Fig. 6: Megakaryocytes show nuclear marginalization and enucleation prior to fragmentation.

Megakaryocytes (MKs) from C57BL/6 mice, labelled with CD41-PE antibody (red) and Hoechst 33342 (blue), were passaged repeatedly through the pulmonary vasculature of a C57BL/6 mouse ex vivo. Lungs were ventilated with air throughout. a Representative images of MKs derivatives during platelet generation: nuclear polarization and enucleation, where the nucleus is marginalized, of irregular shape or in the process of ejection from the cell; naked nuclei, where the ejected nucleus is >20 µm in diameter and free from the parent cell and/or partially encased in thin/patchy plasma membrane; MKs without nucleus, where the MKs have an approximately circular shape but without nuclei; and large anuclear objects, where ghost cells are of irregular shape and >10 µm in their longer axis. Scale bar: 5 µm. b Cells were imaged from samples of perfusates after passage numbers 1, 2, 3, 6 and 9. Five subgroups of MKs and their derivatives, as described above, were quantified as a percentage of total number of cells. Quantification was from at least 250 fields of view, counting at least 230 cells in total for each of the subgroups. N = 7, 5, 6, 9, 8 and 6 independent experiments for passage numbers 0, 1, 2, 3, 6 & 9. Data are mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01,***p < 0.001 and ****p < 0.0001. c–g Nuclear lobes of MKs fragment into small condensed sub-nuclei. c Representative images, from n = 3 independent experiments, of naked nuclei generated after multiple passages (as indicated) of mouse MKs through pulmonary vasculature. Scale bars: 5 µm. d–g The depth (d), aspect ratio (e), major axis (f) and minor axis (g) of sub-nuclei decreased substantially with increasing passages. These parameters were measured from after 3 passages to after 18 passages: depth 8.9–5.5 µm, aspect ratio 1.8–1.1, major axis 14.1–6.5 µm, minor axis 8.1–5.7 µm. Numbers above each column indicate significant difference to other passages. 19 sub-nuclei from n = 3 independent experiments were collected. Data are mean ± SEM. Two-tailed Mann–Whitney test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Source data are provided in the Source Data file.