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1.
Disparate effects of interleukin 11 and thrombopoietin on megakaryocytopoiesis in vitro 总被引:6,自引:0,他引:6
The effects of interleukin-11 (IL-11) and thrombopoietin (TPO) on murine megakaryocytopoiesis were studied using a serum-free culture system. Acting alone, both IL-11 and TPO increased the number of acetylcholinesterase (AchE)(+)cells (megakaryocytes), the latter being more potent than the former. TPO, but not IL-11, increased the mean AchE activity per megakaryocyte (AchE activity/megakaryocyte). TPO increased both the number of megakaryocytes with high ploidy, and of those with low ploidy. In contrast, IL-11 increased only the number of megakaryocytes with high ploidy. The effect of TPO on megakaryocyte ploidy was stronger than that of IL-11. Both IL-11 and TPO increased the proportion of large megakaryocytes, but the latter was more potent than the former. While the stimulatory effects of IL-11 and TPO on the number of megakaryocytes were enhanced by IL-3 or stem cell factor (SCF), synergism of IL-11 or TPO with IL-3 or SCF in stimulating AchE activity/megakaryocyte was inconsistent. IL-11 and TPO stimulated the formation of colony-forming units of megakaryocyte in the presence of IL-3, but not alone, with similar maximum colony numbers for both cytokines. Our findings thus demonstrate that IL-11 principally stimulates megakaryocyte maturation rather than the proliferation of megakaryocytes, whereas TPO stimulates both. 相似文献
2.
To examine the influence of insulin on megakaryocytopoiesis, we tested its effect on murine bone marrow cultures in a liquid culture system. In the presence of pokeweed mitogen-stimulated spleen cell conditioned medium in culture, insulin markedly enhanced megakaryocyte colony formation and increased the number and size of free megakaryocytes seen after 7 days. Many of the cells in cultures with insulin, however, were classified as immature, since they had a basophilic cytoplasm, a low cytoplasmic/nuclear ratio and low acetylcholinesterase activity. It is suggested that insulin potentiates murine marrow megakaryocytopoiesis in vitro, but that this is not accompanied by differentiation of the cells from the immature to mature state. 相似文献
3.
Kinetic analysis of megakaryocyte numbers and ploidy levels in developing colonies from mouse bone marrow cells 总被引:8,自引:0,他引:8
Abstract. The kinetics of megakaryocyte formation from mouse bone marrow cells in semi-solid medium was studied directly in the culture dish by staining the cells for acetylcholinesterase after drying the cultures. A WEHI-3 cell-conditioned medium (WEHI-3 CM) was used as a general source of stimulus for megakaryocyte colony formation. The addition of peritoneal exudate supernatant as well as WEHI-3 CM increased the frequency of megakaryocyte colonies detected. Colonies containing acetylcholinesterase-positive cells were first detected at day 3. Maximum numbers of 25–40 megakaryocyte colonies per 105 nucleaet mouse bone marrow cells were observed from days 7 to 11. The mean number of cells within each colony increased progressively with time of culture, and a modal range of 11–20 cells was obtained by day 7. Between 3 and 200 cells per colony were generally detected. A continuous distribution of the number of megakaryocytes per colony suggests that the clonable precursor cells are not synchronized either with respect to maturation stage or with respect to their capability to undergo nuclear endoreduplication. The addition of peritoneal exudate supernatant to the cell cultures increased the DNA levels of megakaryocytes grown in the presence of WEHI-3 CM but did not affect the number of cells per colony. The DNA content of colony megakaryocytes was measured after staining the cells with Feulgen reagent. A modal DNA value of 8 N was observed between days 4 and 7 for megakaryocytes stimulated with WEHI-3 CM. In the presence of both WEHI-3 CM and peritoneal exudate supernatant, the DNA content of megakaryocytes increased with the time of cell culture. Modal DNA values increased from 8 N at days 4 and 5, to 16 N by day 6. In these optimally stimulated cultures, 44% of colony megakaryocytes were 32 N or greater, a proportion higher than in normal bone marrow, but similar to that seen in the marrow of acutely thrombocytopenic animals. It is concluded that megakaryocytopoiesis in cell cultures is not a strictly controlled process with respect to cell division and endomitosis and that when certain culture conditions are employed, megakaryocyte development in vitro might reflect that seen in a stressed animal condition. 相似文献
4.
An assay describing conditions for the maturation of single immature megakaryocytes in vitro is reported. Enriched populations of small, relatively immature megakaryocytes have been found to develop into single, mature megakaryocytes by 60 hours in semisolid agar cultures. Continued incubation of these cells did not lead to the formation of colonies within 5–7 days. Maturation was indicated by increasing cell size and cytoplasmic and acetylcholinesterase content. Factors stimulating the development of immature megakaryocytes were found in preparations of human embryonic kidney cell-conditioned media (a source of in vivo Thrombopoietic Stimulatory Factor), peritoneal exudate cell-conditioned medium, lung-conditioned medium, or bone marrow cellular sources of activity (adherent cells or cells that sediment at 5–6 mm hr-1). Immature megakaryocytes cultured serum free responded to sources of an auxiliary megakaryocyte potentiating activity by developing into single, large megakaryocytes but did not respond to a megakaryocyte colony-stimulating factor devoid of detectable potentiator activity present in WEHl-3-conditioned medium. In contrast, serum-free proliferation of the megakaryocyte progenitor cell required both megakaryocyte colony-stimulating factor and the auxiliary potentiator activity. In the presence of megakaryocyte colony-stimulating factor alone, progenitor cells did not form colonies of easily detectable megakaryocytes. However, groups of cells comprised entirely of small acetylcholinesterase containing immature megakaryocytes were observed, thus establishing that megakaryocyte colony development passes through a stage of immature cells prior to detectable megakaryocyte development and that some acetylcholinesterase-containing cells can undergo cellular division. 相似文献
5.
Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells 总被引:22,自引:0,他引:22
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. 相似文献
6.
Separation of murine megakaryocytes and their progenitors on continuous gradients of Percoll 总被引:2,自引:0,他引:2
Murine bone marrow was separated on continuous Percoll density gradients to analyze the distribution of cells of the megakaryocyte lineage. Eighty-seven percent of the recovered megakaryocytes were found in fractions of density less than 1.058 g/cm3, with 63% of these cells found between 1.020 and 1.036 g/cm3. When megakaryocytes were classified according to size, 92% of the large (greater than or equal to 18 micron) acetylcholinesterase (AchE) positive cells were found in the least dense fractions (1.016-1.039 g/cm3), whereas 86% of the small (less than or equal to 10.6 micron) AchE positive cells were found in fractions of higher density (1.039-1.078 g/cm3). The distribution of enzymatic AchE activity of the separated fractions corresponded to the location of the histochemically positive cells. When ploidy measurements were made of various fractions, most of the high ploidy (32N and 64N) cells were found at low density (1.028-1.036 g/cm3), whereas no cells greater than 4N were found at density greater than 1.071 g/cm3. Thus, large AchE positive cells and the cells of highest ploidy were found at lower densities of Percoll, while small AchE positive cells and cells of low ploidy were found at higher densities. An exception to this inverse relationship was found in fractions of lowest density (less than 1.030 g/cm3) where an anomalous distribution of size and ploidy was found. The majority of megakaryocytic colony-forming cells (CFU-MK) were found at high density, as were the granulocyte-macrophage colony-forming cells (CFU-GM; approximately 1.074 g/cm3). The density distribution of the incorporation of tritiated thymidine into liquid marrow cultures was concordant with the high density distribution of colony-forming cells. The data show that megakaryocytic maturity and Percoll density varies inversely and that fractionation of marrow on continuous Percoll gradients may be a useful method for the separation and/or enrichment of megakaryocytes at different stages of differentiation. 相似文献
7.
P A de Alarcon 《Blood cells》1989,15(1):173-185
Megakaryocyte colony-stimulating activity (Mk-CSA) is required for in vitro megakaryocyte colony formation. Its in vivo significance in megakaryocytopoiesis is unknown. We studied 12 patients undergoing bone marrow transplantation (BMT) at our institution. The bone marrow megakaryocyte progenitor cells (CFU-Mk), the serum level of Mk-CSA, and the platelet count on the 28th day after BMT were studied. Patients with elevated Mk-CSA levels had less CFU-Mk in their bone marrow than did patients with a normal or decreased Mk-CSA (p less than 0.01). Animal experiments using murine models have documented that several purified molecules including erythropoietin, multi-CSF and GM-CSF possess Mk-CSA. The in vitro Mk-CSF of WEHI-3-conditioned medium is multi-CSF. The in vivo significance for megakaryocytopoiesis of these factors is not clear. In the human system, Mk-CSA is increased in conditions with decreased bone marrow megakaryocytes. Recombinant human or primate CSFs have in vitro Mk-CSA utilizing both human and murine cells as targets. However, the presence of these activities does not fully explain the Mk-CSA in human serum rich in Mk-CSA. The precise regulation of human blood cell levels and the studies discussed suggest that there is a specific Mk-CSF that responds to in vivo changes in megakaryocyte numbers. Proof of its physiologic role awaits the isolation of a pure factor. 相似文献
8.
The in vitro cloning technique for detecting megakaryocyte precursor cells was employed to compare stimuli known to influence megakaryocytopoiesis. Preparations of thrombopoietic stimulating factor (TSF) did not directly stimulate the growth of megakaryocyte colonies (CFU-m) but increased the frequency of CFU-m when TSF was added to the cultures with a constant amount of megakaryocyte colony stimulating factor. Platelets or platelet homogenates did not influence the frequency of CFU-m or the size of individual colonies. Analysis of cell surface properties of megakaryocytes obtained either by isolation from bone marrow or from in vitro colonies revealed species differences. The possibility that megakaryocytopoiesis and platelet release are regulated both within the marrow as well as by humoral factors is discussed. 相似文献
9.
S A Burstein 《Blood cells》1986,11(3):469-484
A fluorescence assay for the quantitation of acetylcholinesterase (AchE) has been adapted for measurement of megakaryocytic maturation in short-term serum-free cultures of murine marrow. When marrow cells were cultured for 3 days in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) under serum-free conditions, AchE production was found to be related to the concentration of PWM-SCM. Interleukin 3 (IL3), a purified glycoprotein promoting the proliferation of several early hematopoietic progenitors including megakaryocytic colony-forming cells, also induced AchE production in a dose-responsive manner. The response to IL3 was linearly related to the number of cells cultured. When marrow was first subjected to plastic adherence and the nonadherent cells then separated on Percoll gradients, a small megakaryocyte-enriched population markedly depleted of colony-forming cells and large megakaryocytes, responded to IL3 in a similar dose-responsive manner. A significant amount of AchE was produced in the absence of any added factors. The data show that AchE production can be measured in 3-day serum-free cultures, and suggest that IL3, a factor promoting megakaryocytic proliferation in vitro, also promotes maturation. 相似文献
10.
Erythropoietin and megakaryocytopoiesis 总被引:1,自引:0,他引:1
To determine if erythropoietin influences megakaryocytopoiesis, the purified recombinant human hormone (rEpo) was added to serum-free liquid cultures of murine marrow. A dose-related increment in acetylcholinesterase (AchE) production was observed. To assess if increments in this relatively megakaryocyte-specific enzyme marker were mediated by a direct hormone-megakaryocyte interaction rather than via an accessory cell population, rEpo was added to cultures of isolated single megakaryocytes. A significant, dose-related increase in cell size was noted in the presence of the hormone, accompanied by a high probability of an increase in cellular DNA content. The data show that rEpo does directly influence some aspects of megakaryocytic maturation, although the physiologic significance of this effect remains unknown. 相似文献
11.
M Podolak-Dawidziak 《Folia haematologica (Leipzig, Germany : 1928)》1990,117(2):237-250
Regulation of megakaryocyte and platelet production remains poorly understood. In culture system two separate activities are needed for maximum production of megakaryocyte progenitors: promotor of clonal expansion and promoter of maturation, other growth factors and cells also contribute to regulation of megakaryocytopoiesis. Increased proliferation of megakaryocytes is observed in myeloproliferative disorders and idiopathic thrombocytopenic purpura, and decreased proliferation is found in aplastic anaemia and hypomegakaryocytic thrombocytopenia. Dysmegakaryocytopoiesis is present in myelodysplastic syndromes and acute leukaemia, and a proliferation of immature megakaryocytes in acute megakaryoblastic leukaemia. Increased understanding of human megakaryocytopoiesis is beginning to help in rational clinical management. 相似文献
12.
Multiple levels of regulation of megakaryocytopoiesis 总被引:3,自引:0,他引:3
A working hypothesis for the regulation of megakaryocytopoiesis is described on the basis of current data. The hypothesis proposes that in vivo megakaryocytes are generated by 1) the expansion of clonable progenitor cells into immature megakaryocytes by locally produced (and regulated) interleukin-3 (IL-3) and 2) the development and maturation of immature megakaryocytes by a dual system; by a lineage specific mechanism involving thrombopoietic stimuli in the steady state and thrombocytopenic conditions, and by a lineage nonspecific mechanism via IL-3 in damaged or reconstituting marrow. The hypothesis predicts that if IL-3 is a significant in vivo regulator of megakaryocyte formation and development, receptor for IL-3 should be present on megakaryocytes and may be vestigially on platelets. Small but significant levels of 125I IL-3 were found to bind to platelets from normal mice. The level of binding on platelets was found to be enhanced sevenfold from mice that had received high levels of irradiation followed by bone marrow transplantation. This contrasted with a twofold increase in the level of binding to platelets from mice made acutely thrombocytopenic with antiplatelet serum. The data suggest that IL-3 may be involved in the in vivo regulation of murine megakaryocytopoiesis and may be a significant factor in rebound thrombopoiesis following bone marrow damage. 相似文献
13.
M Podolak-Dawidziak 《Folia haematologica (Leipzig, Germany : 1928)》1990,117(1):179-183
The ability of plasma from ITP patients (before and after splenectomy) to support the growth of megakaryocyte progenitors was compared with that from healthy subjects. Plasma Factor Index-Megakaryocyte PFI-Mk (ITP) which expressed resultant colony growth was significantly lower before splenectomy, but it normalized after splenectomy. (PFI-Mk) (ITP) did not relate neither to megakaryocyte nor to platelet counts. A positive correlation has been observed between megakaryocyte and platelet numbers in healthy subjects and in ITP patients after splenectomy, but not before splenectomy. The proportion of immature megakaryocytes was markedly higher in ITP marrow before splenectomy. This study indicates, that in ITP apart from antibodies directed to platelets and megakarocytes a low plasma stimulatory activity affected megakaryocytopoiesis. 相似文献
14.
《Cell cycle (Georgetown, Tex.)》2013,12(7):1342-1353
It has been demonstrated that stromal cell precursors exist in human umbilical cord blood. After being cultured in vitro, these cells are called human umbilical cord blood-derived stromal cells (hUCBDSCs). However, the role of hUCBDSCs in hematopoiesis is still unclear. We have previously shown that hUCBDSCs are superior to human bone marrow stromal cells (hBMSCs) at enhancing the expansion of megakaryocyte colony forming units (CFU-Meg). Based on this observation, we postulated that hUCBDSCs might promote megakaryocytopoiesis. To test this hypothesis, we developed a megakaryocyte/hUCBDSC co-culture model and a hematopoietic microenvironment injury model in nude mice. We explored the ability and mechanisms by which hUCBDSCs promoted the proliferation of megakaryocytes in vitro, and we also explored their capacity to restore the hematopoietic microenvironment in vivo. As expected, hUCBDSCs were more effective than hBMSCs at enhancing the proliferation of megakaryocyte lines from HEL cells and restoring megakaryocytopoiesis in a hematopoietic microenvironment injury model in nude mice. Thrombopoietin (TPO) and stromal cell derived factor-1 (SDF-1) are two of the key factors underlying this capacity. We also found that gap junction intercellular communication (GJIC) between HEL cells and hUCBDSCs might be partially absent. Our data provide the first evidence that hUCBDSCs play a regulatory role during megakaryocytopoiesis, which might be important for designing treatments for patients with megakaryocytic injury. 相似文献
15.
本文采用液体培养体系结合酶细胞化学方法,对体外培养不同发育阶段的小鼠肾髓巨核细胞乳酸脱氢酶、苹果酸脱氢酶、谷氨酸脱氢酶的活性变化进行了动态观察。在9天培养期间,巨核细胞的增殖数在5—7天达到高峰,并随时间有不同程度分化。对培养3、5、7、9天的巨核细胞进行酶细胞化学研究,结果表明,巨核细胞在发育成熟前,三种酶活性均有增高。提示巨核细胞在分化过程中,糖酵解及三羧酸循环代谢均有增强。 相似文献
16.
《Cell biology international reports》1977,1(5):445-452
In the rat bone marrow, cells of the megakaryocyte line of differentiation are the only hemopoietic cells which show cytochemically demonstrable Acetylcholineesterase activity (AchE; EC 3.1.1.7).A small number of morphologically unrecognizable cells exhibit AchE activity in their Golgi complexes. These cells are identified as committed precursor cells of the MK compartment. 相似文献
17.
S Ebbe 《Radiation research》1991,127(3):278-284
Megakaryocytic macrocytosis was evaluated in mice after irradiation with 6.5 Gy 60Co gamma rays. During the second and third months after sublethal irradiation, one or more of the following abnormalities of thrombocytopoiesis was present: thrombocytopenia, megakaryocytopenia, macromegakaryocytosis, a shift to higher ploidies, and enlargement of cells within ploidy groups. After transfusion-induced thrombocytosis, reductions in megakaryocyte size were delayed or absent relative to non-irradiated mice, and there was more of a tendency to shift to lower values for megakaryocyte ploidy. Mice with radiation-induced megakaryocytopenia failed to show rebound thrombocytosis during recovery from immunothrombocytopenia, in spite of further increases in megakaryocyte size and ploidy. The findings support the hypotheses that numbers of megakaryocytes may influence the regulation of megakaryocytopoiesis even when there is an excess of platelets and that ploidy distribution is not the sole determinant of the average size of a population of megakaryocytes. After irradiation, persistent megakaryocytopenia may not severely affect platelet production under steady-state conditions, but the ability of the marrow to respond to homeostatic regulation is compromised. 相似文献
18.
Kumi Sumiyoshi Satoshi Kubota Rika A. Furuta Kazuta Yasui Eriko Aoyama Harumi Kawaki Kazumi Kawata Toshihiro Ohgawara Takashi Yamashiro Masaharu Takigawa 《Journal of cell communication and signaling》2010,4(1):5-14
CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage
in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration
process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2
in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells
nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression
or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated
around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets.
To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte
cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly
enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor
stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such
thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts
endochondral ossification. 相似文献
19.
In vivo inhibition of megakaryocyte and platelet production by platelet factor 4 in mice. 总被引:1,自引:0,他引:1
Z C Han S Bellucci E Bodevin H Y Wan Z X Shen J P Caen 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1991,313(12):553-558
The in vivo effect of human platelet factor 4 (PF4) on murine megakaryocytopoiesis and thrombopoiesis was studied. Administration of PF4 induced a dose-dependent decrease in the numbers of megakaryocytes and their progenitor cells (CFU-MK), continuing for 1 week after the injection. However, the size of megakaryocytes and their colonies was not changed following PF4 injection. Platelet levels were significantly decreased at days 3-4. The number of CFU-GM was decreased at days 1-2. White blood cells and hemoglobin were unaffected by PF4. These data indicate that PF4 inhibits megakaryocyte and platelet production in vivo by acting on the early stage of megakaryocyte development. 相似文献
20.
造血干细胞分化生成巨核细胞是一个十分复杂的过程,包括造血干细胞动员及其向巨核系祖细胞分化,巨核系祖细胞增殖、分化生成未成熟巨核细胞,巨核细胞的成熟和血小板释放等过程。研究发现,造血干细胞动员及其向各系细胞分化的大部分过程都在一种称为"龛"的结构中进行,多种龛内信号分子参与了造血干细胞的动员和分化调控。该文对造血干细胞龛内参与造血干细胞动员和分化生成巨核细胞的几种重要细胞因子及其调控作用进行综述。 相似文献