Recombinant GM-CSF/IL-3 fusion protein: its effect on in vitro human megakaryocytopoiesis.

An evaluation of the effectiveness of a genetically engineered recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin 3 (IL-3) fusion protein (FP) as a means of delivering cytokine combinations to megakaryocyte (MK) progenitor cells was performed, utilizing a serum-depleted clonal assay system and a long-term bone marrow culture system. The effects of the FP, alone and in combination with a variety of other cytokines, on the primitive MK progenitor cell, the megakaryocyte burst-forming unit (BFU-MK), and the more differentiated megakaryocyte colony-forming unit (CFU-MK) were assessed. Subpopulations of bone marrow cells (CD34+ DR- for BFU-MK and CD34+ DR+ for CFU-MK) served as sources of these two classes of MK progenitor cells. The FP was equivalent to a combination of optimal concentrations of GM-CSF and IL-3 in promoting both the number and size of BFU-MK-derived colonies. The GM-CSF/IL-3 combination, however, promoted the formation of far greater CFU-MK-derived colonies than did the FP alone. The size of MK colonies formed in the presence of the FP or GM-CSF/IL-3 was similar. The ability of the FP to stimulate BFU-MK- but not CFU-MK-derived colony formation was also further augmented by the addition of interleukin 1 alpha (IL-1 alpha). The addition of c-kit ligand (KL) increased both FP-stimulated CFU-MK- and BFU-MK-derived colony numbers but only BFU-MK-derived colony size. In addition, the FP alone sustained long-term megakaryocytopoiesis in vitro to a level equivalent to that of the GM-CSF/IL-3 combination and was superior in this regard to either GM-CSF or IL-3 alone. These data indicate that FP is capable of supporting various stages of human megakaryocytopoiesis. We conclude that such genetically engineered molecules as the FP may prove to be effective means of pharmacologically delivering the biological effects of specific cytokine combinations.     

Basic fibroblast growth factor promotes the proliferation of human megakaryocyte progenitor cells

Basic fibroblast growth factor (bFGF), a multifunctional growth factor produced by bone marrow stromal cells, is known to be a potent modulator of hematopoiesis. Because bFGF is present in both human megakaryocytes (MKs) and platelets, we have hypothesized that this growth factor might affect human megakaryocytopoiesis. To test this hypothesis, either low density bone marrow (BM) cells (LDBM), a human BM subpopulation (CD34+ DR+) enriched for the colony-forming unit megakaryocyte (CFU-MK) or a BM subpopulation (CD34+ DR-) enriched for the more primitive burst-forming unit megakaryocyte (BFU-MK) were assayed in the presence of this growth factor. The effect of bFGF on MK colony formation differed according to the cell population assayed. bFGF alone had on MK colony-stimulating activity (MK-CSA) when either CD34+ DR+ or CD34+ DR- BM cells were cloned, but exhibited MK-CSA equivalent to that of interleukin-3 (IL-3) when LDBM cells were used as the target cell population. The MK-CSA of bFGF was inhibited by the addition of neutralizing antisera to either IL-3 and/or granulocyte- macrophage colony-stimulating factor (GM-CSF) but not IL-6. The addition of excess amounts of either IL-3 or GM-CSF to cultures containing bFGF plus anti-IL-3 or anti-GM-CSF reversed the inhibition by the corresponding antisera. The addition of bFGF and IL-3 to assays containing CD34+ DR+ or CD34+ DR- cells increased the size of both CFU- MK- and BFU-MK-derived colonies, respectively, when compared with assays containing IL-3 alone. This increase in MK colony size mediated by bFGF was not affected by addition of either an anti-GM-CSF or anti- IL-6 neutralizing antisera. When LDBM cells were assayed, bFGF alone increased CFU-MK-derived colony size when compared with control values. However, this potentiation of MK colony size by bFGF could be reversed by the addition of either anti-IL-3 or anti-GM-CSF but not anti-IL-6 antisera. In addition, the effects of bFGF and IL-3 on the size of MK colonies cloned from LDBM were not additive. These results suggest that bFGF affects human megakaryocytopoiesis by directly promoting MK progenitor cell proliferation and stimulating BM accessory cells to release growth factor(s) with MK-CSA, such as IL-3 and GM-CSF. We conclude that bFGF, likely produced by cellular components of the BM microenvironment, plays an important role in the control of human 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.     

Cytokine regulation of the human burst-forming unit-megakaryocyte

The human burst-forming unit-megakaryocyte (BFU-MK) is a primitive megakaryocytic progenitor cell. A marrow cell population enriched for BFU-MK (CD34+ DR-) was obtained by monoclonal antibody labeling and fluorescence-activated cell sorting. CD34+DR- cells were assayed in a serum-depleted, fibrin clot culture system. Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), recombinant interleukin-3 (rIL-3), and megakaryocyte colony-stimulating factor (MK-CSF), partially purified from human plasma, were each individually capable of promoting BFU-MK-derived colony formation. Recombinant erythropoietin, rG-CSF, rIL-4, rIL-6, and thrombocytopiesis stimulating factor, partially purified from human embryonic kidney cell conditioned media, had no stimulatory effect on BFU-MK-derived colony formation when added alone or in various combinations with either GM-CSF, IL-3, or MK-CSF, GM-CSF and IL-3, GM-CSF and MK-CSF, but not IL-3 and MK-CSF had additive actions in promoting BFU-MK-derived colony formation, rIL-1 alpha had no influence alone on BFU-MK cloning efficiency, but had a dose-dependent, synergistic effect with IL-3, but not with GM-CSF or MK-CSF. The synergistic relationship between IL-1 alpha and IL-3 was abrogated by addition of an IL-1 alpha neutralizing antibody but not by a GM-CSF neutralizing antiserum, suggesting that IL-1 alpha acts directly on the BFU-MK and not by stimulating marrow auxiliary cells to secondarily release additional cytokines. Information presented here indicates that the regulatory influence, acting on the different stages of megakaryocyte development, are stage-specific and accomplished by multiple cytokines.     

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.     

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.     

Effect of recombinant and purified human haematopoietic growth factors on in vitro colony formation by enriched populations of human megakaryocyte progenitor cells

Nonadherent low density T-lymphocyte depleted (NALT-) marrow cells from normal donors were sorted on a Coulter Epics 753 Dye Laser System using Texas Red labelled My10 and phycoerythrin conjugated anti HLA-DR monoclonal antibodies in order to obtain enriched populations of colony forming unit-megakaryocyte (CFU-MK). The CFU-MK cloning efficiency (CE) was 1.1 +/- 0.5% for cells expressing both high densities of My10 and low densities of HLA-DR (My10 DR+). This procedure resulted in an 18-fold increase in CE over NALT- cells. The effect of purified or recombinant human haematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1 alpha (IL-1 alpha), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF or CSF-1) and interleukin MK colony formation by My10 DR+ cells was determined utilizing a serum depleted assay system. Neither Epo, TSF, CSF-1, IL-1 alpha nor G-CSF alone augmented MK colony formation above baseline (2.5 +/- 0.8/5 x 10(3) My10 DR+ cells plated). In contrast, the addition of GM-CSF and IL-3 each increased both CFU-MK colony formation and the size of colonies with maximal stimulation occurring following the addition of 200 units/ml of IL-3 and 25 units/ml of GM-CSF. At maximal concentration, IL-3 had a greater ability to promote megakaryocyte colony formation than GM-CSF. The stimulatory effects of GM-CSF and IL-3 were also additive in that the effects of a combination of the two factors approximated the sum of colony formation in the presence of each factor alone. The CFU-MK appears, therefore, to express HPCA-1 and HLA-DR antigens. These studies also indicate that GM-CSF and IL-3 are important in vitro regulators of megakaryocytopoiesis, and that these growth factors are not dependent on the presence of large numbers of macrophages or T cells for their activity since the My10 DR+ cells are largely devoid of these accessory cells.     

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells.

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.     

Homing and clonogenic outgrowth of CD34(+) peripheral blood stem cells: a role for L-selectin?

OBJECTIVE: After transplantation of hematopoietic stem cells, adhesion molecules play a major role in the multistep process of engraftment in which L-selectin is suggested to be of relevance. A positive correlation previously was found between the number of reinfused L-selectin(+) stem cells and platelet recovery. In the present study, we determined the role of L-selectin in different engraftment steps, i.e., adhesion to endothelial cells, migration, and clonogenic outgrowth by in vitro assays that closely mimic the in vivo situation. MATERIALS AND METHODS: Flow adhesion and migration experiments were performed using the human bone marrow endothelial cell line 4LHBMEC and isolated peripheral CD34(+) cells with or without blocking of L-selectin-ligand interaction. Various clonogenic assays, including serum-free colony-forming unit-megakaryocytes (CFU-MK) and burst-forming unit-megakaryocytes (BFU-MK), were performed with sorted L-selectin(+)L-selectin(-) cells or in the presence of antibodies. RESULTS: Blocking of L-selectin on CD34(+) cells did not significantly affect rolling over and firm adhesion to 4LHBMEC. In addition, no role for L-selectin was found in transendothelial migration experiments. Finally, in clonogenic outgrowth of sorted or anti-L-selectin monoclonal antibody-incubated CD34(+) cells, no key role for L-selectin expression could be defined in BFU-MK and CFU-MK assays. CONCLUSION: Using in vitro assays for CD34(+) stem cell adhesion, migration, and clonogenic capacity, we were not able to define a major role for L-selectin.     

Low frequency of myeloid progenitor cells in chronic idiopathic neutropenia of adults may be related to increased production of TGF-beta1 by bone marrow stromal cells.

Previous studies in our laboratory have shown that patients with chronic idiopathic neutropenia of adults (CINA) have increased serum levels of inflammatory cytokines including IL-1beta. Since IL-1beta may affect bone marrow stromal cell function, a study was designed to investigate the capacity of patients' stromal cells to produce adequate amounts of haemopoietic growth factors or excessive amounts of inhibitors of myelopoiesis in long-term bone marrow cultures (LTBMCs). The study was carried out on 52 CINA patients and 19 normal controls. We found that CINA patients had significantly low numbers of marrow lineage-specific CD34+ cells, including CFU-GM and CD34+/CD33+ cells. Stromal cells from patients' LTBMCs failed to stimulate CFU-GM colony formation by normal marrow cells in a manner comparable to that of stromal cells of controls. Patients' LTBMC supernatants had normal or increased amounts of G-CSF. Detectable amounts of supernatant GM-CSF were found in 35% of patients and 19% of controls. IL-3 and MIP-1alpha were not detected in any supernatant fluid. Moreover, supernatants from patients' LTBMCs had increased concentrations of IL-6 and TGF-beta1, which strongly correlated with serum IL-1beta. About 82% of our patients had TGF-beta1 values higher than the upper limit of values found in the controls. Individual TGF-beta1 values inversely correlated with the number of circulating neutrophils and the frequency of marrow CD34+/CD33+ cells. We suggest that increased levels of serum IL-1beta, resulting from an underlying low-grade chronic inflammatory process, may stimulate marrow stromal cells to produce both haemopoietic growth factors and inhibitors of myelopoiesis. Since steady-state myelopoiesis results from a balance between negative- and positive-acting cytokines, it seems very probable that the increased production of TGF-beta1 by bone marrow microenvironment in CINA patients may suppress myelopoiesis and contribute, to some extent, to the pathogenesis of neutropenia in affected subjects.     

Effects of thrombocytopoiesis-stimulating factor on terminal cytoplasmic maturation of human megakaryocytes

Aplastic anemia serum (AAS) contains humoral factors that alter both proliferation and maturation of human megakaryocytes (MK). The ability of AAS to augment MK colony formation (colony-forming unit, CFU-MK) was neutralized by an antiserum against MK colony-stimulating factor (MK-CSF), a glycoprotein isolated from AAS. The adsorbed AAS still retained the ability to accelerate cytoplasmic maturation of recognizable MK. Similar experiments were done with thrombocytopoiesis-stimulating factor (TSF) and an anti-TSF antiserum to further define the activity in AAS responsible for accelerating cytoplasmic maturation. Bone marrow fractions enriched for recognizable human MK, but devoid of CFU-MK, were obtained by centrifugal elutriation and placed in short-term liquid cultures. MK progressed through identifiable maturation stages (1-4) more quickly in the presence of either TSF or AAS. TSF slightly enhanced the cloning efficiencies of CFU-MK, but did not alter the number of MK in individual colonies derived from non-adherent, low-density, T-cell-depleted bone marrow. In contrast, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and crude AAS substantially augmented both MK colony formation and cells per colony. TSF also doubled the percent 35S incorporation into platelets of immunothrombocythemic mice, but stimulation was completely abolished by anti-TSF. Anti-TSF antiserum was then used to analyze the promotion of MK colony formation by cytokines. Cloning efficiencies of CFU-MK were reduced to baseline values when TSF was pretreated with anti-TSF; however, the MK colony-stimulating activity (MK-CSA) of GM-CSF, IL-3, or AAS was not altered by adsorption with anti-TSF. In contrast, the cytoplasmic maturation of recognizable MK was slower, and fewer mature stage-4 cells were present at days 1-3 in AAS adsorbed with anti-TSF than MK cultured in AAS treated with normal rabbit serum or untreated AAS. Therefore, TSF appears to be a major factor in AAS that accelerates terminal maturation of human MK. TSF primarily affects megakaryocytopoiesis by promoting MK maturation rather than enhancing CFU-MK proliferation.     

Effect of interleukin 6 on in vitro human megakaryocytopoiesis: its interaction with other cytokines

The effects of human recombinant interleukin 6 (rIL-6) on in vitro human megakaryocytopoiesis were studied utilizing a serum-depleted culture system. Recombinant IL-6 increased both the number of megakaryocyte (MK) colonies formed and the number of cells comprising individual MK colonies cloned from normal low-density bone marrow (LDBM) cells. This stimulation of MK colony number and size was significantly less than that observed following the addition of recombinant interleukin 3 (rIL-3) or granulocyte-macrophage colony-stimulating factor (rGM-CSF). The addition of either rIL-3 or rGM-CSF, but not rIL-6 promoted MK colony formation by nonadherent, low-density, T-cell-depleted (NALDT-) marrow cells. Recombinant interleukin 1 alpha (rIL-1 alpha) and interleukin 4 (rIL-4) failed either to promote LDBM MK colony formation when added alone or to significantly increase rIL-6-promoted MK colony formation. MK colony formation promoted by optimal doses of rIL-6 was, in fact, significantly inhibited by rIL-1 alpha at all concentrations tested. Addition of either recombinant erythropoietin (rEpo) or purified thrombocytopoiesis-stimulating factor (TSF) to assays containing rIL-6 also resulted in significant inhibition of MK colony formation. The effect of suboptimal concentrations of rIL-6 on MK colony formation was additive to that of rIL-3 but not rGM-CSF. The addition of transforming growth factor beta (TGF-beta) resulted in a 58% reduction of rIL-6-promoted MK colony formation by LDBM. These data suggest that rIL-6 can promote in vitro megakaryocytopoiesis and that this effect can be either augmented or inhibited by the addition of several other cytokines. Recombinant IL-6, however, might affect the MK colony-forming unit (CFU-MK) by acting through bone marrow accessory cells or requiring the presence of as yet unidentified additional cytokines.     

Bone marrow progenitor cell reserve and function and stromal cell function are defective in rheumatoid arthritis: evidence for a tumor necrosis factor alpha-mediated effect

Based on previous reports for impaired hematopoiesis in rheumatoid arthritis (RA), and in view of the current interest in exploring the role of autologous stem cell transplantation (ASCT) as an alternative treatment in patients with resistant disease, we have evaluated bone marrow (BM) progenitor cell reserve and function and stromal cell function in 26 patients with active RA. BM progenitor cells were assessed using flow cytometry and clonogenic assays in short-term and long-term BM cultures (LTBMCs). BM stroma function was assessed by evaluating the capacity of preformed irradiated LTBMC stromal layers to support the growth of normal CD34(+) cells. We found that RA patients exhibited low number and increased apoptosis of CD34(+) cells, defective clonogenic potential of BM mononuclear and purified CD34(+) cells, and low progenitor cell recovery in LTBMCs, compared with healthy controls (n = 37). Patient LTBMC stromal layers failed to support normal hematopoiesis and produced abnormally high amounts of tumor necrosis factor alpha (TNF alpha). TNF alpha levels in LTBMC supernatants inversely correlated with the proportion of CD34(+) cells and the number of colony-forming cells, and positively with the percentage of apoptotic CD34(+) cells. Significant restoration of the disturbed hematopoiesis was obtained following anti-TNF alpha treatment in 12 patients studied. We concluded that BM progenitor cell reserve and function and BM stromal cell function are defective in RA probably due, at least in part, to a TNF alpha-mediated effect. The role of these abnormalities on stem cell harvesting and engraftment in RA patients undergoing ASCT remains to be clarified.     

Expression of CD41 and c-mpl does not indicate commitment to the megakaryocyte lineage during haemopoietic development

Haemopoietic progenitors with the phenotype expected of early megakaryocyte precursors (CD34+ CD41+) were isolated from normal human bone marrow or induced in culture from CD34+ CD41- bone marrow cells by treatment with thrombopoietin (TPO) or IL-3. We found that although this population included the majority of cells that can form CFU-MK in culture, it also contained both erythroid and myeloid progenitors. The clonogenic potential of the CD34+ CD41+-induced cells was greater than that of isolated CD34+ CD41+ cells in that the isolated cells only formed CFU-MK and BFU-e, whereas the induced cells formed myeloid colonies as well. Glycophorin was found on isolated CD34+ CD41+ cells, not on induced cells. Its presence distinguished between MK and erythroid progenitors. Separation of a CD34+ CD41+ glycophorin A+ population resulted in the isolation of a highly purified population of BFU-e. A major portion of the cells that expressed CD34+ CD41+, in either cohort, were of the erythroid lineage. True MK progenitors were present in the CD34+ population in greater proportion than in whole marrow and were further enriched amongst CD34+ populations that expressed CD41. The presence of the thrombopoietin (TPO) receptor, c-mpl, did not correlate with inducibility of the gpIIbIIIa complex since essentially all CD34+ progenitors, including the earliest identifiable human haemopoietic progenitors (CD34+ CD38- cells), expressed c-mpl mRNA detectable by PCR regardless of their ultimate fate. Thus neither the expression of CD41 nor the expression of c-mpl was predictive of commitment to the MK lineage.     

Effects of interleukin-3 and granulocyte-macrophage colony-stimulating factor on thrombopoiesis in congenital amegakaryocytic thrombocytopenia

Amegakaryocytic thrombocytopenia (AMT) is a rare and often fatal disorder of infancy and childhood presenting with isolated thrombocytopenia that progresses to marrow failure. The defect in thrombopoiesis is not well understood nor is the etiology of the progressive marrow failure. No standard modality of treatment exists. Here, we evaluated the capacity of marrow cells isolated from five patients with AMT and progressive marrow failure to generate megakaryocyte progenitor cells (CFU-MK). These in vitro studies demonstrated assayable numbers of CFU-MK from all patient bone marrows that responded in vitro to the addition of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or the combination of both. These findings suggest that the defect in AMT might be partially correctable by the administration of these cytokines. A Phase I/II trial of in vivo administration of these same hematopoietins in the identical patients was conducted in which no significant toxicity was observed. IL-3 but not GM-CSF administration resulted in improved platelet counts in two patients and decreased bleeding and transfusion requirement in the remaining three. No clinical benefit was observed when GM-CSF was administered after IL-3 pretreatment. Prolonged IL-3 administration has resulted in platelet increases in an additional two patients. In vitro responsiveness of CFU- MK to either cytokine did not predict the degree of clinical response. Although the optimal dose and schedule of IL-3 either alone or in combination remains to be established, this study suggests that IL-3 may contribute to the treatment of patients with AMT.     

Interleukin-6 and interleukin-11 act synergistically with thrombopoietin and stem cell factor to modulate ex vivo expansion of human CD41+ and CD61+ megakaryocytic cells

BACKGROUND AND OBJECTIVE: Thrombopoietin (TPO), the ligand for the c-mpl receptor, regulates in vivo platelet production and increases the number of colony-forming unit megakaryocytes (CFU-MK). Other cytokines including interleukin (IL) -3, IL-6, IL-11 and stem cell factor (SCF) can stimulate megakaryopoiesis. The aim of this study was to evaluate the effects of different combinations of cytokines involved in megakaryocytopoiesis on stroma-free liquid cultures of purified human CD34+ cells. DESIGN AND METHODS: Peripheral blood cells were collected after mobilization with granulocyte colony-stimulating factor (G-CSF). Purified CD34+ cells were then cultured with different combinations of TPO, SCF, IL-3, IL-6 and IL-11. RESULTS: The addition of TPO and SCF alone generated a population positive for the antigens CD41 (5.5+/-2.9%) and CD61 (6. 1+/-2.2%) but induced a low amplification of cell number (8.1+/-0.9 fold expansion). The presence of IL-6 or IL-11 was associated with MK progenitor cell expansion, and up to 7-10% of cultured cells were found to be CD41 and CD61 positive by flow cytometry. Conversely, the addition of IL-3 to this cytokine combination was associated with a prominent expansion of the myeloid lineage (70+/-10% of CD33+ cells) but only 0.9% and 2% of cultured cells were positive for CD61 and CD41 respectively. INTERPRETATION AND CONCLUSIONS: Our study supports the idea that IL-6 and IL-11 play crucial roles in the proliferation of MK progenitors and the use of SCF, TPO, IL-6 and IL-11 for ex vivo expansion of this cell population.     

Platelet-derived growth factor enhances ex vivo expansion of megakaryocytic progenitors from human cord blood.

Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is a strategy for shortening the duration of thrombocytopenia after haematopoietic stem cell transplantation. The cell dose after expansion has emerged as a critical factor for achieving the desired clinical outcomes. This study aimed to establish efficient conditions for the expansion of the MK lineage from enriched CD34(+) cells of umbilical cord blood and to investigate the effect of platelet-derived growth factor (PDGF) in this system. Our results demonstrated that thrombopoietin (TPO) alone produced a high proportion of CD61(+)CD41(+) cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of interleukin-1 beta (IL-1 beta), Flt-3 ligand (Flt-3L) and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400-fold with the highest yield observed in the presence of TPO, IL-1 beta, IL-3, IL-6 and Flt-3L. CD34(+) cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61(+)CD41(+) cells, CFU-MK and CD34(+) cells increased by 101%, 134% and 70%, respectively. On day 14, the CD61(+) population consisted of diploid (86.5%), tetraploid (11.8%) and polyploid (8N--32N; 1.69%) cells. Their levels were not affected by PDGF. TPO, IL-1 beta, IL-3, IL-6, Flt-3L and PDGF represented an effective cytokine combination for expanding MK progenitors while maintaining a moderate increase of CD34(+) cells. This study showed, for the first time, that PDGF enhanced the ex vivo expansion of the MK lineage, without promoting their in vitro maturation. PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications.     

Modulation of megakaryocytopoiesis by human basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) may act to modulate hematopoiesis in addition to its effects on mesenchymal cells. We studied the effects of bFGF on human and murine primary marrow megakaryocytes. bFGF modestly enhanced the size of the human megakaryocyte colony-forming unit (CFU-MK) and cell numbers per colony, in combination with interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF). Adhesion of human megakaryocytes to bone marrow (BM) stromal fibroblasts was enhanced when either stromal fibroblasts or megakaryocytes were treated with bFGF. This resulted in significantly increased proliferation of megakaryocytes. In addition, bFGF augmented secretion of the cytokines tumor necrosis factor alpha and IL-6 by human primary BM megakaryocytes. Immature murine megakaryocytes showed a significant growth response to bFGF as measured by the single cell growth assay. This effect was abrogated by specific antibodies for bFGF and combination of anti-IL-6 and anti-IL-1 beta antibodies. bFGF has no effect on murine CFU-MK formation, but significantly potentiated CFU-MK formation in the presence of IL-3 or GM-CSF. These results indicate that the effect of bFGF on various megakaryocyte populations is different and that bFGF may affect megakaryocytopoiesis via modulation of megakaryocyte-stromal interactions and via augmentation of cytokine secretion from megakaryocytes.     

Study of megakaryocytic progenitors (CFU-MK) in human long-term bone marrow cultures (LTBMC): adjuvant effect of plasma from aplastic patients

Human long-term bone marrow cultures (LTBMC) provide a very interesting tool for studying the events that are involved in stem cell commitment. At the present time, megakaryocyte (MK) progenitor cells have never been demonstrated in this system. In an effort to detect this cell lineage, we modified the culture medium by substituting fetal calf serum (FCS) and horse serum (HS) mix with human plasma obtained from treated aplastic leukemic patients. This plasma was harvested between days 15 and 21 following induction chemotherapy or conditioning regimen for autograft or allogeneic bone marrow transplantation. Using LTBMC, 17 normal marrows were cultivated for 11 weeks in Iscove's modified Dulbecco's medium containing either 20% human aplastic plasma or control FCS/HS mixture. In this plasma medium we observed the development of an adherent layer morphologically comparable to that observed with standard medium. We demonstrated presence of MK cells at all stages of maturation for 10 weeks and MK colony-forming cells (CFU-MK) for 11 weeks in the culture supernatants. An increased production of nonadherent cells and granulocyte-macrophage progenitors (CFU-GM) was also observed. LTBMC in aplastic plasma medium provide a new method for studying megakaryocytopoiesis, especially in human hematological diseases.     

Lack of compensatory megakaryocytopoiesis in HIV-1-seropositive thrombocytopenic individuals compared with immune thrombocytopenic purpura patients.

In this study we demonstrate that HIV-1-seropositive thrombocytopenic individuals, in contrast with immune thrombocytopenic purpura (ITP) patients, fail to have a compensatory increase of megakaryocytopoiesis. The in vitro growth of bone-marrow megakaryocyte progenitors (CFU-MK) and the production of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 and IL-6 by bone-marrow mononuclear adherent cells and peripheral blood (PB) light-density mononuclear cells were studied in 12 HIV-1-seropositive thrombocytopenic individuals with respect to 12 ITP patients and 15 normal controls. In HIV-1-seropositive thrombocytopenic individuals, CFU-MK size (number of megakaryocytes per colony) was similar to normal controls but significantly lower (P less than 0.05) than in ITP patients. IL-1 and IL-6 production was similar in the three groups of subjects. On the other hand, GM-CSF production by bone-marrow mononuclear adherent cells in HIV-1-seropositive thrombocytopenic individuals was similar to normal controls but significantly (P less than 0.05) lower than in ITP patients, whereas GM-CSF production by PB light-density mononuclear cells was markedly (P less than 0.05) defective compared with both normal controls and ITP patients. The positive correlation between number and size of CFU-MK and production of GM-CSF by bone-marrow mononuclear adherent cells, observed in all three groups of subjects, demonstrates the central role of GM-CSF in the control of megakaryocytopoiesis.