Ultrastructural Evidences of Caspase-Dependent Platelet Generation in ES Cell-Differentiation System.

Platelets are essential for thrombosis and hemostasis, and the elucidating the unique mechanism for platelet production from megakaryocyte is of major importance. However, extensive data on platelet generation have yet to be fully documented because it has been extremely difficult to obtain the sufficient amounts of hematopoietic stem cells. In this study, we therefore used murine embryonic stem (ES) cells that can proliferate and differentiate to megakaryocyte in the presence of thrombopoietin in vitro. ES-derived megakaryocytes and platelets were studied in detail by morphological analyses, and we especially focused on the relationship between cell death of megakaryocytes and platelet production in sequential experiments. A coculture system with ES/OP9 cells, stromal cells derived from M-CSF deficient mice, in the presence of thrombopoietin was used to differentiate mesodermal-like hematopoietic progenitors, immature megakaryocytes, mature megakaryocytes, and functional platelets from ES cells on days 5, 8, 12, and 15 of the culture, respectively, and the peak of cell count was observed on the day 12 for megakaryocytes and the day 15 for platelets. These were confirmed by morphology, Wright-Giemsa staining, CD41expression or fibrinogen binding assays. Interestingly, electron microscopy and immuno-electron microscopy during platelet production showed morphological changes supported by both “proplatelet theory” and “explosive fragmentation theory”, which have been controversial issues related to platelet biogenesis. On the days 8 and 12, megakaryocyte with tube-like (proplatelet-like) extensions of the periphery showed the expression of CD41, CD42c, or von Willebrand factor, in contrast, megakaryocyte-like large cells with smooth periphery had no expression of CD41 and CD42c. On the days 12 and 15, global fragmentation of megakaryocyte cytoplasm into individual platelets was frequently observed. For cell death of megakaryocytes, cells exhibit clear morphological evidence of nuclear change: chromatin condensation, typical of early apoptosis, on the day 8 and extensive condensation and apoptotic nucleus surrounded by a shortrim of cytoplasm in continuity with a portion of granulated cytoplasm on the days 12 and 15. These proceedings of cell death were confirmed by the TUNEL assay in each stage. Also, low production of platelet was observed by adding Z-DEVD-fmk, an inhibitor of caspases −3 and −7, on the day 8. Next, we examined the caspase activation in the different stage of the platelet production by western blotting, and anti-CD41 antibody was used to test the purity of the meg-lineage cells in each stage. Peak expressions of activated caspases −12, −9, and −7 or caspase 10 levels were observed on the day 8. Peak expression of activated caspase 3 was observed on the day 12. On the contrary, no different levels of caspase 6 were shown between the days 8 and 15. Together, present studies in the sequential experiments for megakaryocyte differentiation and platelet production develop previous theories for platelet generation and also suggest that the process for platelet production is focally associated with caspases −12, −10, −9, −7, and −3 dependent cell death of mrgakaryocytes.