Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34(+) (c luster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along th e megakaryocytic lineage in liquid cultures by the addition of the megakary ocyte-specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6- 8 days, virtually all of the CD34-derived cells expressed the early megakar yocytic CD61 antigen, while, after 15-16 days, most cells also expressed th e late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obta ined after 7 days of culture showed aspects typical of developing megakaryo cytes (MX), such as formation of platelet territories and cytoplasmic fragm entation. At later (15-16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphologica l features of apoptosis. Analysis of DNA fragmentation in these cells revea led that apoptosis in megakaryocytes occurred in the absence of the internu cleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytomet ry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing. Anat Rec 25 8:90-99, 2000. (C) 2000 Wiley-Liss, Inc.