The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34(+) (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte-specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6-8 days, virtually all of the CD34-derived cells expressed the early megakaryocytic CD61 antigen, while, after 15-16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. 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 morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing.

The effect of thrombopoietin (TPO) on the functional activity of surface αIIbβ3 (GPIIbIIIa) was investigated in both primary human megakaryocytic cells, derived from peripheral blood CD34+ cells, and HEL hematopoietic cell line. TPO (100 ng/mL) induced a sixfold to ninefold enhancement of adhesion of both primary megakaryocytic and HEL cells to plates coated with either fibrinogen or fibronectin and a parallel increase of immunoreactivity to the PAC1 monoclonal antibody (MoAb) and fluorescein isothiocyanate-fibrinogen, both of which recognize an activated state of αIIbβ3 . The enhanced adhesion to fibrinogen or fibronectin was mediated by the Arg-Gly-Asp (RGD) recognition sequence of αIIbβ3 , as it was abolished by pretreatment of cells with saturating concentrations of RGDS peptide. A MoAb specific for the αIIb subunit of αIIbβ3 also inhibited cell attachment to fibrinogen or fibronectin, while MoAb to anti-αvβ3 or anti-α5 integrins were completely ineffective, clearly indicating that αIIbβ3 participates in this association. A role for PI 3 kinase (PI 3-K) in the TPO-mediated increase in αIIbβ3 function in megakaryocytic cells was suggested by the ability of the PI 3-K inhibitor wortmannin (100 nmol/L) and antisense oligonucleotides directed against the p85 regulatory subunit of PI 3-K to completely block the TPO-induced increase in αIIbβ3 integrin activity upon TPO stimulation. The modulation of adhesiveness to extracellular matrix proteins containing the RGD motif mediated by TPO likely plays a physiologic role in megakaryocytopoiesis, as pretreatment of CD34+ cells with RGDS or anti-αIIb MoAb significantly reduced the number of megakaryocytic colonies obtained in a fibrinclot semisolid assay.

Summary.Mammalian megakaryocyte development is characterized by a progressive accumulation of cells exhibiting a polylobated nucleus with a polyploid DNA content. In this study human megakaryocytes were obtained from CD34 þ haemopoietic progenitors by in vitro liquid culture in the presence of 100 ng/ml of recombinant thrombopoietin (TPO). Ultrastructural examination of polyploid megakaryocytes showed the presence of a large number of centrioles, the breakdown of the nuclear envelope, and the progressive chromatin condensation, all aspects characteristic of mitosis. At both indirect immunofluorescence and Western blot analyses, cyclin B and its related cyclindependent kinase (CDK)1, which forms the mitosis promoting factor (MPF), showed an increased expression in maturating megakaryoblasts and megakaryocytes (day 8 of culture) with respect to freshly isolated CD34 þ progenitors. This expression tended to decline in fully developed megakaryocytes (day 15 of culture). The amount of cyclin D and of the related CDK4, governing the G1 phase of the cell cycle, increased during megakaryocyte development, maintaining high levels of expression also in mature megakaryocytes. These results indicate that megakaryocyte polyploidization depends on a true, although incomplete, mitotic process, and that cyclin D/CDK4 probably plays a crucial role throughout megakaryocytopoiesis.