Megakaryocyte Colony-forming Units Research Articles

Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.

Role of Cytokines in Sustaining Long-Term Human Megakaryocytopoiesis In Vitro

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.

Influence of interleukin-3 on zidovudine (AZT)-induced in vitro toxicity to human hematopoietic progenitors.

Zidovudine (AZT), the antiviral drug used in the treatment of acquired immunodeficiency syndrome (AIDS), produces some toxicity to the hematopoietic system. Although several hematopoietic growth factors are currently undergoing clinical trials to evaluate their ability to modulate antiviral toxicity, there are scant data which support their ability to ameliorate AZT toxicity on hematopoietic progenitor cells when combined in vitro. We describe in this report the results of studies designed to evaluate in vitro the capacity of the cytokine interleukin-3 (IL-3), in dose-escalation fashion, to modulate AZT toxicity on normal human marrow derived granulocyte/erythroid/macrophage/megakaryocyte colony-forming units (CFU-GEMM), CFU-granulocyte/macrophage (CFU-GM) and erythroid burst-forming units (BFU-E). Colony formation for each progenitor was increased in the presence of IL-3 compared to cultures plated in its absence. In the presence of AZT (ID50 dose, used for each progenitor), IL-3 reduced AZT toxicity, with the most significant response observed for CFU-GEMM, indicating IL-3 may exert an effect on early, less differentiated hematopoietic progenitors. These studies indicate IL-3 may be an effective agent in reversing the hematopoietic toxicity associated with AZT; however, further in vivo studies are required before clinical use of IL-3 is advocated.

Efficacy of combined autologous of bone marrow and peripheral blood The International Journal of Cell Cloning on hematological recovery following high-dose chemotherapy

We have conducted high-dose chemotherapy (HDCT) with autologous bone marrow transplant (ABMT) to overcome drug resistance and to obtain higher anti-tumor efficacy for malignant lymphoma and breast cancer. However, the delay of platelet recovery was major problem following ABMT and patient was received a lot of platelet transfusion. When we analized CFU-Megakaryocyte (CFU-Meg) formation in vitro on peripheral blood mononuclear cells obtained from patient receiving conventional chemotherapy, overshoot of CFU-Meg was observed during the period of bone marrow recovery phase. Based on these data, we added peripheral blood stem cell transplant(PBSCT) to ABMT, whether the recovery of platelet could be enhanced due to infuse a lot of CFU-Meg. As the result of applying combined stem cell autotransplant (ABMT+PBSCT), the recovery of platelet was significantly enhanced and we could reduce the number of platelet transfusion.

In vitro megakaryocyte colony formation in patients with idiopathic thrombocytopenic purpura: differences between acute and chronic ITP.

In vitro megakaryocyte colony formation from the bone marrow of patients with acute idiopathic thrombocytopenic purpura (ITP) or chronic ITP was compared using a plasma clot system. The number of megakaryocyte colony-forming units (CFU-Meg) was significantly higher (p less than 0.05) in acute ITP compared to chronic ITP (54.3 +/- 68.4 vs. 12.9 +/- 15.3/10(5) nonadherent mononuclear cells, mean +/- SD), and significantly lower (p less than 0.05) in chronic ITP compared to controls (12.9 +/- 15.3 vs. 22.8 +/- 15.9). A significant correlation was observed between platelet recovery 7 and 30 days after culture, and the number of CFU-Meg (r = 0.49 and 0.45, respectively, p less than 0.05). An inverse correlation was observed between platelet count at the time of culture and the number of Megs per colony (r = -0.48, p less than 0.05). These results indicated a difference between acute and chronic ITP in the ability to promote in vitro Meg colony formation and may suppose a different immune mechanism for thrombocytopenia in these two disorders.

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.

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.

ACCELERATION OF THE HEMOPOIETIC RECONSTITUTION IN MICE UNDERGOING BONE MARROW TRANSPLANTATION BY RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR

We have investigated the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on hemopoietic reconstitution after bone marrow transplantation (BMT) following lethal irradiation in mice. Mice received a daily administration of 10 micrograms/kg rG-CSF or control vehicle one through 21 days after BMT. Spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte colony-forming units (CFU-Meg), and erythroid burst-forming units (BFU-E) increased in both bone marrow and spleen of the rG-CSF-treated mice as compared with the control. This increase was evident during the administration period. In spite of the increase in the progenitor cells in bone marrow and spleen, only a recovery of neutrophils was accelerated in peripheral blood. Thus rG-CSF accelerated granulopoietic recovery in the BMT mice, with an enhanced recovery of the stem cells and the progenitors for erythrocytes and megakaryocytes. These results indicate the potential clinical usefulness of rG-CSF in the treatment of patients undergoing BMT.

In vitro cloning of murine megakaryocyte progenitors (CFU-Meg)

Convenient and reproducible culture systems for murine megakaryocyte progenitor cells (colony-forming unit-megakaryocytes, CFU-Meg) are described. Mouse bone marrow cells are cultured in fibrin clots supplemented with Iscove's modified Dulbecco's medium and fetal bovine serum or in fibrin clots supplemented with serum-free IMDM, bovine serum albumin, transferrin, cholesterol, and L-α-phosphatidylcholine. In the presence of murine interleukin-3 or pokeweed mitogen-stimulated murine spleen cell-conditioned medium, these cultures support CFU-Meg colony formation effectively. The cultivation and counting of colonies in these culture systems is considerably easier when compared with previously reported culture systems.

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.

Essential thrombocythemia: Impaired regulation of megakaryocyte progenitors

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

In Vivo Stimulation of Megakaryocytopoiesis by Recombinant Murine Granulocyte-Macrophage Colony-Stimulating Factor

Murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) was injected in mice, and the effects on bone marrow, splenic megakaryocytes, megakaryocyte precursors (megakaryocyte colony-forming units [CFU-Meg]) were evaluated. In mice injected three times a day for 6 days with 12,000 to 120,000 U rGM-CSF, no significant modification of both platelet levels and mean platelet volume was observed, while there was a twofold increase in blood neutrophils. However, the rate of platelet production, as assessed by the measurement of 75selenomethionine incorporation into blood platelets, was On the contrary, administration of up to 384,000 U rGM-CSF two times a day for 2 days, as for a typical "thrombopoietin assay," failed to modify platelet production. A significant dose-related increase in the number of splenic megakaryocytes occurred in mice receiving 60,000 to 120,000 U rGM-CSF, while a slight increase in the number of bone marrow megakaryocytes was observed in mice injected with 120,000 U rGM-CSF. The proportion of bone marrow megakaryocytes with a size less than 18 microns and greater than 35 microns resulted significantly higher in mice receiving rGM-CSF in comparison with controls; an increase in the percentage of splenic megakaryocytes greater than 35 microns was also observed. A statistically significant increase in the total spleen content of CFU-Meg was observed after administration of 90,000 and 120,000 U rGM-CSF three times a day for 6 days, while no effect on bone marrow CFU-Meg was recorded, irrespective of the dose delivered. Finally, 24 hours after a single intravenous injection of rGM-CSF, there was a significant increase in the proportion of CFU-Meg in S-phase, with the splenic progenitors being more sensitive than bone marrow-derived CFU-Meg. These data indicate that rGM-CSF has in vivo megakaryocyte stimulatory activity, and are consistent with previous in vitro observations. However, an effective stimulation of megakaryocytopoiesis in vivo, bringing about an increase in the levels of blood platelets, may require interaction of rGM-CSF with other cytokines.

In vivo stimulation of megakaryocytopoiesis by recombinant murine granulocyte-macrophage colony-stimulating factor

Murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) was injected in mice, and the effects on bone marrow, splenic megakaryocytes, megakaryocyte precursors (megakaryocyte colony-forming units [CFU-Meg]) were evaluated. In mice injected three times a day for 6 days with 12,000 to 120,000 U rGM-CSF, no significant modification of both platelet levels and mean platelet volume was observed, while there was a twofold increase in blood neutrophils. However, the rate of platelet production, as assessed by the measurement of 75selenomethionine incorporation into blood platelets, was On the contrary, administration of up to 384,000 U rGM-CSF two times a day for 2 days, as for a typical “thrombopoietin assay,” failed to modify platelet production. A significant dose-related increase in the number of splenic megakaryocytes occurred in mice receiving 60,000 to 120,000 U rGM-CSF, while a slight increase in the number of bone marrow megakaryocytes was observed in mice injected with 120,000 U rGM-CSF. The proportion of bone marrow megakaryocytes with a size less than 18 microns and greater than 35 microns resulted significantly higher in mice receiving rGM-CSF in comparison with controls; an increase in the percentage of splenic megakaryocytes greater than 35 microns was also observed. A statistically significant increase in the total spleen content of CFU-Meg was observed after administration of 90,000 and 120,000 U rGM-CSF three times a day for 6 days, while no effect on bone marrow CFU-Meg was recorded, irrespective of the dose delivered. Finally, 24 hours after a single intravenous injection of rGM-CSF, there was a significant increase in the proportion of CFU-Meg in S- phase, with the splenic progenitors being more sensitive than bone marrow-derived CFU-Meg. These data indicate that rGM-CSF has in vivo megakaryocyte stimulatory activity, and are consistent with previous in vitro observations. However, an effective stimulation of megakaryocytopoiesis in vivo, bringing about an increase in the levels of blood platelets, may require interaction of rGM-CSF with other cytokines.

Platelet factor 4 inhibits human megakaryocytopoiesis in vitro

Platelet factor 4 (PF4) is a multifunctional protein specific to platelets, synthesized in megakaryocytes and stored in alpha granules. This report of our work shows that PF4 potently inhibits human megakaryocyte colony formation in vitro. Colony formation by megakaryocyte progenitor cells from normal bone marrows was studied using the plasma clot culture system and indirect immunoperoxidase staining. Nonadherent mononuclear cells were co-cultured with various concentrations (0 to 20 micrograms/mL) of highly purified human PF4. Statistically significant inhibition of three classes of megakaryocyte progenitor cells, the mixed colony forming unit-megakaryocytes (mCFU- MK), the burst forming unit-megakaryocytes (BFU-MK), and the colony forming unit-megakaryocytes (CFU-MK), was seen at a PF4 concentration of 2.5 micrograms/mL or greater. PF4 had no effect on erythroid (BFU-E) and granulocyte/macrophage (CFU-GM) colony formation except at high concentration (5 micrograms/mL for BFU-E and 10 micrograms/mL for CFU- GM). When a concentration of 5 micrograms/mL PF4 was added at various time points during marrow culture, a reduction of megakaryocyte colony formation also occurred. In the presence of PF4 2.5 micrograms or 5 micrograms/mL, the percentage of mature type of colonies was found to be decreased compared with cultures with no added PF4. These data demonstrate that PF4 inhibits both proliferation and maturation of megakaryocyte progenitor cells in vitro and suggest that PF4 may play a role in autoregulating human megakaryocytopoiesis.

Platelet Factor 4 Inhibits Human Megakaryocytopoiesis In Vitro

Platelet factor 4 (PF4) is a multifunctional protein specific to platelets, synthesized in megakaryocytes and stored in alpha granules. This report of our work shows that PF4 potently inhibits human megakaryocyte colony formation in vitro. Colony formation by megakaryocyte progenitor cells from normal bone marrows was studied using the plasma clot culture system and indirect immunoperoxidase staining. Nonadherent mononuclear cells were co-cultured with various concentrations (0 to 20 micrograms/mL) of highly purified human PF4. Statistically significant inhibition of three classes of megakaryocyte progenitor cells, the mixed colony forming unit-megakaryocytes (mCFU-MK), the burst forming unit-megakaryocytes (BFU-MK), and the colony forming unit-megakaryocytes (CFU-MK), was seen at a PF4 concentration of 2.5 micrograms/mL or greater. PF4 had no effect on erythroid (BFU-E) and granulocyte/macrophage (CFU-GM) colony formation except at high concentration (5 micrograms/mL for BFU-E and 10 micrograms/mL for CFU-GM). When a concentration of 5 micrograms/mL PF4 was added at various time points during marrow culture, a reduction of megakaryocyte colony formation also occurred. In the presence of PF4 2.5 micrograms or 5 micrograms/mL, the percentage of mature type of colonies was found to be decreased compared with cultures with no added PF4. These data demonstrate that PF4 inhibits both proliferation and maturation of megakaryocyte progenitor cells in vitro and suggest that PF4 may play a role in autoregulating human megakaryocytopoiesis.

Anti-thoracic duct lymphocyte globulin stimulates human megakaryocytopoiesis in vitro

Anti-thoracic duct lymphocyte globulin (ALG) therapy is effective in patients with aplastic anemia. We examined the effect of ALG on human megakaryocyte progenitor cells (colony-forming unit-megakaryocyte, CFU-Meg) in vitro. Normal human bone marrow mononuclear cells (MNC) were cultured in plasma clots with varying concentrations of ALG or non-immunized horse IgG. After 12 days of culture, significant megakaryocyte colony formation was observed in cultures containing ALG but not in cultures containing non-immunized horse IgG. The peak stimulatory effect seemed to occur with 10-25 micrograms/ml of ALG. When marrow MNC, depleted of adherent and T cells, were cultured in plasma clots with ALG, its stimulatory effect on megakaryocytopoiesis decreased markedly. Finally, it was demonstrated that ALG stimulated marrow MNC to produce a factor stimulatory for CFU-Meg. The in vitro megakaryocytopoietic stimulatory effect of ALG may be related to its clinical efficacy in some patients with aplastic anemia.

Human GM-CSF in vivo: Identification of the target cells and of their kinetics of response

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was given for three days (8 micrograms/kg/day) to 14 subjects who had solid tumors and normal hemopoiesis. The treatment induced a rapid 3- to 5-fold increase in the number of circulating neutrophils, eosinophils and monocytes. Lymphocytes, platelets and reticulocytes were unmodified during treatment. Activation of circulating neutrophils during GM-CSF treatment was demonstrated by a significant, increased release of neutrophil-derived platelet-activating factor after stimulation with N-formyl-methionyl-leucyl-phenylalanine, tumor necrosis factor-alpha or phagocytosis. The granulomonocytosis was dependent on increased bone marrow production of mature cells. Using the thymidine suicide technique, we observed that GM-CSF more than doubled the percentage of granulocyte-macrophage and megakaryocyte colony-forming units (CFU-gm and CFU-meg) and erythroid burst-forming units (BFU-e) in the S phase of the cell cycle. However, at the level of morphologically recognizable cells with autoradiography, we observed that GM-CSF increased the labeling index of the granulo-monopoietic cells, whereas that of the erythroblasts was unchanged. These data suggest that in accordance with in vitro observations, GM-CSF exerts its activity through all granulo-monopoietic lineages, whereas other cytokines (erythropoietin, thrombopoiesis-stimulating factors) may be needed to fully exploit the proliferative stimulus of GM-CSF on BFU-e and CFU-meg. After treatment discontinuation, the proliferative activity drops to values lower than before treatment, suggesting a period of relative refractoriness of marrow progenitors to the cytocidal effect of cell cycle-specific antineoplastic agents. This hypothesis is under evaluation in a controlled clinical trial where GM-CSF is given prior to chemotherapy.

Endotoxin-induced tumor necrosis factor formation in man and its reversal by pentoxifylline: [formula omitted] Forschungsinstitut Borstel, D-2061 Borstel, FRG and Hoechst Werk Albert, D-6000 Wiesbaden, FRG

The BMT program at Princess Margaret Hospital performed 105 transplants using cryopreserved peripheral blood stem cells (PBSC) from related allogeneic donors. The outcomes were compared with those of a historic control of 106 patients transplanted with freshly procured PBSC. The infusions were tolerated with limited toxicity related to nausea/vomiting or bradycardia, correlated with the total amount of DMSO infused. The average viability of the total nucleated cell (TNC) population after thawing was 71%. The survival of clonogenic progenitors amounted to 75% for colony-forming unit-granulocyte-macrophage (CFU-GM), 69% for burst-forming units erythroid (BFU-E), and 78% for colony-forming units granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM). In contrast, colony-forming units megakaryocyte (CFU-MEG) was significantly more cryosensitive with recovery rates of 39%. The number of viable CD34+ cells transplanted was correlated with the number of transplanted viable CFU-GM (P < .001), BFU-E (P < .001), CFU-MEG (P < .001), and CFU-GEMM (P = .049), but not with the TNC dose. The number of transplanted CD34+ cells was correlated with engraftment of neutrophils (P = .012) and platelets (P = .013). The outcomes of cryopreseved or fresh PBSC transplants (PBSCT) with respect to engraftment of neutrophils (P = .178) and platelets (P = .785), lymphocyte recovery (P = .926), acute (P = .113), and chronic graft-versus-host disease (P = .673), recurrence (P = .295), nonrelapse mortality (P = .340), and overall survival (P = .668) were not significantly different. It is therefore reasonable to consider the option of cryopreserved allografts.

Characterization of the human burst-forming unit-megakaryocyte

Two classes of human marrow megakaryocyte progenitor cells are described. Colony-forming unit-megakaryocyte (CFU-MK)-derived colonies appeared in vitro after 12-day incubation; burst-forming unit- megakaryocyte (BFU-MK)-derived colonies appeared after 21 days. CFU-MK- derived colonies were primarily unifocal and composed of 11.6 +/- 1.2 cells/colony; BFU-MK-derived colonies were composed of 2.3 +/- 0.4 foci and 108.6 +/- 4.4 cells/colony. CFU-MK and BFU-MK were separable by counterflow centrifugal elutriation. CFU-MK colony formation was diminished by exposure to 5-fluorouracil (5-FU); BFU-MK colony formation was unaffected. CFU-MK and BFU-MK were immunologically phenotyped. CFU-MK expressed the human progenitor cell antigen-1 (HPCA- 1, CD34, clone My10) and a major histocompatibility class II locus, HLA- DR, and BFU-MK expressed only detectable amounts of CD34. BFU-MK colony formation was entirely dependent on addition of exogenous hematopoietic growth factors. Recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) possessed such colony- stimulating activity, whereas recombinant erythropoietin (Epo), G-CSF, IL-1 alpha, IL-4, and purified thrombocytopoiesis-stimulating factor did not. These studies indicate the existence of a human megakaryocyte progenitor cell, the BFU-MK, which has unique properties allowing it to be distinguished from the CFU-MK.

Characterization of the Human Burst-Forming Unit-Megakaryocyte

AbstractTwo classes of human marrow megakaryocyte progenitor cells are described. Colony-forming unit-megakaryocyte (CFU-MK)-derived colonies appeared in vitro after 12-day incubation; burst-forming unit-megakaryocyte (BFU-MK)- derived colonies appeared after 21 days. CFU-MK-derived colonies were primarily unifocal and composed of 11.6 ± 1.2 cells/colony; BFU-MK-derived colonies were composed of 2.3 ± 0.4 foci and 108.6 ± 4.4 cells/colony. CFU-MK and BFU-MK were separable by counterflow centrifugal elutriation. CFU-MK colony formation was diminished by exposure to 5-fluorouracil (5-FU); BFU-MK colony formation was unaffected. CFU-MK and BFU-MK were immunologically phenotyped. CFU-MK expressed the human progenitor cell antigen-1 (HPCA-1, CD34, clone My10) and a major histocompatibility class II locus, HLA- DR, and BFU-MK expressed only detectable amounts of CD34. BFU-MK colony formation was entirely dependent on addition of exogeneous hematopoietic growth factors. Recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) possessed such colony-stimulating activity, whereas recombinant erythropoietin (Epo), G-CSF, IL-1α, IL-4, and purified thrombocyto- poiesis-stimulating factor did not. These studies indicate the existence of a human megakaryocyte progenitor cell, the BFU-MK, which has unique properties allowing it to be distinguished from the CFU-MK.