The action of dipyridamole on megakaryocytopoiesis in regenerating and stationary bone marrow cell populations]

The effect of dipyridamole on megakaryocytopoiesis in regenerating and stationary populations of mouse bone marrow cells has been studied by heterotopic transplantation of the bone marrow using histological, electron microscopic and biochemical techniques. It is shown that drug administration induced destruction of megakaryocytes. In megakaryocytic cytoplasm giant lipid granules were found whose growth and number increase resulted in megakaryocytes kill. Gas-liquid chromatography was used to evaluate the effect of dipyridamole on distribution of lipid fatty acids of the stationary and regenerating populations of the bone marrow cells. A marked increase of the percentage of docosahexaenoic acid was found in lipids of the stationary population. Chronic dipyridamole administration caused an increase of percentage of myristic, palmitic oleic acids, and decrease of percentage of arachidonic and eicosapentaenoic acids in lipids of regenerating bone marrow cells population.     

Emperipolesis and interrelations of megakaryocytes and neutrophilic granulocytes in the bone marrow of healthy dogs

Emperipolesis is an ability of one cell to penetrate into cytoplasm of another cell and to stay there for some time, or to pass through it. Unlike a common phagocytosis, not a cell interacting is damaged. Penetration of neutrophilic granulocytes into cytoplasm of megakaryocytes is also considered as emperipolesis, that is revealed in the bone marrow of healthy persons. Its occurrence becomes greater at various pathological states of tumorous and non-tumorous etiology. Histological investigation of the bone marrow of the rib, breast bone, vertebral body and iliac bone crest, performed in 10 healthy dogs makes it possible to state, that there are possible specific interrelations between megakaryocytes and neutrophilic granulocytes, that terminate in death of the former. Therefore, the phenomenon observed cannot be identified with emperipolesis. In this case we, evidently, deal with a morphological demonstration of cell cytotoxicity, when neutrophils, being killers, act as effector-cells, and megakaryocytes - as target-cells. Cell cytotoxicity is a natural phenomenon, that under certain physiological conditions is evidently connected with thrombocytopoiesis.     

Emperipolesis--a peculiar feature of megakaryocytes as evaluated in chronic myeloproliferative diseases by morphometry and ultrastructure

A combined morphometric and ultrastructural study was performed on so called emperipolesis or internal wandering of myeloid cells in the cytoplasm of large mature megakaryocytes. Measurements were made on material from a total of 115 patients comprising a normal control group and 5 groups with subtypes of chronic myeloproliferative diseases, including primary (essential, idiopathic) thrombocythemia (PTH). A significant increase in this peculiar phenomenon was noted in myeloproliferative disorders and especially in PTH where the frequency of emperipolesis showed a positive correlation with the number of anuclear cytoplasmic fragments sectioned, with the circular deviation of the shapes of megakaryocytes and with the extent to which the peripheral thrombocyte count was elevated. Electron microscopy in selected cases displayed the integrity of the plasma membranes of engulfed hematopoietic cells within the dilated cavities of the megakaryocytic demarcation membrane system (DMS) and no evidence of phagocytosis. Moreover there was a close relationship between engulfed myeloid cells and the presumptive sites of platelet shedding which had their openings from the cisternal lumina of the DMS. Our results demonstrate that emperipolesis of hematopoietic cells within megakaryocytes should not be regarded as a special nosological feature, but as an indication of enforced thrombocytogenetic activity which is expressed particularly in PTH.     

Satellite nucleoli in the megakaryocytic lineage of rats

The present study was undertaken to provide more information on the incidence of satellite nucleoli in developmental stages of the megakaryocytic lineage. Satellite nucleoli representing solitary silver stained nucleolus organizer regions (AgNORs) present in nuclei in addition to other nucleolar types were observed in all stages of megakaryocytic development. However, the incidence of satellite nucleoli was more frequent in mature megakaryocytes than in less differentiated immature megakaryoblasts and naked megakaryocytic nuclei representing the terminal stages of megakaryocytic development after loss of the cytoplasm transformed to thrombocytes. There is a possibility that the increased incidence of satellite nucleoli in mature megakaryocytes might be due to the loss of AgNORs from active nucleoli characteristic for immature cells. The decreased incidence of satellite nucleoli in naked megakaryocytic nuclei might reflect their disintegration in the terminal stages of the megakaryocytic development.     

Effects of gangliosides on cell maturation of murine megakaryocytes in a liquid culture system

Formation of platelet-producing megakaryocytes, the cytoplasm of which showed the terminal stage of cell maturation, heavy granulation and platelet-fields delineated with demarcation membranes, was observed in a short-term culture system, using megakaryocyte-enriched bone marrow cell suspension. Approximately 6-8% of the megakaryocytes changed to the platelet-producing megakaryocytes during 12-hour incubation. In the presence of inhibitors of energy metabolism, formation of the platelet-producing megakaryocytes was inhibited, suggesting that the process is dependent on energy producing systems. Ganglioside GD1a increased both the number of total megakaryocytes and the ratio of the platelet-producing megakaryocytes to total megakaryocytes, while GM1 did not influence the number of total megakaryocytes, but increased the ratio. Gangliosides GM2, GM3 and GD1b showed little effect on either the number of total megakaryocytes or the ratio. The results suggest that ganglioside GD1a stimulates at least two steps of megakaryocyte maturation, the change of megakaryocytic progenitors to megakaryocytes and the subsequent maturation of megakaryocytes to the platelet-producing megakaryocytes, while GM1 stimulates only the latter step of the maturation.     

Modulation of megakaryocyte emperipolesis by phlebotomy: megakaryocytes as a component of marrow-blood barrier

The presence of marrow cells within the cytoplasm of megakaryocytes has been documented and is attributed to the phenomenon of emperipolesis. In a previous study most patients demonstrating this phenomenon had or were suspected of having blood loss. To find out if these are causally related, we induced acute and chronic blood loss in rats and quantitatively measured the emperipolesis index (EI). EI dropped after acute blood loss but rose after chronic blood letting, indicating that blood loss can modulate this phenomenon. In the marrow, megakaryocytes are located preferentially on the abluminal side of sinus endothelium and it is postulated that in the state of heightened demand for cell delivery from marrow into the circulation, some cells take a transmegakaryocytic route to enter the circulation. This concept incorporates megakaryocytes as a component of the marrow-blood barrier.     

Lycramine brilliant blue JL: a new stain for human megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte cytoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

Lycramine Brilliant Blue Jl: A New Stain for Human Megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte ctyoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

Stromal inhibition of megakaryocytic differentiation correlates with blockade of signaling by protein kinase C-epsilon and ERK/MAPK

Contact with bone marrow stromal cells maintains normal and leukemic hematopoietic progenitors in an undifferentiated state. Recently, stromal contact has been shown to diminish the yield of megakaryocytes in cultures of primary human hematopoietic stem cells. This inhibition may explain the poor megakaryocytic engraftment frequently observed after bone marrow transplantation. In the current study, stromal co-culture is shown to render K562 cells refractory to megakaryocytic induction. This stromal inhibition correlated with the selective down-regulation in K562 cells of protein kinase C-epsilon (PKC-epsilon), which has recently been implicated in regulation of megakaryocytic lineage commitment. In addition, the stromal inhibition correlated with inactivation of the ERK/MAPK pathway, which has also been implicated in promoting megakaryocytic development. Forced expression of PKC-epsilon by retroviral transduction was insufficient to reverse the stromal blockade of ERK/MAPK signaling or of megakaryocytic induction. Thus stromal interruption of ERK/MAPK signaling occurred independently of PKC-epsilon levels and correlated more closely with megakaryocytic blockade. These findings provide potential mechanisms for stromal inhibition of hematopoietic differentiation and possibly for the poor megakaryocytic engraftment seen after bone marrow transplantation.     

Comparative study of myelodysplastic syndromes and normal bone marrow biopsies with conventional staining and immunocytochemistry

OBJECTIVE: To study the histomorphometric features of megakaryocytic elements in bone marrow trephine biopsies of various subtypes of myelodysplastic syndrome (MDS). STUDY DESIGN: Comparative image morphometry using hematoxylin-eosin-stained and CD 61-stained trephine biopsies was carried out on 40 cases of MDS and 10 normal subjects to analyze the megakaryocytes objectively. The various variables analyzed were number of megakaryocytes and micromegakaryocytes, area, perimeter and diameter of the megakaryocytic elements. RESULTS: The mean number of megakaryocytes was lower in cases of MDS as compared to the normals in all except for a single case of hypoplastic MDS, in which the megakaryocytes were more abundant (3.6 per high-power field [hpf]). No micromegakaryocytes were observed in the 2 cases of chronic myelomonocytic leukemia. The mean area, perimeter and diameter of megakaryocytes decreased significantly on immunostaining with CD 61. CONCLUSION: The mean number of megakaryocytes per hpf was lower in the cases of MDS as compared to normal cases on hematoxylin-eosin. However, on CD 61 staining the number of megakaryocytes per hpf increased in cases of MDS. Micromegakaryocytes were seen in scanty numbers in the normals but increased in MDS cases and increased significantly on CD 61 immunostaining. The mean area, perimeter and diameter of megakaryocytes decreased significantly on immunostaining with CD 61, indicating the increased numbers of micromegakaryocytes in MDS. Hence, immunostaining is an efficient method of detecting increased numbers of megakaryocytes and micromegakaryocytes that would ordinarily be missed by using routine hematoxylin-eosin staining.     

Spleen and bone marrow megakaryocytes as targets for inhaled vanadium

An increased incidence in ischemic and thromboembolic events in the population of cities with rising air suspended particle pollution has suggested the interaction of some of the components of these particles in the coagulation system. A previous report from our laboratory identified thrombocytosis as a consequence of the subacute and chronic inhalation of vanadium. With this preceding information we decided to evaluate the effects of this element in the spleen and bone marrow in a mouse experimental model. CD-1 male mice inhaled V2O5 0.02 M for one hour twice a week for twelve weeks. The spleen and bone marrow were processed for light microscopy. The increase in quantity and size of megakaryocytes (MKs) in the exposed group in both organs was striking. Also, modifications in the cytoplasm, granule content and nuclear ultrastructure were evident. Our results indicate the influence of vanadium on megakaryopoyesis, an effect which could be the onset of the thrombocytosis previously reported by our group. The modifications in MKs described here suggest that inhaled vanadium could induce megakaryocytic proliferation, which may result in increased production of platelets and increased risk for thromboembolic events.     

Effect of gangliosides on murine megakaryocytopoiesis in a liquid culture system

Bovine brain gangliosides were applied to primary cultures of murine bone marrow cells to examine the role of gangliosides in development of megakaryocytes. Megakaryocytes in the cultures were detected by staining for a cytoplasmic enzyme, acetylcholinesterase, and divided into two types, 1) immature megakaryocytes which were stained less intensely, and 2) mature ones which were stained intensely. A medium containing total ganglioside fraction from bovine brain increased the number of both immature and mature megakaryocytes in the presence of pokeweed mitogen-stimulated murine spleen cell conditioned medium. Between the two cell types, the number of the mature cells was more significantly increased than the immature cells. The ganglioside GD1a could substitute for the total ganglioside mixture. The results suggested that bovine brain gangliosides potentiated both megakaryocytic proliferation and maturation in vitro.     

Identification of young megakaryocytes by immunofluorescence and cytophotometry.

The DNA-content of fluoresceine-labeled platelet antigen containing cells of mouse bone marrow was measured. For immunofluorescence highly specific anti-mouse-platelet-serum and fluoresceine-conjugated antigammaglobuline was used, applying the "sandwich" technique. Three hundred panoptically identifable megakaryocytes served as control group. The DNA-polyploidization pattern of megakaryocytes and immunofluorescence positive cells was almost identical. However, among the immunofluorescence positive cells a considerable amount of cells showed DNA-values lower than 4c, whereas the megakaryocytes of the Pappenheim stained smears revealed no DNA-values lower than 4c. The percentages of diploid and tetraploid cells, respectively, was 6 and 7% compared with 0 and 1% of panoptically identifiable megakaryoctyes. The results suggest that young megakaryocytic cells with diploid and tetraploid DNA-values can be detected by immunofluorescence technique, indicating that the flow from the uncommited to the committed megakaryocytic precursor cell appears at this early stage of megakaryocyte production.     

Ultrastructure of chronic megakaryocytic-granulocytic myelosis.

To study chronic megakaryocytic-granulocytic myelosis, bone marrow biopsies from 5 patients were obtained. Ultrastructural quantitative and qualitative assessments demonstrate proliferation of both the megakaryocytic and granulocytic cell lines. Factors indicative of malignant growth in megakaryocytes included atypical maturation, nuclear-cytoplasmic asynchrony, nuclear inclusions and production of micromegakaryocytes. Abnormal thrombocyte delineation provoked giant platelet production. The neutrophil series also presented atypia as generally observed in chronic myelogenous leukemia. Even in cases without evidence of myelofibrosis under the light microscope, megakaryoblasts were associated with fibrillar structures. These cells may be responsible for the initial step in fibrillogenesis by providing a medium conductive to the collagen formation found in later stages of this disease.     

小鼠巨核系祖细胞增殖,分化特性的研究

小鼠骨髓细胞经7d培养后进行细胞形态学观察,可见不同发育阶段的巨核细胞及不同大小的巨核细胞集落。通过计数每个集落中的细胞数,可确定相应祖细胞的有丝分裂能力。结果表明,具有不同有丝分裂能力的祖细胞的体外增殖动力学有所不同。祖细胞的数量与其有丝分裂次数呈负相关(r=-0.986)。进行0、1、2和3次有丝分裂的祖细胞的阿糖胞苷自杀率分别为48.9,58.7,48.0和41.2％;放射敏感性的D_O值(Gy)分别为1.71,1.24,1.03和0.77,D_O值的大小与有丝分裂次数呈负相关(r=-0.958)。经3Gy全身照射后CFU-Meg与CFU-GM的恢复动态过程具有不同特点。     

A clinicopathological study of non-Hodgkin's malignant lymphomas in terms of the Kiel classification.

A group of 25 patients with non-Hodgkin's lymphoma was investigated. Patients diagnosed earlier were reclassified according to the Kiel system. A correlation between age distribution and histological malignancy was found. The time between the onset of symptoms and diagnosis was 1 year on an average. The majority of the patients belonged to stage IV. The survival rate was higher in the low-grade malignancy group than in the high-grade group. When assessing the prognosis, the histological classification as well as the clinical staging ought to be considered. The bone marrow was the most frequent extranodal site of involvement in stage IV. Cytopenic changes were almost invariably accompanied by bone marrow infiltration. All the 7 cases analysed for lymphocyte surface markers proved to be B cell type. No significant difference was seen between the results of single agent and combined chemotherapy.     

Bone marrow stromal proteoglycans regulate megakaryocytic differentiation of human progenitor cells

Adherence of hematopoietic progenitor cells (HPCs) to stroma is an important regulatory step in megakaryocytic differentiation. However, the mechanisms through which megakaryocytic progenitors are inhibited by stroma are poorly understood. We examined the role of sulfated glycoconjugates, such as proteoglycans (PGs), on human bone marrow stroma (hBMS). To this end, PG structure was altered by desulfation or enzymatic cleavage. PGs participated in adhesion of human HPC, as desulfation resulted in about 50% decline in adhesion to hBMS. Heparan sulfate proteoglycans (HSPGs) were found to be responsible by showing about 25% decline in adhesion after pre-incubation of HPC with heparin and about 15% decline in adhesion after enzymatic removal of HSPGs from hBMS. Furthermore, PGs were involved in binding cytokines. Both desulfation and enzymatic removal of stromal HSPGs increased release of megakaryocytopoiesis-inhibiting cytokines, that is, interleukin-8 (IL-8, 1.9-fold increase) and macrophage inflammatory protein-1alpha (MIP-1alpha, 1.4-fold increase). The megakaryocytic output of HPC grown in conditioned medium of desulfated stroma was decreased to 50% of the megakaryocytic output in CM of sulfated stroma. From these studies, it can be concluded that PGs in bone marrow, in particular HSPGs, are involved in binding HPC and megakaryocytopoiesis-inhibiting cytokines. Bone marrow stromal PGs thus reduce differentiation of HPC toward megakaryocytes.     

Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.     

The use of the anti-factor VIII method on trephine biopsies of the bone marrow for the identification of immature and atypical megakaryocytes in myeloproliferative diseases and allied disorders. A morphometric study

A morphometric analysis was performed on trephine biopsies of the bone marrow to identify atypical megakaryocyte proliferation following PAS staining and the immunohistological demonstration of factor VIII. This study includes nine patients with a megakaryoblastic crisis in chronic myeloid leukemia (CML), four with acute megakaryoblastic leukemia (AM) and three with myeloid dysplasia later evolving into overt acute leukemia. Comparison and statistical evaluation of the PAS reaction with anti-factor VIII staining reveals that the latter technique not only facilitates the recognition of immature and abnormal megakaryocytes, but leads to a significantly increased count for all megakaryocytic elements in the bone marrow. Thus our retrospective investigation of routinely processed and paraffin-embedded trephine biopsies shows that the diagnosis of a megakaryoblastic crisis in CML as well as AM may be easily established with the aid of the anti-factor VIII method. In all cases of megakaryoblastic proliferation in CML and AM, the appearance of blasts was associated with moderate to pronounced myelofibrosis which could be also determined by morphometry.     

Microvesicles promote megakaryopoiesis by regulating DNA methyltransferase and methylation of Notch1 promoter

Megakaryopoiesis is the process of formation of mature megakaryocytes that takes place in the bone marrow niche resulting in the release of platelets into the peripheral blood. It has been suggested that cell to cell communication in this dense bone marrow niche may influence the fate of the cells. Numerous studies point to the role of exosomes and microvesicles not only as a messenger of the cellular crosstalk but also in growth and developmental process of various cell types. In the current study, we explored the effects of megakaryocyte-derived microvesicles in hematopoietic cell lines in the context of differentiation. Our study demonstrated that microvesicles isolated from the induced megakaryocytic cell lines have the ability to stimulate noninduced cells specifically into that particular lineage. We showed that this lineage commencement comes from the change in the methylation status of Notch1 promoter, which is regulated by DNA methyltransferases.