Human Megakaryocyte Colony Research Articles

Effect of human recombinant erythropoietin on human marrow megakaryocyte colony formation in vitro.

The effect of human recombinant erythropoietin (EP) on the growth of human marrow megakaryocyte colony forming units (CFU-M) in vitro was investigated by the use of a plasma clot assay. EP as a single stimulating factor or as an additional factor to optimal concentration of leucocyte conditioned medium (PHA-LCM) had no effect on the number of CFU-M derived colonies. However, addition of EP (0.5-1 U/ml) to cultures with suboptimal concentrations of PHA-LCM increased megakaryocytic colony formation by 50-90% but had no effect on the number of granulocytic-monocytic colonies (CFU-GM). Exposure of marrow cells to EP for 24-48 h in liquid suspension cultures, followed by removal of the hormone and assaying the cells for CFU-M in plasma clots, resulted in a 50-100% increase of megakaryocyte colony formation in vitro. The augmenting effect of EP on CFU-M growth in vitro was abolished when EP was added to the medium after the third day of culture. The presence of factors in human serum and in PHA-LCM was an absolute requirement for the hormone to exert its potentiating effect on human CFU-M growth in vitro. Recombinant EP potentiates the growth of human marrow CFU-M and this effect seems to be exerted during the early stages of CFU-M development in vitro.

Suppression of maturation of megakaryocyte colony forming unit in vitro by a platelet-released glycoprotein.

The suppressive role of platelets on the growth of human marrow megakaryocyte colony forming units (CFU-M) in vitro was investigated by the use of a plasma clot assay. An inverse correlation was established between the number of megakaryocytic colonies grown and the platelet concentration of the plasma or the resultant serum used in the culture system. The suppressive effect of platelets on megakaryocyte colony formation reached a plateau at normal human blood platelet concentration and was specific for CFU-M growth, since marrow cell erythroid burst formation (BFU-E) and granulocytic-monocytic colony formation (CFU-GM) remained unaffected. The inhibitory activity was detectable in the supernatants of platelet suspensions aggregated by thrombin or ADP, and the inhibitory activity released from ADP-stimulated platelets was blocked by pretreatment of platelets with monoclonal antibody HuPl-m1. Partial purification of this activity was achieved by diethylaminoethyl (DEAE)-ion exchange and phytohemagglutinin (PHA)-E agarose affinity chromatography. This inhibitor is a glycoprotein with a molecular weight of 12-17K daltons. This platelet released glycoprotein does not affect the early proliferative phase of CFU-M in vitro but acts on a day 6-8 CFU-M-derived cell by adversely affecting its maturation into recognizable megakaryocytes. These findings demonstrate that a glycoprotein released from platelets suppresses the maturation of CFU-M into megakaryocytes.

Spermine and spermidine are non-specific inhibitors of in vitro hematopoiesis

The polyamine spermine has been reported to be the inhibitor of in vitro erythropoiesis present in uremic serum. We have employed a panel of hematopoietic colony-forming assays to evaluate the specificity of the inhibitory activity. Spermine and its precursor spermidine when added to culture inhibited mouse and human erythroid (CFU-E and BFU-E), granulocyte-macrophage, and megakaryocyte colony growth in a non-specific, dose-dependent fashion. Erythroid and non-erythroid colony growth were equally sensitive to spermine- and spermidine-induced inhibition. Increasing concentrations in culture of erythropoietin and mitogen-stimulated leukocyte-conditioned medium, a source of colony-stimulating activity, failed to overcome the in vitro inhibition. Although anemia is characteristic of chronic renal failure (CRF), leukopenia and thrombocytopenia are not. Therefore, we conclude that the non-specific inhibitory activity of spermine and spermidine, as defined by in vitro colony assays, is either of no pathophysiologic significance in the anemia of CRF, or else there are unrecognized repair mechanisms in vivo which maintain granulopoiesis and thrombopoiesis at normal levels.

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.

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.

Apparent heterogeneity of human megakaryocyte colony- and thrombopoiesis-stimulating factors: studies on urinary extracts from patients with aplastic anaemia and idiopathic thrombocytopenic purpura.

Megakaryocyte colon-stimulating factor (MEG-CSF) in the urinary extracts from patients wit aplastic anaemia (AA) revealed two distinct peaks of activity on Sephadex G-200 gel filtration with apparent molecular weights of 155,000 and 76,000. Both fractions induced significant thrombocytosis in peripheral blood and megakaryocytosis in the spleen of rats. Heterogeneity of MEG-CSF was also found in the extracts from the urine of patients with idiopathic thrombocytopenic purpura. The higher molecular weight MEG-CSF was significantly reduced when the gel filtration was performed under the dissociating conditions. Ion-exchange chromatography indicated that the higher molecular weight MEG-CSF had a different charge from the lower molecular weight MEG-CSF. These results suggest that the apparent heterogeneity of MEG-CSF is due to interaction of MEG-CSF with other proteins in the urinary extracts.

Immunofluorescent identification of human megakaryocyte colonies using an antiplatelet glycoprotein antiserum

The development of a satisfactory in vitro assay system for human megakaryocyte colony forming progenitor cells has been delayed by the lack of a suitable marker for cells of human megakaryocyte lineage. For this purpose we raised an antiserum directed against a purified human platelet glycoprotein preparation. In conjunction with indirect immunofluorescent staining of human bone marrow, this antiserum labeled only platelets, megakaryocytes, and an infrequent population of small mononuclear cells. These small mononuclear cells, not otherwise identifiable as members of the megakaryocyte series, constituted 22.9% of the total fluorescein positive nucleated bone marrow cells. This antiserum was also used to label colonies cultured from human peripheral blood mononuclear cells using a modified plasma clot technique. A mean of 123 fluorescein-labeled colonies were cloned per 10(6) mononuclear cells cultured. Granulocyte-macrophage and erythroid burst colonies did not label using this method. No augmentation of colony numbers was found with varying concentrations of erythropoietin, human embryonic kidney cell conditioned media (a source of thrombopoietin), or media conditioned by a human T lymphoblast cell line (a source of both colony stimulating and burst promoting activities). Immunofluorescent labeling for platelet glycoproteins is a convenient phenotypic marker for cells of human megakaryocyte lineage useful in the study of in vitro human megakaryocytopoiesis.

Immunofluorescent Identification of Human Megakaryocyte Colonies Using an Antiplatelet Glycoprotein Antiserum

The development of a satisfactory in vitro assay system for human megakaryocyte colony forming progenitor cells has been delayed by the lack of a suitable marker for cells of human megakaryocyte lineage. For this purpose we raised an antiserum directed against a purified human platelet glycoprotein preparation. In conjunction with indirect immunofluorescent staining of human bone marrow, this antiserum labeled only platelets, megakaryocytes, and an infrequent population of small mononuclear cells. These small mononuclear cells, not otherwise identifiable as members of the megakaryocyte series, constituted 22.9% of the total fluorescein positive nucleated bone marrow cells. This antiserum was also used to label colonies cultured from human peripheral blood mononuclear cells using a modified plasma clot technique. A mean of 123 fluorescein-labeled colonies were cloned per 10(6) mononuclear cells cultured. Granulocyte-macrophage and erythroid burst colonies did not label using this method. No augmentation of colony numbers was found with varying concentrations of erythropoietin, human embryonic kidney cell conditioned media (a source of thrombopoietin), or media conditioned by a human T lymphoblast cell line (a source of both colony stimulating and burst promoting activities). Immunofluorescent labeling for platelet glycoproteins is a convenient phenotypic marker for cells of human megakaryocyte lineage useful in the study of in vitro human megakaryocytopoiesis.

Megakaryocyte Colony Formation From Human Bone Marrow Precursors

We report the growth in plasma clot culture of megakaryocyte colonies from adult bone marrow cells with the use of four different sources of erythropoietin (Ep) as the stimulating factor. A major proportion of the megakaryocyte colonies (75%) were pure, while the others were mixed, involving erythroblasts and megakaryocytes. Ultrastructural studies have shown that the maturation of megakaryocytes was essentially normal and that platelet shedding occurred. Megakaryocyte colony formation required a large number of plated cells (greater than 3 × 105/ml). In the absence of erythropoietin, rare spontaneous megakaryocyte colonies could be observed, while no erythroid colonies were present. However, erythropoietin induced a fivefold increase in the total number of colonies. These data suggest that erythropoietin is involved in the differentiation of human megakaryocytes, but that it does not act alone, since another factor related to the number of seeded cells appears essential for the formation of human megakaryocyte colonies.

Megakaryocyte colony formation from human bone marrow precursors

We report the growth in plasma clot culture of megakaryocyte colonies from adult bone marrow cells with the use of four different sources of erythropoietin (Ep) as the stimulating factor. A major proportion of the megakaryocyte colonies (75%) were pure, while the others were mixed, involving erythroblasts and megakaryocytes. Ultrastructural studies have shown that the maturation of megakaryocytes was essentially normal and that platelet shedding occurred. Megakaryocyte colony formation required a large number of plated cells (greater than 3 X 10(5)/ml). In the absence of erythropoietin, rare spontaneous megakaryocyte colonies could be observed, while no erythroid colonies were present. However, erythropoietin induced a fivefold increase in the total number of colonies. These data suggest that erythropoietin is involved in the differentiation of human megakaryocytes, but that it does not act alone, since another factor related to the number of seeded cells appears essential for the formation of human megakaryocyte colonies.