In Vitro Senescence and Apoptotic Cell Death of Human Megakaryocytes (original) (raw)
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Immunological study of in vitro maturation of human megakaryocytes
British Journal of Haematology, 1984
Human megakaryocyte colonies were grown from the bone marrow in plasma clot or methyl cellulose cultures. Maturation of the megakaryocytic cells was sequentially studied from day 5 to day 16 of culture by fluorescent labelling with a panel of monoclonal and polyclonal antibodies against different platelet glycoproteins (Gp), P1 A , antigen, factor VIII RAg platelet factor 4 (PF 4), fibrinogen and platelet-derived growth factor (PDGF). Expression of Gp Ib was also studied by immunogold technique at electron microscopy. The first cells identifiable by these antibodies were found at day 5 of culture. They had the size of a lymphocyte. These small megakaryocyte precursors already expressed all the platelet antigens, HLA-DR and transferrin receptors and were devoid of erythroid or myeloid markers. Among the platelet antigens, Gp IIIa was the most sensitive marker for the identification of these precursors. However, double-fluorescent labelling demonstrated that the different platelet markers were coexpressed in a large majority of cells. Interestingly, cytoplasmic markers demonstrated that these small megakaryocyte precursors were themselves heterogenous by morphological criteria. During maturation, expression of Gps, particularly of Gp Ib, increased while the labelling pattern of anti factor VIII RAg and anti PF 4 antibodies switched from diffuse to granular staining. PDGF could also be detected in the megakaryocytes grown in culture. The megakaryocytic line is the most difficult haemopoietic lineage to study because of its scarcity in normal bone marrow. Two recently developed techniques, purification of the megakaryocytic population (Rabellino et a/. I 979) and megakaryocyte cultures (Vain
Immature megakaryocytes undergo apoptosis in the absence of thrombopoietin
Experimental Hematology, 1999
We examined withdrawal effects of recombinant mouse Tpo (rm-Tpo) on the apoptosis of mature and immature megakaryocytes in in vitro experiments. Apoptotic megakaryocytes were detected by double staining for acetylcholinesterase and by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. When the purified mature megakaryocytes were cultured with or without rm-Tpo, the numbers of viable megakaryocytes, apoptotic megakaryocytes, and megakaryocytes with cytoplasmic processes were not significantly different between the two groups. In contrast, purified immature megakaryocytes underwent apoptosis when rm-Tpo was absent from the culture system. Murine bone marrow cells were cultured with rm-Tpo (50 U/mL) on days 1-7 to generate immature megakaryocytes and subsequently were cultured with different concentrations of rm-Tpo (0-50 U/mL) on days 8-14. The number of viable megakaryocytes was decreased and that of apoptotic megakaryocytes was increased by rm-Tpo in a dose-dependent manner. These results indicated a clear relation between the rm-Tpo level and the apoptosis of immature megakaryocytes.
Blood, 2004
To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP), ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% ± 14% of ITP megakaryocytes, which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 compared with normal (28% ± 4% versus 0%). No difference, however, was observed in the number of bone marrow megakaryocyte colony-forming units (ITP, 118 ± 93/10 5 bone marrow cells; versus controls, 128 ± 101/10 5 bone marrow cells; P = 0.7). To demonstrate that circulating antibodies might affect megakaryocytes, suspension cultures of CD34 + cells were performed with ITP or normal plasma. Morphology compatible with (para-)apoptosis could be induced in cultured megakaryocytes with ITP plasma (2 of 10 samples positive for antiplatelet autoantibodies). Finally, the plasma glycocalicin index, a parameter of platelet and megakaryocyte destruction, was increased in ITP (57 ± 70 versus 0.7 ± 0.2; P = 0.009) and correlated with the proportion of megakaryocytes showing (para-)apoptotic ultrastructure (P = 0.02; r = 0.7). In conclusion, most ITP megakaryocytes show ultrastructural features of (para-)apoptosis, probably due to action of factors present in ITP plasma.
Human megakaryocytes. V. Changes in the phenotypic profile of differentiating megakaryocytes
The Journal of experimental medicine, 1985
Human megakaryocytes were studied for phenotypic changes occurring throughout differentiation using a panel of monoclonal antibodies raised against marrow megakaryocytes and blood platelets. 11 monoclonal antibody preparations were selected for restricted specificity against megakaryocytes and/or platelets after screening by immunofluorescence, complement-mediated cytolysis, and solid phase enzyme-linked immunosorbent assay. The expression of the cellular epitopes recognized by these reagents enabled the identification of three levels of megakaryocyte maturation characterized by distinct immunologic phenotypes. Based upon their reactivities against megakaryocytic cells at different ontogenetic levels, monoclonal antibodies were operationally categorized into three groups. Group A consisted of six different monoclonal antibodies that recognized antigens on the colony-forming unit-megakaryocyte (CFU-Mk), in vitro grown colony megakaryocytes, and early immature marrow megakaryocytes, o...
Comparative analyses of megakaryocytes derived from cord blood and bone marrow
British Journal of Haematology, 2000
Thrombocytopenia is typically observed in patients undergoing cord blood transplantation. We hypothesized that delayed recovery of the platelet count might be caused by defects in the megakaryocytic differentiation pathway of cord blood progenitors. To test this hypothesis, we compared the features of in vitro megakaryocytopoiesis between cord blood progenitors and those in bone marrow cells after isolation of CD34+ cells as progenitors. The proliferative responses of the progenitors in cord blood are higher than those in bone marrow cells in the presence of interleukin (IL)-3, stem cell factor (SCF) and thrombopoietin (TPO). However, the ability to generate mature megakaryocytes was higher in bone marrow progenitors than in cord blood in the same in vitro culture system, when examined by the expression of CD41, polyploidy and proplatelet formation. Furthermore, an earlier induction of c-mpl protein, a receptor for TPO, was observed in the progenitors from bone marrow than in those from cord blood in the presence of SCF and IL-3. Therefore, the ability to generate mature megakaryocytes in bone marrow progenitors is superior to that in cord blood, and the delayed engraftment of platelets after cord blood transplantation might be attributed to the features of cord blood megakaryocyte progenitors.
Journal of Clinical Pathology, 2009
Aims: Essential thrombocythaemia (ET) and primary myelofibrosis (PMF) share some clinical and pathological features, but show different biological behaviour and prognosis. The latest contributions to understanding the nature of these disorders have focused on bone marrow microenvironment remodelling and proliferative stress, recognising megakaryocytes (MKCs) as ''key-cells''. The aim of this study was to investigate the apoptotic profile of ET and PMF MKCs in order to further characterise the biology of these disorders. Methods: Bone marrow biopsy samples from 30 patients with ET, and 30 patients with PMF, were immunophenotypically studied for the expression of pro-apoptotic (Fas, Fas-L, Bax, Bad) and anti-apoptotic (Bcl-2, Bcl-XL, hTERT (human telomerase reverse transcriptase)) molecules and the ''executioner'' molecule caspase-3. The fraction of MKCs undergoing apoptosis was assessed by deoxynucleotidyl transferase-mediated dUTP nick-end labelling. Results: Only the mitochondrial pathway seemed to be involved in MKC apoptosis. The anti-apoptotic molecule Bcl-XL was predominantly found in ET MKCs (50.5% of ET MKCs versus 35% of PMF MKCs; p = 0.036), while pro-apoptotic molecules Bax and Bad showed a prevalent expression in PMF MKCs (30.5% of ET MKCs versus 55% of PMF MKCs; 41% of ET MKCs versus 52% of PMF MKCs; p = 0.001 and p = 0.068, respectively). A significant fraction of PMF MKCs were committed to apoptosis according to caspase-3 expression and TUNEL, while only few ET cells were committed to apoptosis. hTERT was significantly more expressed in PMF (32% of ET MKCs versus 46% of PMF MKCs; p = 0.022), in agreement with the proliferative nature of this disease. Conclusions: It was found that ET and PMF MKCs, which barely differ in terms of morphology and aggregation, are characterised by markedly different apoptotic profiles. The rather high apoptotic fraction of PMF was able to support the fibrotic nature of this process, while the anti-apoptotic profile of ET cells fits well with their ''steady'' maturative state.
Journal of Experimental Medicine, 1979
Human marrow megakaryocytes have been isolated with high purity and yield by processing marrow cells sequentially through density centrifugation and velocity sedimentation. Analysis of the isolated cells for various platelet-associated components by immunofluorescence demonstrated that fibrinogen, plasma factor VIII antigen (factor VIII:AGN) platelet myosin, platelet glycoproteins I and III are present on the membrane and in the cytoplasm of over 90% of marrow megakaryocytes. Parallel studies of human and mouse megakaryocytes and platelets for IgG receptor (FcR), complement receptor type one (CR1) (C3b receptor), complement receptor type two (CR2) (C3d receptor), and Ia antigen by fluorescence and (or) rosette formation methods were performed. FcR were present on most human megakaryocytes and platelets. The Ia antigen was detected on a proportion (10-15%) of human megakaryocytes but it was undetectable on human platelets. CR1 was found on 20-40% of mouse megakaryocytes and also on a...
Leukemia, 2006
Platelet production requires compartmentalized caspase activation within megakaryocytes. This eventually results in platelet release in conjunction with apoptosis of the remaining megakaryocyte. Recent studies have indicated that in low-risk myelodysplastic syndromes (MDS) and idiopathic thrombocytopenic purpura (ITP), premature cell death of megakaryocytes may contribute to thrombocytopenia. Different cell death patterns have been identified in megakaryocytes in these disorders. Growing evidence suggests that, besides apoptosis, necrosis and autophagic cell death, may also be programmed. Therefore, programmed cell death (PCD) can be classified in apoptosis, a caspase-dependent process, apoptosis-like, autophagic and necrosis-like PCD, which are predominantly caspase-independent processes. In MDS, megakaryocytes show features of necrosis-like PCD, whereas ITP megakaryocytes demonstrate predominantly characteristics of apoptosis-like PCD (para-apoptosis). Triggers for these death pathways are largely unknown. In MDS, the interaction of Fas/Fas-ligand might be of importance, whereas in ITP antiplatelet autoantibodies recognizing common antigens on megakaryocytes and platelets might be involved. These findings illustrate that cellular death pathways in megakaryocytes are recruited in both physiological and pathological settings, and that different forms of cell death can occur in the same cell depending on the stimulus and the cellular context. Elucidation of the underlying mechanisms might lead to novel therapeutic interventions.