Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures

We previously showed that basic fibroblast growth factor (bFGF) is a potent mitogen for human bone marrow (BM) stromal cells and significantly delays their senescence. In the present study, we demonstrated that low concentrations of bFGF (0.2 to 2 ng/mL) enhance myelopoiesis in long-term human BM culture. Addition of bFGF to long-term BM cultures resulted in an increase in (a) the number of nonadherent cells (sixfold), particularly those of the neutrophil granulocyte series; (b) the number of nonadherent granulocyte colony-stimulating factor (G-CSF)- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive progenitor cells; (c) the number of adherent foci of hematopoietic cells (10-fold); and (d) the number of progenitor cells in the adherent stromal cell layer. These effects were not noted with higher concentrations of bFGF (20 ng/mL). Thus, low concentrations of bFGF effectively augment myelopoiesis in human long-term BM cultures, and bFGF may therefore be a regulator of the hematopoietic system in vitro and in vivo.     

Thrombin-induced release of active basic fibroblast growth factor- heparan sulfate complexes from subendothelial extracellular matrix

The angiogenic factor, basic fibroblast growth factor (bFGF), is sequestered and protected by binding to heparan sulfate proteoglycans (HSPG) in the subendothelial extracellular matrix (ECM). Release of ECM- bound bFGF provides a novel mechanism for regulation of cell proliferation and neovascularization in normal and pathologic situations. Exposure of ECM to thrombin, the final activation product of the clotting cascade, resulted in release of high molecular weight HSPG-bFGF complex, as indicated by its immunoprecipitation with anti- bFGF antibodies, susceptibility to degradation by bacterial heparinase, and inhibition of its mitogenic activity in the presence of neutralizing anti-bFGF antibodies. The ECM-resident bFGF-HSPG complex was not released by thrombin in the presence of hirudin or antithrombin III, or by catalytically blocked thrombin preparations. A threefold to fivefold higher mitogenic activity was released by thrombin from ECM that was preheated (1 hour, 80 degrees C), as compared with native ECM. This difference is attributed to heat stable bFGF-HSPG complexes that are more readily released after heat treatment of the ECM and to activation and release of ECM-resident transforming growth factor-beta (TGF-beta) activity. Our results indicate that the large reservoir of proteolytic activity present in plasma in the form of prothrombin may participate in release from the subendothelial ECM of biologically active bFGF and TGF-beta, depending on the accessibility of thrombin. Thrombin may gain access to the subendothelium on clot formation after tissue injury and as a result of the conversion of prothrombin to thrombin induced by the ECM itself.     

Effects of fibroblast growth factor-4 (k-FGF) on long-term cultures of human bone marrow cells

Fibroblast growth factor-4 (FGF-4), a highly mitogenic protein encoded by the k-fgf/hst oncogene, stimulates the growth of a variety of cells of mesenchymal and neuroectodermal origin. Addition of FGF-4 to human long-term bone marrow cultures increased both the cell density of the stromal layer and the number of hematopoietic colony forming cells in the cultures in a dose-dependent manner. Hematopoiesis in the stromal layer persisted for up to 8 months. Erythropoiesis was maintained for up to 4 weeks, but granulocytes were the predominant nonadherent cell type. Cultures treated with FGF had increased numbers of monocytes compared with control cultures and some CD14+, CD45+ monocytes could still be detected after 8 months of continuous culture. The addition of the growth factor increased the rate of growth of the stromal layer and appeared to delay its senescence. Subcultures made in the presence of FGF-4 had up to 10-fold increases in plating efficiency and grew as relatively uniform monolayers. These subcultures retained the capacity to support hematopoiesis for several months, while untreated subcultures, made without FGF-4, grew erratically and generally lost the capacity to support hematopoiesis within 4 to 6 weeks. The improved growth after subculture greatly enhanced the reliability of limit- dilution assays of multipotential hematopoietic stem cells that use stromal cell monolayers. The primary effect of FGF-4 appeared to be on the stromal cells of the long-term bone marrow cultures, but a direct effect on hematopoietic progenitors could not be ruled out.     

Plasma basic fibroblast growth factor and bone marrow fibrosis in clonal myeloproliferative disorders

Basic fibroblast growth factor (bFGF) is an important growth factor involved in clonal hematopoietic expansion, neoangiogenesis, and bone marrow fibrosis, all of which are important pathobiologic features of clonal chronic myeloproliferative disorders (CMPD) and myelodysplastic syndromes (MDS). The aim of this study was to assess circulating bFGF concentrations in patients with CMPD and MDS with respect to the presence of bone marrow fibrosis in histopathologic examination. The study group comprised 18 patients with CMPD (six female, 12 male; median age 50 years), seven patients with MDS (one female, six male; median age 66 years) and 10 healthy adults as controls (four female, six male; median age 29 years). CMPD group included six chronic myelogenous leukemia (CML), seven essential thrombocythemia (ET), three polycythemia vera (PV), two agnogenic myeloid metaplasia (AMM). All seven MDS patients were the FAB subtype of refractory anemia (RA). Bone marrow biopsy sections stained with hematoxylin and eosin (H & E) and for reticulin were examined for the presence of fibrosis. The median plasma bFGF level was 18.2 pg/ml (interquartile range, IQR: 15.2-26.7) in patients with CMPD, 18.0 pg/ml (IQR: 15.8-26.4) in patients with MDS, 13.6 pg/ml (IQR: 9.9-20.0) in the control group. The bFGF levels were significantly higher in patients with CMPD in comparison with the healthy control group (P = 0.031). Circulating bFGF tended to be significantly lower in relation to the development of marrow fibrosis (P = 0.028). The complicated interactions of bFGF and fibrosis in the context of CMPD may be either 'cause' or 'effect'. The bFGF might represent an important link between angiogenesis, fibrosis, and clonal neoplastic hematopoiesis during the development of CMPD.     

Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor.

Expression cloning of cDNAs encoding a basic fibroblast growth factor (FGF) binding protein confirms previous hypotheses that this molecule is a cell-surface heparan sulfate proteoglycan. A cDNA library constructed from a hamster kidney cell line rich in FGF receptor activity was transfected into a human lymphoblastoid cell line. Clones expressing functional basic FGF binding proteins at their surfaces were enriched by panning on plastic dishes coated with human basic FGF. The amino acid sequence deduced from the isolated cDNAs revealed several interesting features, including hydrophobic signal and transmembrane domains that flank an extracellular region containing six potential attachment sites for glycosaminoglycan side chains. The structure also contains a short hydrophilic cytoplasmic tail sequence homologous to previously reported actin binding domains. Binding of basic FGF to cells expressing the binding protein could be inhibited by heparin and heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. In addition to binding basic FGF, this protein or related surface proteins may function as an initial cellular attachment site for other growth factors and for viruses, such as herpes simplex virus.     

Circulating basic fibroblast growth factor declines during Cy/TBI bone marrow transplantation.

Basic fibroblast growth factor (bFGF) inhibits radiation-induced apoptosis, and radioprotects haematopoietic, cartilage growth plate, pulmonary and gastrointestinal tissues. Conversely, chronic overexpression of bFGF may promote fibrosis. We measured the endogenous circulating bFGF in blood of patients undergoing conditioning TBI. Twenty-six patients with haematopoietic malignancies were conditioned with cyclophosphamide/TBI for allogeneic BMT. Daily blood samples were collected each morning prior to, during, and for several days after TBI. bFGF levels in plasma of normal volunteers are 0.8-26 pg/ml. bFGF was below detectability in 22%, 30% and 45% of patients pre-TBI, during TBI or post-TBI respectively. Mean circulating plasma levels of bFGF decreased from a median of 52 pg/ml pre-TBI to 26 pg/ml during TBI, and to 5 pg/ml post-TBI. Among the 26 patients, 13 had more than one non-detectable plasma bFGF level, an additional five had at least one non-detectable level, and only eight patients had detectable levels in all daily samples. Naturally high levels of bFGF were observed in some patients undergoing fractionated TBI. In contrast, as many as 79% of patients had low bFGF levels in one or more samples. The impact of endogenous bFGF on the tolerance of normal tissues to irradiation is unknown, and warrants further study.     

Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.     

Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells

We have shown previously that basic fibroblast growth factor (bFGF) is a mitogen for human bone marrow (BM) stromal cells and that bFGF stimulates myelopoiesis in primary BM cultures. In this article, we demonstrate the presence of bFGF in two cell lineages in human BM and peripheral blood as well as the deposition of bFGF into the extracellular matrix of BM stromal cell cultures. In immunofluorescence experiments on BM and peripheral blood smears, megakaryocytes and platelets stained strongly for bFGF, whereas weaker staining was observed in immature and mature cells of the granulocyte series. The presence of bFGF in platelets was confirmed by enzyme-linked immunosorbent assay as well as by immunoprecipitation followed by immunoblotting. bFGF was synthesized by BM stromal cell cultures and was found either cell associated or localized in the nucleus and the nucleoli, and its location was dependent on the fixation procedure used. Addition of exogenous bFGF to stromal cells showed the presence of extracellular binding molecules for this cytokine. bFGF could be released from these sites by soluble heparin or phosphatidylinositol- specific phospholipase C. This study supports the role of bFGF as a stromal cell mitogen and stimulator of myelopoiesis. The data indicate that the stromal cells produce bFGF and that their extracellular matrix can serve as a reservoir for this growth factor. In addition, the results suggest a possible involvement of bFGF in platelet function as well as in megakaryocytopoiesis.     

A method to establish pure fibroblast and endothelial cell colony cultures from murine bone marrow

Studies on the effect of the microenvironment on hematopoiesis would benefit from the availability of pure populations of nontransformed cells of each of the stromal cell types. The adherent murine bone marrow stromal cell population in this study consisted of fibroblasts, endothelial cells, and macrophages. Fibroblasts were segregated from the phagocytic endothelial cells and macrophages by allowing the phagocytic cells to ingest magnetic beads, with subsequent exposure to a magnetic field, effecting cell separation. Pure colony cultures of fibroblasts and endothelial cells were formed by varying the bead-to-cell ratio and incubation period of the cells. For complete purification of the fibroblasts, subsequent passaging was also necessary. Near confluent growth of each type was obtained with subsequent passages and sustained culture. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance endothelial cell growth. We were not able to obtain pure populations of bone marrow macrophages in near confluent culture. The three cell types were identified by cellular morphology, acid and alkaline phosphatase staining, binding with the lectins Ulex europaeus and Bandeiraea simplicifolia, and the capacity to stain for the factor VIII-related antigen (von Willebrand's Factor).     

Regulation of proteolytic activity in human bone marrow stromal cells by basic fibroblast growth factor, interleukin-1, and transforming growth factor beta.

Plasminogen activators (PAs) and/or plasmin may be involved in hematopoietic regulation. These enzymes release biologically relevant cytokines such as basic fibroblast growth factor (bFGF) from matrix and cell surfaces. In addition, transforming growth factor beta (TGF beta) and interleukin-1 beta (IL-1 beta) are converted from inactive to active forms by plasmin. Therefore, we studied the regulation of PAs and their specific inhibitors, PA inhibitor 1 (PAI-1) and PA inhibitor 2 (PAI-2), in human bone marrow stromal fibroblasts by IL-1 beta, bFGF, and TGF beta. All three cytokines stimulated PA secretion. IL-1 beta at 10(4) U/mL increased urokinase (u-PA) levels approximately 10-fold, bFGF at 0.2 ng/mL also increased production 10-fold, but increased predominantly tissue PA (t-PA) expression. TGF beta at 0.2 ng/mL increased u-PA production up to 300-fold. PAI-1 and PAI-2 are also regulated by these cytokines. IL-1 beta decreased PAI-1 levels by 50% and stimulated PAI-2 levels sixfold. bFGF had minimal effects on PAI-1 and TGF beta increased PAI-1 levels twofold. Neither of these agents had an effect on PAI-2 levels. Thus, three cytokines relevant to bone marrow physiology regulate PA and inhibitor production by human bone marrow stromal fibroblasts. In this manner PA and plasmin generation in specific microenvironments in the bone marrow may be one of the factors orchestrating the complex series of events, which results in an efficient exquisitely regulated hematopoietic process.     

Cellular distribution of platelet-derived growth factor, transforming growth factor-beta, basic fibroblast growth factor, and their receptors in normal bone marrow

We performed an immunohistochemical study on 5 normal marrow samples and 3 fibrotic marrow samples to investigate the cellular distribution of various isoforms of platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (bFGF), and their corresponding receptors. Immature hematopoietic precursors strongly expressed PDGF-B, all the TGF-beta isoforms, PDGFRbeta, TGFbetaRI and II, and FGFR1, 3, and 4. Megakaryocytes stained primarily for PDGF-B, TGF-beta2-3, and PDGFRbeta. Histiocytes displayed intense TGF-beta1, bFGF, and FGFR2 expression. Fibroblasts and endothelial cells carried receptors for all the aforementioned cytokines. The last 2 cell types also expressed the ligand cytokines to varied degrees.     

Human herpesvirus-6-associated suppression of growth factor-induced macrophage maturation in human bone marrow cultures

Cultures of marrow mononuclear cells were exposed to medium derived from cell cultures infected with several different strains of human herpesvirus-6 (HHV-6) immediately before the addition of either of two growth factors, granulocyte-macrophage colony-stimulating factor and interleukin-3. Exposure to any of the viral preparations suppressed the outgrowth of nonspecific esterase-positive adherent macrophages induced by the factors by more than 90%. The nonadherent cell populations in the infected cultures were numerically similar to those in uninfected control cultures, demonstrating the absence of a nonspecific cytotoxic effect of the viral materials. Infectious virus was not necessary for the macrophage outgrowth suppression. These findings suggest that HHV-6 either encodes or induces a soluble mediator or mediators that can interfere with the responses of bone marrow to growth factors and possibly block the normal differentiation of macrophages from marrow precursors.     

Interleukin-2 inhibits growth of fibroblasts derived from human bone marrow

The adherent layer that forms in human bone marrow suspension cultures contains fibroblast colonies which arise from colony-forming cells (CFU-F). We have demonstrated that recombinant interleukin-2 (r-IL2) inhibits growth of CFU-F by preventing their entry into S phase. This inhibition is not mediated by gamma-interferon, nor by T lymphocytes, and can be abrogated by anti-IL2 receptor antibody. r-IL2 may influence haematopoiesis by its effect on bone marrow CFU-F.     

Production of T lymphocyte colony-forming units from precursors in human long-term bone marrow cultures

T cell differentiation in human marrow was studied in Dexter type long- term bone marrow cultures. In these cultures, T lymphocyte colony- forming units (TL-CFU), E rosette-forming cells (E+), and T3+, T4+, and T8+ cells (assayed by indirect immunofluorescence) were found to be present for at least 7 weeks. It was investigated whether the existence of T cells in long-term culture resulted from the persistence of inoculated T lymphocytes or from the production by immature progenitors. No significant numbers of E+, T3+, T4+, or T8+ cells were detected in cultures that were established from E+ lymphocyte-depleted bone marrow, indicating little or no production of T lymphocytes from E- negative precursors. On the other hand, bone marrow cells purged of E+ lymphocytes did not contain TL-CFU, but appeared to regain high numbers of TL-CFU during Dexter culture; this suggested that an earlier step in T cell differentiation may take place in this culture system. The generation of TL-CFU in the E-negative long-term marrow cultures only occurred when an adherent stroma layer had been established in the culture flask; it did not require added mitogens or detectable interleukin 2 in the culture medium. TL-CFU in fresh marrow (TL-CFU II) are mature (E+, T3+) T cells and are capable of producing helper (T4+) and suppressor/cytotoxic (T8+) phenotype cells in colonies. The TL-CFU newly formed in E-depleted Dexter cultures (TL-CFU I) are distinct from this population, as they are E-negative and give rise to colonies of the helper type only. T3 cell depletion of the marrow inoculum prior to culture did not prevent the appearance of TL-CFU I in long-term culture; this suggests that TL-CFU I are derived from an E- and T3- precursor (pre-TL-CFU).     

Regulation of lymphoid colony growth by factors derived from human bone marrow

We have previously demonstrated the ability of soluble factors derived from cultured murine and human bone marrow supernatants to modulate a variety of lymphoid functions, including DNA synthesis. In the present report, we show that human marrow supernatants contain a suppressor factor (BSF) that suppresses T-lymphoid colony formation, and an augmenting factor (BEF) that enhances T-colony growth. BSF suppression exhibits no tissue specificity, affecting marrow-derived and peripheral T colonies similarly. The suppressive activity occurs prior to mitogenic activation by TCGF. In contrast, a preferential augmentation of the size and number of marrow-derived T-cell colonies, as compared to peripheral T-cell colonies, was observed in the presence of BEF. BEF required prior mitogen activation of the colony inocula to effect colony enhancement. In addition, the response to BEF was greater for E- than for E+ colony-forming cells, indicating the target of BEF activity to be an early T cell. The active subfraction of BEF with colony-enhancing activity was found to be between 8000 and 30,000 daltons.     

Stromal colonies can be grown from the non-adherent cells in human long-term bone marrow cultures

The absence of a pluripotent stem cell assay for human cells means that we must rely on assays of committed progenitors as a means for examining the ability of long-term bone marrow culture (LTBMC) to support haemopoiesis. The CFU-GM assay has been employed by many authors to demonstrate that the proliferative capacity and differentiation potential has been retained in LTBMC. In this study the non-adherent cells from human LTBMC were set up using a liquid culture technique for the clonal proliferation of stromal colonies (CFU-F). Weekly plating of the non-adherent cells produced CFU-F for up to 7 wk. Morphological, cytochemical and antigenic characterisation of these colonies revealed that they were identical to those grown from bone marrow de novo. LTBMC can now be used to identify the interactions between the haemopoietic inductive microenvironment and stromal progenitors and allow us to investigate the precise role of cell-cell interactions or cytokine influences on the proliferative capacity of stromal cell progenitors.     

Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow

Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, their multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm(2)) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FACS analysis demonstrated that stationary cultures contained a major population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS-1 cells. During the lag phase, the RS-1 cells gave rise to a new population of small and densely granular cells (RS-2 cells). During the late log phase, the RS-2 cells decreased in number and regenerated the pool of RS-1 cells found in stationary cultures. In repeated passages in which the cells were plated at low density, they were amplified about 10(9)-fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation.     

Cloned stromal cell lines derived from human Whitlock/Witte-type long-term bone marrow cultures.

We report a human bone marrow culture technique that initially parallels the murine Whitlock/Witte culture system. As in the murine system, B cells predominate over other cell types, and all differentiation stages from pre-B to plasma cell are observed. Although these human long-term cultures pass through stages resembling phases I to III of murine Whitlock/Witte cultures, no outgrowth of nonadherent cells was seen after cultures had reached the "crisis" phase unless Epstein-Barr virus (EBV)-transformants appeared. The stromal cells persisted well beyond crisis, but they could not be maintained and passaged as cell lines, limiting their use in molecular analysis. Transfection of these stromal cells with plasmid DNA containing the simian virus 40 (SV40) early region yielded 124 cloned cell lines. Analysis of these lines showed that all expressed SV40 large T antigen, but they retained most phenotypic markers found on non-transformed stromal cells. When adherent and T-cell-depleted bone marrow cells were cultured on either nontransformed stromal layers or transformed cell lines they proliferated actively and soon yielded predominantly lymphoid nonadherent populations. Moreover, prolonged survival of acute lymphoblastic leukemia cells of pre-B phenotype was regularly achieved on both normal and transformed adherent cell layers. Although the liquid culture system favored lymphocytes, transformed stroma supported colony formation by both human and murine hemopoietic progenitors when marrow was added in agar medium. This was not explained by colony-stimulating factor (CSF) production, because striking heterogeneity in the levels of granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) secretion by the lines was noted. Some lines that did not produce detectable CSF demonstrated good support of fresh bone marrow growth and acute lymphoblastic leukemia (ALL) cell survival. The heterogeneity of these cell lines and their capacity to support hemopoiesis suggest that they will be useful in studying the molecular basis of in vitro lymphohemopoiesis in man.     

Angiogenic therapy of acute myocardial infarction by intrapericardial injection of basic fibroblast growth factor and heparin sulfate: An experimental study

To examine whether angiogenesis and myocardial salvage occur, 30 μg basic fibroblast growth factor (bFGF) and 3 mg heparin sulfate (HS) were injected through the right atrium into the pericardial cavity by a thin needle-tipped catheter in a canine model of acute myocardial infarction. One month later infarcted weight/left ventricle weight was 24% ± 5.2%, 25% ± 4.0%, 18% ± 2.4%, and 10% ± 1.8% (mean + SE) in saline solution, HS, bFGF alone, and bFGF plus HS groups, respectively. Vascular number in the infarcted area of the outer layer was 13 ± 3.3, 20 ± 2.2, 47 ± 8.3, and 136 ± 26.3/200 × 200 μm² in saline solution, HS, bFGF alone, and bFGF plus HS groups, respectively. Thus the vascular number was the largest in the bFGF plus HS group. The vascular number was larger in the subepicardial than in the subendocardial infarcted areas. Vessels directed from the epicardium toward the subepicardial infarcted area were also observed. The transcatheter intrapericardial injection of bFGF plus HS caused angiogenesis and myocardial salvage. This method might bring about a selective therapeutic and preventive modality of myocardial infarction irrespective of coronary anatomy and contraindications for coronary interventions and surgery.