection of 20 million washed platelets. The exponential trend of human platelet absolute count decrease over time was used for half-life calculation (mean±s.e.m.; n=5/4 for donor and fopMK (hiPSC#1 and #5) platelets respectively).