Megakaryocyte Colony Formation Research Articles

Interrelationship between mitosis and endomitosis in cultures of human megakaryocyte progenitor cells [published erratum appears in Blood 1987 May;69(5):1547

Sera from dogs rendered aplastic by total-body irradiation stimulate human bone marrow megakaryocyte progenitors to form megakaryocyte colonies in plasma clot cultures. In this investigation, we evaluated the effects of varying concentrations of such sera on both the mitotic and endomitotic phases of human megakaryocyte development in vitro. When low concentrations of aplastic canine sera (2.5% to 5.0% [vol/vol]) were added to cultures of human peripheral blood mononuclear cells in place of normal AB serum, megakaryocyte colony formation was augmented fivefold, cell numbers per colony increased approximately 2.5-fold, and the geometric mean megakaryocyte ploidy almost doubled. Further increasing the aplastic canine serum concentration from 10% to 30% (vol/vol) stimulated no additional colony formation. However, there was a further augmentation of cell numbers per colony associated with a progressive decrease in the mean megakaryocyte ploidy. Megakaryocyte cultures were harvested after 7, 12, 15, and 19 days of incubation, and these demonstrated that the lower mean ploidy values found at the higher concentrations of aplastic canine serum did not result from delayed endoreduplication. At all aplastic serum concentrations evaluated, there existed a strong correlation between nuclear ploidy and cell diameter. We conclude that both the mitotic and endomitotic events in human megakaryocytopoiesis may be influenced by a factor or factors present in aplastic canine serum. At lower in vitro concentrations, such sera stimulate both mitosis and endomitosis, which promotes the development of megakaryocyte colonies composed of larger cells with a higher mean ploidy. With increasing aplastic serum concentrations, colony formation plateaus and mitosis is favored over endomitosis. This results in colonies composed of more numerous but smaller megakaryocytes with a lower mean ploidy. Our data suggest that the size and extent of polyploidization that can be achieved by a developing megakaryocyte may be influenced by the mitotic prior history of its immediate precursor cell.

Interrelationship Between Mitosis and Endomitosis in Cultures of Human Megakaryocyte Progenitor Cells

Abstract Sera from dogs rendered aplastic by total-body irradiation stimulate human bone marrow megakaryocyte progenitors to form megakaryocyte colonies in plasma clot cultures. In this investigation, we evaluated the effects of varying concentrations of such sera on both the mitotic and endomitotic phases of human megakaryocyte development in vitro. When low concentrations of aplastic canine sera (2.5% to 5.0% [vol/vol]) were added to cultures of human peripheral blood mononuclear cells in place of normal AB serum, megakaryocyte colony formation was augmented fivefold, cell numbers per colony increased approximately 2.5- fold, and the geometric mean megakaryocyte ploidy almost doubled. Further increasing the aplastic canine serum concentration from 10% to 30% (vol/vol) stimulated no additional colony formation. However, there was a further augmentation of cell numbers per colony associated with a progressive decrease in the mean megakaryocyte ploidy. Megakaryocyte cultures were harvested after 7, 12, 15, and 19 days of incubation, and these demonstrated that the lower mean ploidy values found at the higher concentrations of aplastic canine serum did not result from delayed endoreduplication. At all aplastic serum concentrations evaluated, there existed a strong correlation between nuclear ploidy and cell diameter. We conclude that both the mitotic and endomitotic events in human megakaryocytopoiesis may be influenced by a factor or factors present in aplastic canine serum. At lower in vitro concentrations, such sera stimulate both mitosis and endomitosis, which promotes the development of megakaryocyte colonies composed of larger cells with a higher mean ploidy. With increasing aplastic serum concentrations, colony formation plateaus and mitosis is favored over endomitosis. This results in colonies composed of more numerous but smaller megakaryocytes with a lower mean ploidy. Our data suggest that the size and extent of polyploidization that can be achieved by a developing megakaryocyte may be influenced by the mitotic prior history of its immediate precursor cell.

Progressive preleukemia presenting amegakaryocytic thrombocytopenic purpura: Association of the 5q- syndrome with a decreased megakaryocytic colony formation and a defective production of Meg-CSF

The authors present a patient with the typical clinical picture of an acquired amegakaryocytic thrombocytopenic purpura. After 16 months of observation, the patient developed acute myelomonocytic leukemia. During the preleukemic phase and after progression to overt leukemia, serial in-vitro analyses of megakaryocytic, granulocytic, erythrocytic and T-lymphocytic colony growth were carried out in a microagar culture system. At presentation, a marked diminution of CFU-M was observed, whereas CFU-E, BFU-E, CFU-C and CFU-TL were in the normal range. The CFU-M number remained at its low level during the whole observation period. The CFU-C number declined steadily during the preleukemic period, while BFU-E, CFU-E and CFU-TL remained constant until January 1985 when the patient developed AML. After progression to overt leukemia, a distinct reduction became evident in all colony-forming cells. Cytogenetic studies performed during the preleukemic phase indicated the presence of a 5q - chromosome. The authors submit evidence here that the patient was not only characterized by defective megakaryocytic colony formation but also by a deficiency of functional megakaryocyte colony-stimulating activity. No humoral or cellular inhibitors of CFU-M colony formation were found. It is concluded that in preleukemia with a 5q - chromosome the megakaryocytic cell lineage may be involved in the process that precedes overt leukemia at an earlier time than cells of granulocytic and erythrocytic lineages. In addition, it is shown here that megakaryocytopoiesis during the preleukemic period can be characterized by two different defects: first, an intrinsic megakaryocytic stem cell defect and, second, a deficiency of functional megakaryocytic colony-stimulating activity.

Megakaryocytic colony formation (CFU-Meg) in essential thrombocythemia: quantitative and qualitative abnormalities of bone marrow CFU-Meg.

For the purpose of examining the mechanisms for the overproduction of megakaryocytes and platelets in essential thrombocythemia (ET), marrow megakaryocytic colony-forming units (CFU-Meg) were characterized using a methylcellulose culture system in nine patients with ET, and they were compared with those of 16 control subjects and four patients with secondary thrombocytosis (ST). The number of CFU-Meg per 10(5) cells in ET was significantly higher (p less than .001) than that in the controls (5.8 times of the controls) or that in ST. ET showed endogenous CFU-Meg (58 +/- 16% of the total CFU-Meg) that could form megakaryocytic colonies without phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) as a source of Meg-CSA. The controls and the patients with St revealed no endogenous CFU-Meg. The dose-response studies with PHA-LCM revealed that CFU-Meg from ET are highly sensitive to low concentrations of PHA-LCM compared to CFU-Meg from the control and ST. Erythropoietic burst-forming units (BFU-E) were monitored as a control progenitor, because the patients with ET studied did not have erythrocytosis. Although five out of nine patients with ET had endogenous BFU-E, the percentages of endogenous BFU-E were small (4-10%). The number of BFU-E in ET was not different from the control value. Thus, ET is associated with an enlarged CFU-Meg compartment that shows abnormal growth patterns when cultured in vitro, while the growth abnormality of BFU-E is small, which may underlie the hematological features of ET. The culture of CFU-Meg should be clinically useful for differentiating ET from ST.

Effect of insulin on murine megakaryocytopoiesis in a liquid culture system.

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.

Lymphokine(s) from isolated T lymphocyte subpopulations support multilineage hematopoietic colony and megakaryocytic colony formation

Conditioned medium derived from peripheral mononuclear low-density cells stimulated with phytohemagglutinin (PHA) supports the growth of noncommitted hematopoietic progenitors from marrow and peripheral blood cells. These immature progenitors (CFU-GEMM) can be identified in culture as multilineage hematopoietic colonies containing erythroblasts, eosinophilic, basophilic and neutrophilic granulocytes, megakaryocytes, macrophages, and T and B lymphocytes. In this report, we describe the effect of lymphokines released from purified T lymphocyte preparations of helper (T4) and suppressor/cytotoxic (T8) phenotype derived from peripheral blood on the growth of multilineage hematopoietic colonies and megakaryocytic colonies. It was found that PHA-stimulated lymphocytes of T4 phenotype and, to a lesser extent, of T8 phenotype elaborate lymphokine(s) that support the growth and development of multilineage colonies (CFU-GEMM), granulopoietic colonies (CFU-C), erythroid bursts (BFU-E) and megakaryocytic colonies (CFU-M) by nonadherent and T cell-depleted bone marrow cells.

Lymphokine(s) From Isolated T Lymphocyte Subpopulations Support Multilineage Hematopoietic Colony and Megakaryocytic Colony Formation

Conditioned medium derived from peripheral mononuclear low-density cells stimulated with phytohemagglutinin (PHA) supports the growth of noncommitted hematopoietic progenitors from marrow and peripheral blood cells. These immature progenitors (CFU-GEMM) can be identified in culture as multilineage hematopoietic colonies containing erythroblasts, eosinophilic, basophilic and neutrophilic granulocytes, megakaryocytes, macrophages, and T and B lymphocytes. In this report, we describe the effect of lymphokines released from purified T lymphocyte preparations of helper (T4) and suppressor/cytotoxic (T8) phenotype derived from peripheral blood on the growth of multilineage hematopoietic colonies and megakaryocytic colonies. It was found that PHA-stimulated lymphocytes of T4 phenotype and, to a lesser extent, of T8 phenotype elaborate lymphokine(s) that support the growth and development of multilineage colonies (CFU-GEMM), granulopoietic colonies (CFU-C), erythroid bursts (BFU-E) and megakaryocytic colonies (CFU-M) by nonadherent and T cell-depleted bone marrow cells.

The influence of T lymphocyte subsets and humoral factors on colony formation by human bone marrow and blood megakaryocyte progenitor cells in vitro.

Cellular and humoral influences of T lymphocytes on human megakaryocyte colony formation in vitro were assessed by using a microagar system. Megakaryocyte colony formation from nonadherent low density T lymphocyte-depleted (NALDT-) bone marrow cells was increased significantly after the addition of aplastic anemia serum (AAS) or purified megakaryocyte colony-stimulating factor (Meg-CSF). The addition of conditioned medium obtained from phytohemagglutinin-stimulated T lymphocytes replaced, at least partially, the requirement for AAS or purified Meg-CSF for the growth of megakaryocyte colonies. The cellular influence of T lymphocytes and T lymphocyte subsets on megakaryocyte colony formation was assessed by removing either T cells from nonadherent peripheral blood mononuclear cells with monoclonal OKT4, OKT8, or OKT3 antibodies plus complement, or by adding back populations of bone marrow or blood T4+ or T8+ lymphocytes, isolated by means of fluorescence-activated cell sorting, respectively, to NALDT--bone marrow or -blood cells. When sorted T cell subpopulations were added to a fixed number of NALDT--bone marrow or -peripheral blood cells in the presence of AAS or Meg-CSF, T4+ cells enhanced megakaryocyte colony formation and T8+ cells decreased it. These studies demonstrate that although the stimulation of megakaryocytic progenitor cells by Meg-CSF may not require the presence of monocytes or T lymphocytes, T4+ lymphocytes enhance and T8+ lymphocytes down-regulate megakaryocyte colony formation induced by Meg-CSF. These observations suggest that the immune system is capable of modulating the proliferative response of human megakaryocytic progenitor cells to Meg-CSF.

Megakaryocytopoiesis in vitro of patients with essential thrombocythaemia: effect of plasma and serum on megakaryocytic colony formation.

To clarify the mechanism of increased numbers of megakaryocytes in patients with essential thrombocythaemia (ET), we studied in vitro megakaryocytopoiesis in ET and other myeloproliferative disorders, using a megakaryocytic colony assay in methylcellulose containing plasma or serum and medium conditioned by phytohaemagglutinin (PHA) stimulated leucocytes (PHA-LCM). Megakaryocytic colony formation was supported well by heparinized or citrated plasma and citrated serum which was harvested after clot formation of citrated plasma. Whole serum was inhibitory for megakaryocytic colony growth. The addition of platelet releasates and partially purified platelet derived growth factor (PDGF) resulted in a decrease in the number of megakaryocytic colonies. These findings suggested that platelet-derived factor(s) in serum was inhibitory to megakaryocytic colony formation. ET plasma supported the megakaryocytic colony formation by normal or ET bone marrow cells better than normal plasma. Moreover, in ET bone marrow cells, spontaneous megakaryocytic colonies were formed in the absence of PHA-LCM. Increased megakaryocytopoiesis in ET may be ascribed to (i) increased megakaryocyte-colony stimulating activity (Meg-CSA) in plasma and (ii) increased sensitivity to Meg-CSA or autonomous proliferation of megakaryocytic progenitor cells.

Cell-Mediated Suppression of Megakaryocytopoiesis in Acquired Amegakaryocytic Thrombocytopenic Purpura

Acquired amegakaryocytic thrombocytopenic purpura (AATP) is a disorder of hematopoiesis characterized by severe thrombocytopenia due to a selective reduction or total absence of megakaryocytes in an otherwise normal-appearing bone marrow. Although the development of autoantibodies directed against cells in the megakaryocyte progenitor cell pool has been implicated in the pathogenesis of this disorder, cell-mediated suppression of megakaryocytopoiesis has not been described. Accordingly, we report two cases of AATP in which in vitro suppression of megakaryocyte colony formation by autologous ancillary marrow cells was demonstrable. Light-density bone marrow mononuclear cells (MNCs) obtained from both patients were either plated directly into plasma clot cultures, or after first being depleted by adherent monocytes (M phi) or T lymphocytes using standard methodologies. In some experiments, the depleted ancillary marrow cells were recovered for autologous co-culture studies with the MNCs from which they had been depleted. Megakaryocyte colony formation was detected in the cultures using an indirect immunofluorescence assay with a rabbit anti-human platelet glycoprotein antiserum. Removal of M phi (n = 6), or T lymphocytes (n = 4) from normal marrow MNCs had no apparent effect on colony formation. In contrast, depleting T lymphocytes from the MNCs of patient 1 significantly augmented megakaryocyte colony formation; a similar effect was observed after depleting M phi from the MNCs of patient 2. This observed augmentation in colony formation could be abrogated by autologous co-culture with the putative suppressor cell at effector cell/target cell ratios of 1:10 in the case of T lymphocytes or 1:5 in the case of M phi. Neither suppression nor stimulation of megakaryocyte colony formation was observed after culturing normal MNCs with autologous T cells (n = 4) or M phi (n = 3) at similar or greater ratios. We also observed inhibition of megakaryocyte colony formation after culturing normal MNCs in the presence of tissue culture medium conditioned by the M phi of patient 2. This effect was shown to be specific for megakaryocytes since this same conditioned medium had no significant effect on BFU-E and CFU-E-derived colony formation by autologous marrow mononuclear cells. These results suggest that: both T cells and M phi are capable of exerting a regulatory effect on the proliferation of human megakaryocyte progenitor cells (CFU-Meg); in the case of M phi, a soluble factor elaborated by these cells may be responsible for suppressing CFU-Meg growth; and aberrant ancillary cell-megakaryocyte progenitor cell interactions may lead to clinically significant disease.

Cell-mediated suppression of megakaryocytopoiesis in acquired amegakaryocytic thrombocytopenic purpura

Acquired amegakaryocytic thrombocytopenic purpura (AATP) is a disorder of hematopoiesis characterized by severe thrombocytopenia due to a selective reduction or total absence of megakaryocytes in an otherwise normal-appearing bone marrow. Although the development of autoantibodies directed against cells in the megakaryocyte progenitor cell pool has been implicated in the pathogenesis of this disorder, cell-mediated suppression of megakaryocytopoiesis has not been described. Accordingly, we report two cases of AATP in which in vitro suppression of megakaryocyte colony formation by autologous ancillary marrow cells was demonstrable. Light-density bone marrow mononuclear cells (MNCs) obtained from both patients were either plated directly into plasma clot cultures, or after first being depleted by adherent monocytes (M phi) or T lymphocytes using standard methodologies. In some experiments, the depleted ancillary marrow cells were recovered for autologous co-culture studies with the MNCs from which they had been depleted. Megakaryocyte colony formation was detected in the cultures using an indirect immunofluorescence assay with a rabbit anti- human platelet glycoprotein antiserum. Removal of M phi (n = 6), or T lymphocytes (n = 4) from normal marrow MNCs had no apparent effect on colony formation. In contrast, depleting T lymphocytes from the MNCs of patient 1 significantly augmented megakaryocyte colony formation; a similar effect was observed after depleting M phi from the MNCs of patient 2. This observed augmentation in colony formation could be abrogated by autologous co-culture with the putative suppressor cell at effector cell/target cell ratios of 1:10 in the case of T lymphocytes or 1:5 in the case of M phi. Neither suppression nor stimulation of megakaryocyte colony formation was observed after culturing normal MNCs with autologous T cells (n = 4) or M phi (n = 3) at similar or greater ratios. We also observed inhibition of megakaryocyte colony formation after culturing normal MNCs in the presence of tissue culture medium conditioned by the M phi of patient 2. This effect was shown to be specific for megakaryocytes since this same conditioned medium had no significant effect on BFU-E and CFU-E-derived colony formation by autologous marrow mononuclear cells. These results suggest that: both T cells and M phi are capable of exerting a regulatory effect on the proliferation of human megakaryocyte progenitor cells (CFU-Meg); in the case of M phi, a soluble factor elaborated by these cells may be responsible for suppressing CFU-Meg growth; and aberrant ancillary cell- megakaryocyte progenitor cell interactions may lead to clinically significant disease.

In vitro actions on hemopoietic cells of recombinant murine GM‐CSF purified after production in Escherichia coli: Comparison with purified native GM‐CSF

Recombinant murine GM-CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM-CSF. Both recombinant and native GM-CSF stimulated granulocyte and/or macrophage colony formation by adult and fetal mouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM-CSF (25 X 10(8) U/mg) was similar to that of the native form (15 X 10(8) U/mg). At high concentrations (greater than 200 U/ml), both forms of GM-CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (greater than 800 U/ml), megakaryocyte and some erythroid and mixed-erythroid colony formation. Recombinant GM-CSF was as effective in stimulating the proliferation of the GM-CSF-dependent cell line FD as the native molecule. Both recombinant and native GM-CSF were able to induce partial differentiation in colonies of WEHI-3B myeloid leukemic cells. Recombinant GM-CSF competed effectively for the binding of 125I-labeled native GM-CSF to hemopoietic cells, and antiserum to recombinant GM-CSF also neutralized the biological activity of native GM-CSF. The bacterially synthesized GM-CSF was a slightly more effective stimulus for megakaryocyte colony formation than the native molecule. The demonstration that purified bacterially synthesized GM-CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.

Megakaryocyte colony formation by bone marrow progenitors in myelodysplastic syndromes.

Megakaryocytic colony formation by precursor cells from the bone marrow was investigated in 10 patients with a myelodysplastic syndrome. All but one exhibited abnormal colony formation: four showed no colony formation at all, while a decreased number of colonies was noticed in five. All of the patients showed defective colony formation by erythroid progenitors, but only four showed clearly abnormal granulocyte-macrophage colony formation. The defect in megakaryocytic progenitors seems to be more akin to the defects occurring in erythroid progenitors than to the defects seen in the granulocyte-macrophage lineage.

Studies of human megakaryocytopoiesis using an anti-megakaryocyte colony-stimulating factor antiserum.

We produced an antiserum by immunizing rabbits with purified human megakaryocyte colony stimulating factor (Meg-CSF). With the use of an anti-Meg-CSF IgG fraction (AM-IgG), we detected immunoreactive Meg-CSF both in human aplastic anemia serum (AAS) and normal serum. Based on our immunological and biological analyses, Meg-CSF appeared to be antigenically as well as functionally distinct from human urinary erythropoietin (EPO) and thrombopoietic stimulating factor. The AM-IgG fraction was able to suppress the ability of both aplastic anemia serum and purified Meg-CSF to promote megakaryocyte colony formation. In addition, the supernatant formed after immune precipitation of the AAS with AM-IgG no longer possessed Meg-CSF-like activity. The AM-IgG did not suppress the ability of EPO, phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LCM), or PHA-LCM + EPO to promote erythroid, granulocyte-macrophage, or mixed colony formation, respectively. The use of this antibody has further defined the dependency of human megakaryocytopoiesis on Meg-CSF.

Effect of ethanol on thrombopoiesis.

Chronic ethanol abuse causes thrombocytopenia but the underlying mechanism is unknown. To determine the target cells involved, we examined the effects of the drug in vitro on both megakaryocyte progenitor cells (CFU-Meg) and isolated, maturing megakaryocytes. In the presence of ethanol concentrations of 0.05-2.0 g/dl, megakaryocyte colony formation by mouse CFU-Meg in soft agar was normal. At 5 g/dl ethanol, colony formation was reduced by 50%; with 7 g/dl ethanol, no megakaryocyte colonies were formed. Acetaldehyde did not inhibit colony formation unless very high concentrations (100 mg/dl) were employed. Isolated guinea-pig megakaryocytes can maintain their viability and incorporate 3H-leucine into TCA-precipitable protein for at least 24 h. Incubation of these maturing megakaryocytes with ethanol did not affect their viability, but at concentrations greater than 120 mg/dl ethanol progressively inhibited protein synthesis. At 0.5 g/dl ethanol, protein synthesis was decreased by 23% while viability was still 93% of control. Like CFU-Meg, maturing megakaryocytes were resistant to the toxic effects of acetaldehyde. To determine the in vivo correlates of these results, guinea-pigs were fed 5 g/dl ethanol in a liquid diet. By 11 d, when blood ethanol levels were 20-150 mg/dl, platelet counts in the animals were reduced by 17-29%, while the number of marrow megakaryocytes was unaltered. These data indicate that the site of action of ethanol in suppressing thrombopoiesis is at the level of the maturing megakaryocyte.

Characterization of human megakaryocytic colony formation in human plasma.

We have analysed the contribution to megakaryocyte colony formation in methylcellulose made by human plasma, serum, media conditioned by phytohemagglutinin (PHA) stimulated leukocytes (PHA-LCM), erythropoietin (EPO) preparations, and platelets. The culture system was used as a bioassay for megakaryocyte colony stimulating activity (Meg-CSA) in plasma samples of patients with perturbed megakaryocytopoiesis. Preparations of heparinized platelet-poor plasma yielded the most consistent results. Platelet-poor plasma of normal subjects will at best facilitate the occasional growth of small megakaryocyte colonies. Colony frequency and size are reproducibly enhanced in the presence of PHA-LCM as a source of exogenous Meg-CSA. Commercially available EPO preparations may vary in their content of activities that influence megakaryocyte colony formation. Addition of these preparations to cultures that contain plasma and PHA-LCM usually does not enhance colony formation. In contrast to platelet-poor plasma, platelet rich plasma and serum are less supportive of megakaryocyte colony growth. It is suggested that this loss of activity may be related to the release of inhibitors by activated platelets or alternatively caused by absorption of activities by platelets. Plasma samples from patients with megakaryocytopoietic dysfunction may contain components that promote colony formation without addition of PHA-LCM or EPO. This phenomenon is consistently observed for patients with severe aplastic anemia and bone marrow transplant recipients after completion of their ablative preparative regimen.

Purification and partial characterization of a megakaryocyte colony-stimulating factor from human plasma.

Human plasma obtained from patients with hypomegakaryocytic thrombocytopenia contains a factor that promotes megakaryocyte colony formation by normal human marrow cells. This megakaryocyte colony-stimulating factor was purified from such a plasma specimen. A four-step purification scheme which included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, affinity chromatography on wheat germ lectin-Sepharose 6MB, and reverse-phase high performance liquid chromatography resulted in a recovery of 16.6% of the initial biological activity and an increase in specific activity by 3,489-fold. The purified protein produced a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified megakaryocyte colony-stimulating factor was capable of promoting megakaryocyte colony formation at a concentration of 7.6 X 10(-8) M. Megakaryocyte colony-stimulating factor was shown to be a glycoprotein and had an apparent 46,000 mol wt. Deglycosylation of megakaryocyte colony-stimulating factor by treatment with trifluoromethane-sulfonate resulted in the loss of its ability to promote megakaryocyte colony formation. Megakaryocyte colony-stimulating factor appears to be an important regulator of in vitro human megakaryocytopoiesis at the level of the colony-forming unit megakaryocyte and may be of importance physiologically.

The Effect of Interleukin 3 and GM-CSA-2 on Megakaryocyte and Myeloid Clonal Colony Formation

Abstract Two separate helper T cell-derived lymphokines, interleukin 3 and granulocyte-macrophage colony-stimulating activity-2, were found to stimulate a broad and similar range of hemopoietic colonies in in vitro soft agar cultures including granulocyte, macrophage, granulocyte- macrophage, megakaryocyte, and mixed megakaryocyte colonies. Both lymphokines were potent stimulators of in vitro megakaryocyte colony formation. At plateau levels of IL-3, megakaryocyte colony formation was increased by biologic activity in pokeweed mitogen spleen- conditioned media.

The effect of interleukin 3 and GM-CSA-2 on megakaryocyte and myeloid clonal colony formation

Two separate helper T cell-derived lymphokines, interleukin 3 and granulocyte-macrophage colony-stimulating activity-2, were found to stimulate a broad and similar range of hemopoietic colonies in in vitro soft agar cultures including granulocyte, macrophage, granulocyte-macrophage, megakaryocyte, and mixed megakaryocyte colonies. Both lymphokines were potent stimulators of in vitro megakaryocyte colony formation. At plateau levels of IL-3, megakaryocyte colony formation was increased by biologic activity in pokeweed mitogen spleen-conditioned media.

Effects of a murine mammary tumor on in vivo and in vitro hemopoiesis.

The effects of an autologous transplanted mammary tumor (RIII-T3) on hemopoiesis in RIII mice are described. Tumor-bearing animals died 30 to 40 days after inoculation and displayed splenomegaly, extreme neutrophilia, and moderately increased monocyte levels in the spleen, peripheral blood, and bone marrow. The precursors of neutrophils and monocytes, granulocyte/macrophage colony-forming cells (GM-CFC) were elevated in the spleen, bone marrow, and peripheral blood. RIII-T3-conditioned medium stimulated bone marrow GM-CFC and caused the myelomonocytic cell line, WEHI-3B, to differentiate in vitro. The conditioned medium did not stimulate erythroid, megakaryocyte, or eosinophil colony formation. When conditioned medium was fractionated, two peaks of activity corresponding to GM-CSF and G-CSF were observed, suggesting that the extreme neutrophilia observed in tumor-bearing animals may result from chronic exposure of the hemopoietic system to these hemopoietic hormones.