Megakaryocyte Colony-stimulating Activity Research Articles

Stimulating effect of catechin, an active component of Spatholobus suberectus Dunn, on bioactivity of hematopoietic growth factor

Hematopoietic growth factor (HGF) is indispensable to hematopoiesis in the body. The proliferation and differentiation of hematopoietic cells must rely on the existence and stimulation of HGF. This study investigated the effect of catechin, an active component extracted from Spatholobus suberectus Dunn (SSD), on bioactivity of granulocyte-macrophage colony-stimulating activity (GM-CSA), burst-promoting activity (BPA) and megakaryocyte colony-stimulating activity (MK-CSA) in spleen condition medium (SPCM) of mice to clarify the hematopoietic mechanism of catechin and SSD. Spleen cells of mice were separated and spleen condition medium (SPCM) was prepared from spleen cell culture. Bone marrow cells of mice were separated and cultured in a culture system including 10% (v/v) SPCM (induced by catechin in vivo or ex vivo) for 6 days. Granulocyte-macrophage colony forming units (CFU-GM), erythrocyte burst-colony-forming units (BFU-E) and megakaryocyte colony-forming units (CFU-Meg) formation were employed to assay the effects of different treatment on the bioactivity of GM-CSA, BPA and MK-CSA in SPCM. SPCM induced by 100 mg/L catechin ex vivo could promote the growth of CFU-GM, BFU-E and CFU-Meg, which indicated that catechin could stimulate the production of GM-CSA, BPA and MK-CSA in SPCM. SPCM prepared at the fourth day of spleen cell culture showed the best stimulating activity. The bioactivity of GM-CSA, BPA and MK-CSA in the SPCM prepared after intraperitoneally injecting catechin into mice was also increased. The number of CFU-GM, BFU-E and CFU-Meg gradually increased as the dose of catechin increased and the time of administration prolonged. CFU-GM, BFU-E and CFU-Meg of the high-dose catechin group were significantly higher than those of the control group (P < 0.01) and reached the maximum at the seventh day after administration. This study suggests that catechin extracted from the active acetic ether part of Spatholobus suberectus Dunn can regulate hematopoiesis by inducing bioactivity of GM-CSA, BPA and MK-CSA in SPCM of mice. This may be one of the mechanisms for the hematopoietic-supportive effect of catechin and Spatholobus suberectus Dunn.

Serum-free culture of human megakaryocyte progenitors.

A serum-free method for the culture of megakaryocyte progenitor cells (burst-forming units megakaryocyte, BFU-Mk) from human bone marrow is described, using recombinant cytokines (interleukin[IL]-3, IL-6, IL-11 and stem cell factor [SCF]) to provide megakaryocyte colony-stimulating activity. IL-3 alone, but not IL-6, IL-11 or SCF alone, promoted BFU-Mk formation. The combination of IL-3 with IL-11 or with IL-6+SCF was necessary for optimal maturation of the megakaryocytes. No significant difference in BFU-Mk numbers was found between the combinations of IL-3+IL-11 and IL-3+IL-6+SCF. The development of BFU-Mk was found to be maximal at 12-14 d. Using these conditions, normal ranges for human bone marrow BFU-Mk were derived which may provide the basis for studying megakaryocyte colony growth in pathological conditions.

Megakaryocytopoietic Activity of a Truncated Variant of Mouse Thrombopoietin

Thrombopoietin (TPO) is a hemopoietic cytokine that specifically stimulates the growth and development of megakaryocytes. In addition to full-length TPO (corresponding to TPO-1), cDNAs of three truncated variants (TPO-2 to 4) have been isolated. However, whether or not these variants have biological activities has not been clarified presumably because of poor secretion into extracellular milieu. To examine the biological significance of the shortest variants, TPO-4, we prepared two fusion cDNAs of TPO-1 and TPO-4, each of which was combined with the finger-growth factor-kringle 1 (FGK1) domains of tissue plasminogen activator (tPA) as a tag and transfected them into baby hamster kidney (BHK) cells. An immunodetection assay for FGK1 antigen revealed that the amount of secreted TPO-4-FGK1 fusion protein was about 2% of that of TPO-1-FGK1. These fusion proteins after affinity purification had comparable activities on a molar basis in both megakaryocyte colony stimulating activity towards mouse bone marrow cells and growth promotion activity towards the TPO-responsive cell line UT-7/GM. These results indicate that the TPO-4 is biologically activein vitro,although its primary structure of the C-terminal half after the 111th amino acid residues is completely different from authentic TPO-1 due to frame shift.

Interferon-gamma enhances megakaryocyte colony-stimulating activity in murine bone marrow cells.

We have demonstrated previously that interferon-gamma (IFN-gamma) accelerates platelet recovery in mice with 5-FU induced-marrow aplasia in vivo. However, the mechanism for the regulation of megakaryocyte development induced by IFN-gamma in bone marrow cells in vivo remains unknown. To further study the effects of IFN-gamma on megakaryocyte development, various steps during IFN-gamma-mediated accelerated differentiation of the megakaryocytes were investigated in serum-free cultures of murine bone marrow cells in vitro. IFN-gamma markedly induced acetylcholine esterase (AChE) activity, a marker of murine megakaryocytic cells, accompanied by increased colony formation of the megakaryocyte lineage. A prominent increase in megakaryocyte number was observed after IFN-gamma treatment. All of these effects were dependent on the presence of IL-3, and, therefore, these results suggest that IFN-gamma acts as a megakaryocyte potentiator (Meg-POT). However, IFN-gamma did not enhance megakaryocyte maturation with respect to increase in cell size. The effects of IFN-gamma on megakaryocyte maturation were similar to those observed after treatment with higher doses of IL-3 alone. Meg-POT is defined as a factor that induces megakaryocyte maturation. Since IFN-gamma enhanced IL-3-dependent megakaryocyte colony formation and proliferation rather than megakaryocyte maturation, the effects on megakaryocyte development, which were induced by IFN-gamma treatment, seem to be different from the effects of a Meg-POT. We, therefore, propose a new function for IFN-gamma as an enhancer of megakaryocyte colony-stimulating factor activity. The effect of IFN-gamma in vitro appears to correlate well with the acceleration of platelet recovery in vivo.

Enhanced Platelet Recovery in Myelosuppressed Mice Treated with Interleukin-1 and Macrophage Colony-Stimulating Factor: Potential Interactions with Cytokines Having Megakaryocyte Colony-Stimulating Activity

Studies were carried out to determine whether the combination of IL-1 + M-CSF, similar to the effect of these cytokines on neutropenia, was able to reduce the duration of thrombocytopenia in the 5-fluorouracil (5-FU)-myelosuppressed mouse. In addition, comparisons were made between the in vivo effects of IL-1 + M-CSF and other "thrombopoietic" cytokines (e.g., IL-3, IL-6, and GM-CSF) that demonstrate some form of megakaryocytopoietic activity in vitro. Of the five cytokines studied, only IL-1 and IL-6, by themselves, were able to effect thrombopoietic recovery in the myelosuppressed mouse. IL-1, either when acting alone or interacting synergistically with M-CSF, was able to reduce significantly the period of thrombocytopenia, but the effects of IL-6 were restricted to enhancing platelet production during the period of rebound thrombocytopenia without altering the kinetics of thrombopoietic recovery. Moreover, none of the cytokine combinations studied were found to interact to reduce further the duration of thrombocytopenia beyond that observed with IL-1 + M-CSF. Nonetheless, IL-3, IL-6, and, to a lesser extent, GM-CSF were each able to interact with IL-1 + M-CSF to extend further the period of enhanced platelet production in the animal. However, scheduling studies suggested that these thrombopoietic cytokines interacted in sequence, rather than in concert, with IL-1 + M-CSF to enhance platelet production during thrombopoietic recovery. Furthermore, the data presented are consistent with the hypothesis that IL-1 + M-CSF initially acts on a multilineage, 5-FU-resistant target cell and that IL-6 (and possibly IL-3 and GM-CSF) serves as a secondary cytokine further to enhance platelet production during rebound thrombopoiesis in the 5-FU-treated mouse.

Recombinant PDGF enhances megakaryocytopoiesis in vitro

The effect of recombinant platelet-derived growth factor (PDGF) on both murine and human megakaryocyte colony formation was studied in the plasma clot culture system. PDGF significantly stimulates megakaryocyte colony formation in a dose-dependent manner. The minimum concentration which had a significant stimulating effect on colony forming unit megakaryocyte (CFU-MK) was 10 ng/ml and maximum stimulation occurred at 50 ng/ml. The effect of PDGF was compared with that of interleukin (IL)-3, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) and acid fibroblast growth factor (aFGF) on megakaryocyte colony formation. The results showed that megakaryocyte colony stimulating activity of PDGF was slightly higher than those of GM-CSF and aFGF, but lower than those of IL-3, IL-6 and EPO. The effect of PDGF in combination with IL-3 or IL-6 on megakaryocyte colony formation was also investigated. No synergistic action was found between PDGF and IL-3 or IL-6, but an additive effect was observed with IL-3 plus IL-6. We also studied the effects of PDGF in combination with anti-IL6, anti-IL-3 or anti-GM-CSF antibody. The increase of megakaryocyte colony formation induced by PDGF was partially inhibited by anti-IL-6 or anti-GM-CSF antibody but not by anti-IL-3 antibody. These results indicate that PDGF is a positive regulator for megakaryocytopoiesis in vitro and IL-6 and GM-CSF may play a role in the mechanism whereby PDGF stimulates megakaryocytopoiesis.

The physiologic role and therapeutic potential of the Mpl-ligand in thrombopoiesis.

The constant and appropriate production of megakaryocytes, and subsequently platelets, is critical for maintenance of hemostasis. Inadequate megakaryopoiesis and/or thrombopoiesis can lead to serious bleeding disorders. The humoral factors regulating these processes have been the subject of study for several decades. Although many cytokines have been shown to influence megakaryocyte development and platelet production, none appeared to do so in a lineage-dominant fashion analogous to the situation with erythrocyte and neutrophil production. More recently, a ligand for the hematopoietic cytokine receptor encoded by the c-mpl gene (Mpl ligand) has been shown to have profound effects on megakaryocyte growth and development. These effects appear to include the expansion of megakaryocyte progenitors (i.e. megakaryocyte-colony stimulating activity), and induction of megakaryocyte maturation to the point of platelet production (i.e. thrombopoietin). Administration of recombinant Mpl-ligand to rodents or primates treated with myelosuppressive agents abrogates or alleviates the severity and the duration of the resultant thrombocytopenias. The in vitro and in vivo data to date indicate that this new cytokine holds tremendous promise as a therapeutic agent for the treatment of thrombocytopenia associated with cancer therapies.

Megakaryocyte colony stimulating activity in allogeneic bone marrow recipients prepared with busulfan and cyclophosphamide

Increased megakaryocyte colony stimulating activity (MK-CSA) has been reported after total body irradiation (TBI) for bone marrow transplant (BMT). We studied the effect of a busulfan (Bu) and cyclophosphamide (Cy) marrow transplant conditioning regimen, without radiation, on MK-CSA production. Initial screening of MK-CSA was done on previously collected and banked sera from 14 BMT patients. MK-CSA was expressed as the ability to stimulate growth of megakaryocyte progenitors (CFU-MK) in standard plasma clot cultures. In the initial samples, MK-CSA peaked at day 7. This preliminary data led to a prospective study of MK-CSA and clinical parameters in seven allogeneic recipients. MK-CSA activity increased from day -7 pre-transplant (2.9 +/- 1.7 CFU-MK/10(5) NATD, mean +/- SD) to day 0 (10.3 +/- 4.7 CFU-MK) and peaked by day 9 post-transplant (20.6 +/- 6.4 CFU-MK). MK-CSA activity decreased in all seven patients by day 21 at which time five of seven patients studied had recovery of platelet counts to greater than 100 x 10(9)/l. MK-CSA activity rose rapidly in both groups of sera after the initiation of this non-irradiation, BMT preparative regimen. High MK-CSA levels, early after transplant, may contribute to the rapid platelet recovery in some patients.

Megakaryocyte colony-stimulating factor (Meg-CSF) is a unique cytokine specific for the megakaryocyte lineage.

The regulation of megakaryocytopoiesis and platelet production has not yet been clearly elucidated. Several cytokines have been shown to be capable of producing megakaryocyte colonies from bone marrow [i.e. Interleukin (IL)-3, granulocyte-macrophage (GM)-colony-stimulating factor (CSF), erythropoietin (Epo)]. In addition, other activities have been reported to stimulate megakaryocyte precursors, yet a megakaryocyte-CSF (Meg-CSF) has not been purified to homogeneity and IL-3, GM-CSF and/or Epo often contaminate purification attempts which could account for the activities. A Meg-CSF has been isolated from the urine of patients with aplastic anaemia and purified by sequential ultrafiltration, cation exchange, G-50 chromatography, preparative PAGE, chromatofocusing and cation exchange HPLC. The activity of this material is 2-4 x 10(4) CFU-Meg/mg as measured in a murine marrow, serum-containing assay. This activity also stimulates CFU-Meg in the absence of adherent accessory cells and in serum-free cultures, indicative of the direct stimulation on CFU-Meg. Immunoassays, colony forming assays, and proliferation assays demonstrate that purified Meg-CSF has no GM-CSF, IL-3, M-CSF, G-CSF or IL-1 alpha, -3, -6, -9 and -11. In confirmation of these results, neutralizing antibody to IL-6 also did not abrogate Meg-CSF activity. Therefore the previously-reported megakaryocyte colony-stimulating activity in purified aplastic anaemia patient urine is due to a unique cytokine: Meg-CSF.

A novel megakaryocyte potentiator produced by MC-1 human lung cancer cell line.

The hematopoietic stimulating activity of a human lung cancer cell line, MC-1, was investigated. The protein fraction (MC-1 protein) was prepared from the serum-free culture supernatant of MC-1 cells using hydroxyapatite and concanavalin A-agarose columns. In serum-containing cultures, MC-1 protein stimulated colony formation by megakaryocyte colony-forming units (CFU), erythroid burst-forming units and granulocyte/macrophage (GM) CFU. The stimulating effect was strongest for megakaryocyte CFU. The factor having megakaryocyte colony-stimulating activity was shown to be a protein whose molecular weight was determined to be 23,000 daltons by gel filtration. By various analyses, this protein was shown to be molecularly different from the heretofore-identified cytokines that may affect megakaryocytopoiesis, i.e., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-7, IL-11, granulocyte colony-stimulating factor (CSF), macrophage CSF, GM-CSF, leukemia inhibitory factor, stem cell factor and tumor necrosis factor. Under serum-free conditions, MC-1 protein augmented murine megakaryocyte colony formation in the presence of murine IL-3 and increased the acetylcholinesterase activity of purified murine megakaryocytes. It was also shown that MC-1 protein stimulated human megakaryocyte colony formation. It was concluded that MC-1 cells produce a megakaryocyte potentiator which is molecularly different from any heretofore-identified cytokines.

Megakaryocytes and Megakaryocyte Progenitors in Human Cord Blood

Thrombocytopenia contributes significantly to morbidity in the sick term or preterm infant. However, few data exist on newborn's megakaryocytes and megakaryocyte progenitor cells (CFU-MK). We therefore studied CFU-MK in term and preterm infant cord blood and compared the results with data on CFU-MK from adult bone marrow and adult peripheral blood in a plasma clot culture with postirradiated aplastic canine serum (PIACS) as a source of megakaryocyte colony-stimulating activity. The number of CFU-MK and the number of cells per CFU-MK were counted with an immunofluorescent method at day 12. The effect of T-lymphocyte depletion on cord blood cultures for CFU-MK was studied with PIACS and a partially purified product of PIACS. We also studied individual megakaryocytes from newborns. The number and sizes of circulating megakaryocytes, isolated from adult peripheral blood and term venous cord blood by elutriation, were compared. Term and preterm cord blood contained more CFU-MK than adult peripheral blood. The numbers of CFU-MK in preterm cord blood were comparable to those in adult bone marrow. When the number of cells per colony were compared, cord blood contained significantly more cells than adult marrow CFU-MK. The depletion of T lymphocytes did not significantly change the growth of CFU-MK compared to nondepleted cultures. A substantial number of circulating megakaryocytes were obtained from venous cord blood, though they were significantly smaller than adult peripheral blood megakaryocytes. Since cord blood is easily obtained and contains large numbers of megakaryocytes and CFU-MK, it may provide a convenient model for studying the regulation of fetal megakaryocytopoiesis.

Effect of c-kit ligand on in vitro human megakaryocytopoiesis

An evaluation of the effects of a recombinant, soluble form of the c- kit ligand alone and in combination with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) on the regulation of human megakaryocytopoiesis was performed using a serum- depleted clonal assay system and a long-term bone marrow culture system. The effects of the c-kit ligand on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit-megakaryocyte (BFU-MK), and the more differentiated colony-forming unit-megakaryocyte (CFU-MK) were determined. The c-kit ligand alone had no megakaryocyte colony- stimulating activity (MK-CSA) but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. The range of synergistic interactions of c-kit ligand varied with the class of MK progenitor cell assayed. In the case of the BFU-MK, the c-kit ligand synergistically augmented the numbers of colonies formed in the presence of IL-3, but not GM-CSF, but increased the size of BFU-MK-derived colonies cloned in the presence of both of these cytokines. However, at the level of the CFU-MK, c-kit ligand synergized with both GM-CSF and IL-3 by increasing both colony numbers and size. Although the c-kit ligand alone exhibited limited potential in sustaining long-term megakaryocytopoiesis in vitro, it synergistically augmented the ability of IL-3, but not GM-CSF, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that the c-kit ligand plays a significant role in this process by amplifying the MK- CSA of both GM-CSF and IL-3.

Effect of c-kit Ligand on In Vitro Human Megakaryocytopoiesis

AbstractAn evaluation of the effects of a recombinant, soluble form of the c-kitligand alone and in combination with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) on the regulation of human megakaryocytopoiesis was performed using a serum-depleted clonal assay system and a long-term bone marrow culture system. The effects of thec-kitligand on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit-megakaryocyte (BFU-MK), and the more differentiated colony-forming unit-megakaryocyte (CFU-MK) were determined. Thec-kitligand alone had no megakaryocyte colony-stimulating activity (MK-CSA) but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. The range of synergistic interactions ofc-kitligand varied with the class of MK progenitor cell assayed. In the case of the BFU-MK, thec-kitligand synergistically augmented the numbers of colonies formed in the presence of IL-3, but not GM-CSF, but increased the size of BFU-MK–derived colonies cloned in the presence of both of these cytokines. However, at the level of the CFU-MK,c-kitligand synergized with both GM-CSF and IL-3 by increasing both colony numbers and size. Although thec-kitligand alone exhibited limited potential in sustaining long-term megakaryocytopoiesis in vitro, it syngeristically augmented the ability of IL-3, but not GM-CSF, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that thec-kit ligand plays a significant role in this process by amplifying the MK-CSA of both GM-CSF and IL-3.

Studies of in vitro megakaryocytopoiesis in adult immune thrombocytopenic purpura (ITP)

In vitro megakaryocytopoiesis was studied in 8 patients with chronic immune thrombocytopenia (ITP). A significant increase of megakaryocyte (MK) colony formation was observed in 5/5 patients studied. Furthermore, the serum of these 8 patients was able to enhance MK colony formation by normal marrow cells. This effect was neither due to a decrease of inhibitors of megakaryocytopoiesis such as betathromboglobulin (beta TG) nor to the IgG fraction of patients' serum. In addition, the level of interleukin 6, which is above all a stimulus for MK maturation, was found within the normal range in 8/8 patients tested. These data suggest that in chronic ITP there is an increase of MK progenitor cell number which may be due to an increased level of MK colony-stimulating activity.

Further examination of the effects of recombinant cytokines on the proliferation of human megakaryocyte progenitor cells

The effect of several recombinant cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL- 6, and IL-1 alpha, on megakaryocyte (MK) colony formation by a normal human bone marrow subpopulation (CD34+ DR+), enriched for the MK colony- forming unit (CFU-MK), was studied using a serum-depleted, fibrin clot culture system. IL-3 and GM-CSF, but not IL-6 or IL-1 alpha, stimulated MK colony formation by CD34+ DR+ cells. However, the addition of IL-1 alpha to CD34+ DR+ cultures containing IL-6 resulted in the appearance of CFU-MK-derived colonies, suggesting that IL-6 requires the presence of IL-1 alpha to exhibit its MK colony-stimulating activity (MK-CSA). Addition of neutralizing antibodies to IL-3 and GM-CSF, but not to IL-6 and IL-1 alpha, specifically inhibited the MK-CSA of IL-3 and GM-CSF, respectively. The addition of either anti-IL-6, anti-IL-1 alpha, or anti-IL-3 antisera to cultures containing both IL-6 and IL-1 alpha totally abolished the MK-CSA of the IL-6/IL-1 alpha combination. However, neither anti-IL-3 nor anti-GM-CSF antisera could totally neutralize the additive effect of the combination of IL-3 and GM-CSF on MK colony formation, indicating that these two cytokines act by affecting distinct effector pathways. These results suggest that while IL-3 and GM-CSF can directly affect CFU-MK-derived colony formation, IL- 1 alpha and IL-6 act in concert to promote de novo elaboration of IL-3 and thereby promote CFU-MK proliferative capacity.

Further Examination of the Effects of Recombinant Cytokines on the Proliferation of Human Megakaryocyte Progenitor Cells

The effect of several recombinant cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, and IL-1 alpha, on megakaryocyte (MK) colony formation by a normal human bone marrow subpopulation (CD34+ DR+), enriched for the MK colony-forming unit (CFU-MK), was studied using a serum-depleted, fibrin clot culture system. IL-3 and GM-CSF, but not IL-6 or IL-1 alpha, stimulated MK colony formation by CD34+ DR+ cells. However, the addition of IL-1 alpha to CD34+ DR+ cultures containing IL-6 resulted in the appearance of CFU-MK-derived colonies, suggesting that IL-6 requires the presence of IL-1 alpha to exhibit its MK colony-stimulating activity (MK-CSA). Addition of neutralizing antibodies to IL-3 and GM-CSF, but not to IL-6 and IL-1 alpha, specifically inhibited the MK-CSA of IL-3 and GM-CSF, respectively. The addition of either anti-IL-6, anti-IL-1 alpha, or anti-IL-3 antisera to cultures containing both IL-6 and IL-1 alpha totally abolished the MK-CSA of the IL-6/IL-1 alpha combination. However, neither anti-IL-3 nor anti-GM-CSF antisera could totally neutralize the additive effect of the combination of IL-3 and GM-CSF on MK colony formation, indicating that these two cytokines act by affecting distinct effector pathways. These results suggest that while IL-3 and GM-CSF can directly affect CFU-MK-derived colony formation, IL-1 alpha and IL-6 act in concert to promote de novo elaboration of IL-3 and thereby promote CFU-MK proliferative capacity.

Thrombocytopenia and Absent Radii Syndrome

Thrombocytopenia and absent radii (TAR) syndrome is a congenital defect with osseous abnormalities and thrombocytopenia. It is inherited as an autosomal recessive trait, but the mechanism of thrombocytopenia in this disorder is not clear. We have had the opportunity to study the mechanism of thrombocytopenia in an infant with TAR syndrome. The infant had normal levels of thrombopoietin and megakaryocyte colony-stimulating activity in spite of marked thrombocytopenia. However, the megakaryocyte progenitor cells in the bone marrow produced abnormal colonies with increased numbers of megakaryocytes per colony and small megakaryocytes similar to the small megakaryocyte seen in vivo. These findings suggest that the TAR syndrome in this infant is due to a failure in the production of thrombopoietin or to an abnormal progenitor cell with a maturational defect.

Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells

Investigations linking human megakaryocyte development and cell biology have been hindered by an inability to obtain large, relatively pure megakaryocyte cell preparations from in vitro stem cell cultures. We report here that such preparations can be generated from liquid cultures of normal human peripheral blood mononuclear cells stimulated by a serum source of megakaryocyte colony stimulating activity (Meg- CSA, the 0% to 60% ammonium sulfate protein fraction of aplastic canine serum). Adherent-depleted peripheral blood mononuclear cells are suspended at 5 x 10(5) to 10(6) cells/mL in supplemented liquid culture medium, platelet-poor human plasma 20% (vol/vol) and 1 to 2 mg/mL serum Meg-CSA protein. After 12 to 14 days of incubation, megakaryocytes constitute 3.0 +/- 2.9% (mean +/- SD, n = 8) of the unseparated cultured cell population. Megakaryocytes can be enriched by counterflow centrifugal elutriation to a purity of 58 +/- 14% (+/- SD) with a recovery of 13 +/- 7% and a viability of 67 +/- 19%. This algorithm results in the average isolation of approximately 3 x 10(5) enriched megakaryocytes from a 100-mL starting volume of peripheral blood. Cultured megakaryocytes exhibit normal light and ultrastructural morphology by Wright-Giemsa staining and electron microscopic analysis. After a 12-day culture interval, enriched megakaryocyte preparations exhibit morphologic stage distributions that are similar to normal human marrow. Stage distributions move rightward with culture duration indicating partial synchrony of megakaryocyte maturation. On cytospin preparations, megakaryocyte diameter averages 30.2 +/- 1.5 microns and increases with maturation stage. Flow cytometric analyses demonstrate the expression of platelet glycoproteins (GP) Ib and IIb/IIIa by the cultured megakaryocytes. The modal ploidy of the enriched cells at day 12 of culture is 16N and most remaining megakaryocytes are 8N or 32N. Liquid culture of serum Meg-CSA-stimulated human peripheral blood mononuclear cells represents a valuable investigative tool that should permit studies of human megakaryocyte biology that have not been possible in the past.

Isolation of Large Numbers of Enriched Human Megakaryocytes From Liquid Cultures of Normal Peripheral Blood Progenitor Cells

Investigations linking human megakaryocyte development and cell biology have been hindered by an inability to obtain large, relatively pure megakaryocyte cell preparations from in vitro stem cell cultures. We report here that such preparations can be generated from liquid cultures of normal human peripheral blood mononuclear cells stimulated by a serum source of megakaryocyte colony stimulating activity (Meg-CSA, the 0% to 60% ammonium sulfate protein fraction of aplastic canine serum). Adherent-depleted peripheral blood mononuclear cells are suspended at 5 x 10(5) to 10(6) cells/mL in supplemented liquid culture medium, platelet-poor human plasma 20% (vol/vol) and 1 to 2 mg/mL serum Meg-CSA protein. After 12 to 14 days of incubation, megakaryocytes constitute 3.0 +/- 2.9% (mean +/- SD, n = 8) of the unseparated cultured cell population. Megakaryocytes can be enriched by counterflow centrifugal elutriation to a purity of 58 +/- 14% (+/- SD) with a recovery of 13 +/- 7% and a viability of 67 +/- 19%. This algorithm results in the average isolation of approximately 3 x 10(5) enriched megakaryocytes from a 100-mL starting volume of peripheral blood. Cultured megakaryocytes exhibit normal light and ultrastructural morphology by Wright-Giemsa staining and electron microscopic analysis. After a 12-day culture interval, enriched megakaryocyte preparations exhibit morphologic stage distributions that are similar to normal human marrow. Stage distributions move rightward with culture duration indicating partial synchrony of megakaryocyte maturation. On cytospin preparations, megakaryocyte diameter averages 30.2 +/- 1.5 microns and increases with maturation stage. Flow cytometric analyses demonstrate the expression of platelet glycoproteins (GP) Ib and IIb/IIIa by the cultured megakaryocytes. The modal ploidy of the enriched cells at day 12 of culture is 16N and most remaining megakaryocytes are 8N or 32N. Liquid culture of serum Meg-CSA-stimulated human peripheral blood mononuclear cells represents a valuable investigative tool that should permit studies of human megakaryocyte biology that have not been possible in the past.

Human serum megakaryocyte colony-stimulating activity appears to be distinct from interleukin-3, granulocyte-macrophage colony-stimulating factor, and lymphocyte-conditioned medium

Sera from patients with bone marrow megakaryocyte aplasia are a rich source of megakaryocyte colony-stimulating activity (Meg-CSA). Other biologic materials exhibiting Meg-CSA include phytohemagglutinin-stimulated human lymphocyte-conditioned medium (PHA-LCM), recombinant interleukin-3 (IL-3), and recombinant granulocyte macrophage colony-stimulating factor (GM-CSF). Neutralizing antisera to both recombinant IL-3 and GM-CSF were used to evaluate the relationship among these sources of Meg-CSA. Varying dilutions of IL-3 and GM-CSF antisera were tested in plasma clot cultures of normal human peripheral blood megakaryocyte progenitors optimally stimulated by either IL-3 (1 U/mL), GM-CSF (1 U/mL), PHA-LCM (2.5% to 5% vol/vol), or aplastic human serum (10% vol/vol). IL-3 antiserum at dilutions up to 1/2,000 totally abrogated megakaryocyte colony growth stimulated by IL-3. A 1/500 dilution of GM-CSF antiserum completely eliminated GM-CSF-induced megakaryocyte colony development. A combination of anti-IL-3 and anti-GM-CSF, each at a 1/500 dilution, inhibited all megakaryocyte colony growth stimulated by optimal concentrations of IL-3 and GM-CSF together. There was no neutralizing crossreactivity between the IL-3 and GM-CSF antisera. At maximally neutralizing concentrations, IL-3 antiserum inhibited 66% of the megakaryocyte colony growth stimulated by PHA-LCM. Residual megakaryocyte colony growth was eliminated by the addition of a 1/500 dilution of anti-GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)