In vitro stimulation of megakaryocyte maturation by megakaryocyte stimulatory factor

Counterflow centrifugal elutriation and Percoll density gradient centrifugation were employed to prepare cell populations from rat bone marrow that were selectively enriched in the cytoplasmically immature megakaryocytes and depleted of the most mature megakaryocytes. The incorporation of [14C]leucine into the platelet-specific alpha-granule protein, platelet factor 4, as well as the incorporation of [35S]sulfate into platelet proteoglycans synthesized by the maturing megakaryocytes were monitored as markers of cytoplasmic maturation. Rat platelet factor 4 was specifically isolated and characterized by its high affinity for heparin-Sepharose and its amino-terminal sequence homology to human and rabbit platelet factor 4. The [35S]sulfate-labeled proteoglycans were primarily composed of chondroitin 4-sulfate glycosaminoglycans and were identified as platelet granule components by their ability to be secreted by megakaryocytes in response to thrombin or A23187. The production of both components was increased as much as 3-fold in a dose-dependent manner by the addition of picomolar concentrations of purified megakaryocyte stimulatory factor, without a concomitant increase in general protein synthesis. The above results suggest that the megakaryocyte stimulatory factor may regulate the synthesis of platelet granule components by megakaryocytes and hence control the rate and/or extent of cytoplasmic maturation during megakaryocyte development.     

Human megakaryocyte ploidy.

We reviewed the literature concerning the history of determination of the ploidy of human megakaryocytes and its relationship with diseases. The ploidy of rabbit megakaryocytes was analyzed by microspectrophotometry in 1964, and the analysis of the ploidy in human megakaryocytes was first performed in 1968. Presently, microphotometry and flow cytometry are the primary methods for the evaluation of the ploidy, but they have their merits and demerits. In the ploidy of human megakaryocytes, a peak has often been reported at 16N in healthy individuals, and the next peaks have been observed at 32N and 8N. The results of ploidy analyses have been reported by many investigators to be comparable between patients with idiopathic thrombocytopenic purpura and normal subjects, but various shifts of the peaks have also been documented. The ploidy is often reported to shift to a larger ploidy class in polycythemia vera and essential thrombocythemia, but it has invariably been reported to shift to a smaller class in chronic myelogenous leukemia. In reactive thrombocytosis, the ploidy pattern was reported to be the same as that in normal individuals by some investigators but to shift to a larger ploidy by others. These differences are considered to be due to heterogeneity of the subjects. In myelodysplastic syndrome, the ploidy shifts mostly to a smaller class, but it may show various patterns. We also reviewed the ploidy in other rare hematological disorders, the relationships of the ploidy with diabetes mellitus and atherosclerotic disorders, and its changes in the ontogeny. Details of the mechanism of polyploidization and its biological significance remain unknown, and further advances in the studies of these topics are anticipated.     

Molecular regulation of human megakaryocyte development

Extracellular regulators of human megakaryocyte development are becoming better defined. How these regulators function at the subcellular and, in particular, the molecular levels remains almost completely unknown. The recent development of molecular micromethodologies such as in situ hybridization, the polymerase chain reaction, and the use of antisense oligodeoxynucleotides now make such studies possible in normal cells. We therefore examined the effect of several recombinant human hematopoietic growth factors and the maturation agonist phorbol myristate acetate on the expression of selected growth-regulated and maturation/function-related genes. We also examined the role of the c-myb proto-oncogene in regulating megakaryocyte proliferative activity and ploidy development. Our results demonstrate that growth factors have complex time and concentration effects on gene expression in morphologically recognizable human megakaryocytes. They also suggest that a more complete understanding of normal megakaryocyte development at the molecular level will soon be possible.     

Alpha-actinin arcs in megakaryocyte spreading

Cultured megakaryocytes, isolated from guinea pig bone marrow, were treated with buffer or adenosine diphosphate (ADP) (10 microM) on plain or coated glass surfaces. Control cells were rounded and non-adherent. The nucleotide induced the cells to spread to several times the initial diameter, and to become flattened and markedly adherent to the substratum. 'Cytoskeletons' were examined by transmission electron microscopy (TEM). Those from unspread cells contained only rare microfilaments and no filament bundles; those from spread cells contained large numbers of microfilaments in nets and many filament bundles, which were largely oriented circumferentially. Interference reflection microscopy demonstrated that the spread cells were attached to the substratum in arc-shaped regions, which corresponded to arcs containing alpha-actinin as seen by specific immunofluorescence of the same cells. However, other arcs of alpha-actinin staining did not correspond to arcs of tight attachment. We conclude that fibrous arcs, which appear to assemble as part of the spreading process, play a role in what are probably transient surface attachment sites. A survey of observations of spreading in other cell types suggests that similar arcs are more widespread than has been realized.     

Flow cytometric analysis of megakaryocyte differentiation

Megakaryocytes were isolated quantitatively from rat bone marrow by centrifugal elutriation (CE). CE-enriched megakaryocytes were stained supravitally for either DNA content with Hoechst 33342, surface membrane immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated antiplatelet antibody, or both. The cells were then measured using a Becton Dickinson FACS IV flow cytometer. The following correlations were analyzed: DNA content and light scatter, light scatter and antiplatelet immunofluorescence, and DNA content and antiplatelet immunofluorescence. Although the range of light scatter increased as a function of DNA content, discrete subpopulations of megakaryocytes with different light scatter properties were detected within each of the three principal ploidy classes (8C, 16C, and 32C). Other discrete megakaryocyte subpopulations were revealed in the analysis of antiplatelet surface immunofluorescence as a function of degree of light scatter. The nonlinear relationship between the latter suggested that the degree of membrane immunofluorescence did not bear a simple relationship to cell size as reflected in light scatter. Megakaryocyte DNA content, on the other hand, varied in a linear fashion with membrane immunofluorescence, supporting the conclusion that there may be a proportional increase in the expression of platelet antigens with DNA content. The use of multiple markers, correlated multiparameter flow cytometry and multivectorial analysis to define differentiation on a single cell basis have revealed new complexities in this process. Flow cytometric analysis holds promise as a useful method for further characterization of megakaryocyte differentiation.     

Roads to polyploidy: the megakaryocyte example.

Polyploidy, recognized by multiple copies of the haploid chromosome number, has been described in plants, insects, and in mammalian cells such as, the platelet precursors, the megakaryocytes. Several of these cell types reach high ploidy via a different cell cycle. Megakaryocytes undergo an endomitotic cell cycle, which consists of an S phase interrupted by a gap, during which the cells enter mitosis but skip anaphase B and cytokinesis. Here, we review the mechanisms that lead to this cell cycle and to polyploidy in megakaryocytes, while also comparing them to those described for other systems in which high ploidy is achieved. Overall, polyploidy is associated with an orchestrated change in expression of several genes, of which, some may be a result of high ploidy and hence a determinant of a new cell physiology, while others are inducers of polyploidization. Future studies will aim to further explore these two groups of genes.     

Stimulators of megakaryocyte development and platelet production

The range of known purified and cloned growth factors and their target cells within the megakaryocytic lineage is described. Data are reviewed outlining that megakaryo-cytopoiesis appears to be controlled at two levels: (i) by feedback control via circulating factors, and (ii) by factors within the marrow itself. Hypotheses are presented about the nature of thrombopoietin, its relationship to known growth factors, especially Interleukin-6 (IL-6), and the specificity of a thrombopoietic response following change in the circulating platelet mass.     

The number of acetylcholinesterase molecules in the rat megakaryocyte.

A megakaryocyte cell series from rat bone marrow has been examined by the isotopic di-isopropyl fluorophosphate (DFP) method for esterases. After complete reaction with 32P-DFP, the numbers of DFP-reacted molecules inindividual cells havebeen determined by beta trackauto-radiography. Previous work has shown the percentage of organophosphate-sensitive sites in these cells which can be taken as active centers of acetylcholinesterase (AChase). Combining these data, the absolute numbers of organophosphate-sensitive esterase molecules and AChase molecules per cell were determined. Histograms show a narrow spread of values within each of four size classes from megakaryoblast to fully mature megakaryocyte, but, with means increasing 4-fold through this series, approximately in proportion to cell volume. A rat megakaryoblast has 2 X 10(6) AChase molecules, and a megakaryocyte (of 48-micro diameter) has 7.6 X 10(6) molecules. The apparent turnover number of the enzyme for intracellular reaction with substrate is calculated and compared with turnover numbers available for other AChases.     

In vitro colony assay for a new class of megakaryocyte precursor: colony-forming unit megakaryocyte (CFU-M).

A plasma culture system has been used successfully to grow and quantitate megakaryocyte colonies from mouse bone marrow following their staining for acetylcholinesterase activity in situ. Colonies averaging about six acetylcholinesterase-positive cells appear with a peak incidence after 4 days in culture with a plating efficiency of one colony formed for every 10(4) nucleated cells plated.     

Aspirin inhibits rat megakaryocyte thromboxane synthesis

This study examines the question of whether the aspirin-induced delay in the recovery of platelet cyclooxygenase pathway activity, as measured by RIA of thromboxane B₂, results from a direct effect on megakaryocyte cyclooxygenase. From our measurement of recovery of TXB₂ and information on megakaryocyte transit time in rats, we propose that thromboxane synthesis may represent a relatively late step in the differentiation of megakaryocytes. Megakaryocyte thromboxane production was depressed by 70% and that of platelets by 85% at two hr after 20 mg/kg oral aspirin dissolved in DMSO. Full megakaryocyte thromboxane recovery occurred by 72 hr and preceded complete platelet thromboxane recovery by 24 hr. Whereas megakaryocyte thromboxane synthesis showed substantial recovery by 36 hr after aspirin, platelet recovery did not begin for 24 hr and achieved a maximal recovery rate over the following 12 hr. This finding is consistent with predictions based upon human data for both megakaryocyte labeling studies and post-aspirin platelet recovery. We conclude from our data and from estimates of megakaryocyte maturation times in marrow, that thromboxane synthesis develops in rat megakaryocytes after approximately 48 hr of cytoplasmic differentiation toward platelet shedding. This metabolic capacity therefore serves as a marker of megakaryocyte differentiation.     

Partial purification of human urinary megakaryocyte colony-stimulating factor

Urinary extracts from patients with aplastic anemia are known to promote murine megakaryocytopoiesis. In this report, we show a simple method for the partial purification of megakaryocyte colony-stimulating factor from human urine. A four-step purification procedure, which included ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-Sepharose chromatography and high-resolution hydroxyapatite chromatography, resulted in an about 430- to 630-fold increase of specific activity. The final fractions were still contaminated with erythropoietin, but the contaminated content of erythropoietin was not enough to stimulate mouse megakaryocytopoiesis in our culture system. We also demonstrate that human urinary extracts stimulated human megakaryocyte colony formation.     

Single-cell level analysis of megakaryocyte growth and development

Several fundamental questions regarding cell growth and development can be answered by recording and analyzing the history of cells and their progeny. Herein, long-term and large-field live cell imaging was used to study the process of megakaryopoiesis at the single cell level (n = 9300) from human CD34+ cord blood (CB) in the presence of thrombopoietin (TPO) or the cytokine cocktail BS1 with or without nicotinamide (NIC). Comparative analyses revealed that the cocktail BS1 increased the mitotic and proplatelet rate of diploid and polyploid cells, respectively. Conversely, only NIC treatment increased the endomitotic rate of megakaryocytes (MKs) leading to the formation of CB-MKs with ploidy level frequently observed with BM-MKs. However, NIC failed to enhance platelet production. Rather, a 7- and 31-fold reduction in proplatelet formation was observed in tetraploid and octaploid CB-MKs, respectively, and ex vivo platelet production output was reduced by half due to a reduction in MK output in NIC cultures. Unexpectedly, a significant fraction of di- and polyploid CB-MKs were seen to undergo complete proplatelet regression. Though rare (< 0.6%), proplatelet reversal led to the formation of regular round cells that could at times resume normal development. The cell tracking data was then used to investigate the impact of "developmental fate" and ploidy on cell cycling time, and to identify potential developmental patterns. These analyses revealed that cell fate and ploidy level have major impacts on the cell cycling time of the cells, and that four recurrent cell lineage patterns could be identified for CD34+ cells undergoing MK differentiation.     

Two-factor requirement for murine megakaryocyte colony formation

WEHI-3 cell-conditioned medium with the capacity to stimulate megakaryocyte colony formation was separated by Sephadex G-150 column chromatography. The development of colonies containing megakaryocytes was observed only when mixing experiments were performed. Individual fractions did not support megakaryocyte colony growth. The two factors in WEHI-3 CM required for megakaryocyte colony growth had apparent average molecular weights of 35,000 daltons (megakaryocyte CSF) and 100,000 daltons (megakaryocyte potentiator). The results were confirmed in serum-free conditions in which colonies were directly identified in the cultures by acetylcholinesterase staining. Two growth factors may be necessary for the genesis of megakaryocytic colonies.     

靶向巨核细胞的原发性骨髓纤维化分化治疗

原发性骨髓纤维化(primary myelofibrosis,PMF)是一种由造血干细胞异常引起的骨髓增殖性疾病。20%的患者可能发展为急性巨核细胞白血病。在患者的骨髓和外周血中发现大量异常分化的巨核细胞和其前体细胞。诱导该类细胞分化能够缓解甚至治愈此疾病。巨核细胞的分化与转录因子GATA1、以及JAK2/STATs、Rho A/ROCK/MLC2、PI3K/AKT/m TOR、NF-κB等信号转导通路密切相关。本综述将讨论PMF患者的巨核细胞分化调控机制以及分化治疗研究进展。     

The enigmatic megakaryocyte gradually reveals its secrets

The recent cloning of thrombopoietin has brought many insights into the cellular and molecular mechanisms of megakaryocyte and platelet development. Thrombopoietin was cloned based on its binding to the product of the proto-oncogene c-mpl and was found to affect all aspects of thrombopoiesis. Many of the molecular pathways that mediate thrombopoietin action have been discerned. Upon hormone binding, the megakaryocyte thrombopoietin receptor homodimerizes, activating members of the JAK family of kinases, which, in turn, phosphorylate the receptor, generating docking sites for second messengers that affect multiple signalling pathways. Ultimately, cellular proliferative and anti-apoptotic mechanisms are initiated, increasing megakaryocyte numbers, as are processes that uncouple DNA synthesis from nuclear and cytoplasmic division, generating polyploid cells. As the net result of thrombopoietin action is an expansion of cells that give rise to mature platelets, the availability of the recombinant hormone has provided new opportunities to manipulate blood cell development for therapeutic benefit. BioEssays 21:353–360, 1999. © 1999 John Wiley & Sons, Inc.     

Essential thrombocythemia: impaired regulation of megakaryocyte progenitors

In this paper, the in vitro growth of bone marrow early (megakaryocyte burst-forming units, BFU-meg) and late (megakaryocyte colony-forming units, CFU-meg) progenitors was evaluated in 18 essential thrombocythemia (ET) patients and 22 normal control subjects. BFU-meg clonality was demonstrated both in normal and ET bone marrows, cultivating these primitive progenitors at limiting dilutions in plasma clot assay: 1 to 7 BFU-meg/2.5 x 10(4) mononuclear non-adherent cells were observed, with a strong correlation in ET [r = 0.955 stimulated by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) plus recombinant human interleukin (rhIL) 3], as well as in normal controls (r = 0.969). In order to clearly elucidate the in vitro response of ET megakaryocyte (meg) progenitors to recombinant growth factors, the interference of accessory cells (i.e., monocytes, T lymphocytes, and natural killer cells) and human serum were avoided by performing experiments on CD34+ cells in a serum-free fibrin clot assay. The number of both early and late meg progenitors in ET was significantly increased in response to rhIL-3, rhIL-3 plus rhIL-6, and rhIL-3 plus rhGM-CSF, but not in response to rhGM-CSF alone. Furthermore, both meg progenitors were investigated for their response to rh transfer growth factor (TGF)-beta 1, tested at concentrations from 0.01 to 10 ng/ml. rhTGF-beta 1 was able to inhibit CFU-meg and BFU-meg in a dose-response manner normal, whereas ET CFU-meg appeared less sensitive to the lower doses investigated (p less than 0.05) and ET BFU-meg were slightly reduced in number only at the higher concentrations of rhTGF-beta 1 (p less than 0.01). Our data suggest that the increased thrombopoiesis in ET may depend on an increased sensitivity of meg progenitors to some of the physiological growth factors and to a disrupted sensitivity to at least one negative regulator of megakaryocytopoiesis. Since these abnormalities involve both meg progenitors, this can be considered a demonstration that the neoplastic event hits the most primitive hemopoietic progenitors.