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Biomedical subjects

M Y Gordon

Publications and source records attributed to M Y Gordon.

At least 19 recordsLinked to original sources
PubMed1992

Binding of primitive hematopoietic progenitor cells to marrow stromal cells involves heparan sulfate.

Blast colony-forming cells (BI-CFC) and pre-colony-forming unit-granulocyte, monocyte (CFU-GM) in human bone marrow bind to marrow-derived stromal layers grown in the presence of methylprednisolone (MP+), but do not bind to stroma grown without MP (MP-). The BI-CFC bind to stroma and form colonies when overlaid with agar; the pre-CFU-GM bind to stroma and release CFU-GM into the supernatant culture medium (delta assay). These two classes of progenitor may represent similar stages of hematopoietic cell development. Their binding to stroma depends on the presence of heparan sulfate proteoglycan (HS-PG) in the extracellular matrix secreted by the stromal cells. Here, we have analyzed the functional and biochemical properties of HS-PG isolated from MP+ and MP- stromal cultures. HS-PG or isolated HS glycosaminoglycan (GAG) side chains partially blocked progenitor cell binding when they were added to the 2-hour binding phase of the BI-CFC or delta assays. Gel electrophoresis of HS-PG resolved more bands in matrix preparations from MP+ cultures than in preparations from MP- cultures. The blocking activity of the eluted MP+ HS-PG bands depended partly on the amount of GAG attached to the protein core and presumably partly on the structure of the core itself. Time course studies demonstrated that the HS-dependent phase of the binding interaction was limited to the first 30 to 60 minutes of the 2-hour binding phase. The different blocking effects of MP+ and MP- HS indicate that they have different biochemical properties. The HS-GAG in MP+ stroma has a higher degree of sulfation and a greater negative charge to mass ratio compared with MP- HS-GAG. Variations in HS may determine specific binding by hematopoietic progenitor cells and a heparan sulfate receptor is envisaged as acting in concert with further cell adhesion molecules (CAMs) on the progenitor cell surface.

Adult↗
PubMed1992

Circulating stem cells in mice treated with cyclophosphamide.

Chemotherapy has been used clinically to mobilize hematopoietic progenitor cells into the peripheral blood so that they can be harvested for autologous transplantation. In humans, this is demonstrated by the presence of circulating granulocyte-macrophage colony-forming cells (CFU-GM) and CD34-positive cells, but it has not been possible to confirm the presence of marrow-repopulating stem cells. In this study, we treated mice with 200 mg/kg cyclophosphamide (CY) and measured the numbers of white blood cells, day 12 CFU-S (CFU-S12), and CFU-GM in the peripheral blood. There was a peak in the numbers of CFU-S12 and CFU-GM 8 days after treatment with cyclophosphamide. Peripheral blood cells taken at this time rescued lethally irradiated mice and engraftment of donor cells was confirmed after 140 days in sex mismatched recipients using a Y chromosome-specific probe. In vitro culture of the blood cells harvested after cyclophosphamide showed that they proliferated in suspension cultures for at least a year in the presence of interleukin-3. The cultured cells rapidly lost their abilities to rescue irradiated mice and to form colonies in vitro, but they did not become leukemic. Also, CY-treated mice were irradiated with a leukemogenic dose of x-rays to coincide with peak circulating cell numbers but these animals did not develop an excess of leukemias over mice given irradiation alone.

Animals↗
PubMed1992

Synthesis and deposition of glycosaminoglycans in the murine hemopoietic stromal line S17: modulators of the hemopoietic microenvironment.

The murine hemopoietic stromal cell line S17 can support either myelopoiesis or lymphopoiesis depending on the culture conditions (i.e., the presence of steroid or mercaptoethanol). The glycosaminoglycans are important components of the extracellular matrix, which influence hemopoietic cell proliferation. Accordingly, glycosaminoglycans have been compared under different growth conditions. Under myeloid conditions (with steroid) a higher proportion of the sulfated glycosaminoglycans was incorporated into the cell layer and the extracellular matrix was increased, whereas synthesis was reduced under lymphoid conditions (with mercaptoethanol). The inclusion of steroid or mercaptoethanol did not alter the nature of the heparan sulfate synthesized as shown by DEAE anion-exchange chromatography, cleavage with specific enzymes and resolution of the digestion products by gel electrophoresis (oligomapping), and glycosaminoglycan size. The major species of sulfated proteoglycan synthesized under the different growth conditions (200 and 110 kd for the culture supernatant and 110, 71, and 38 kd for the cell layer) were shown to be very similar by polyacrylamide gel electrophoresis. Although no qualitative difference was found biochemically between the major glycosaminoglycans/proteoglycans, scanning electron microscopy revealed major differences in the pattern of deposition of the glycosaminoglycans. Under myeloid conditions, a rich fibrous matrix covered the cell layer, whereas under lymphoid conditions glycosaminoglycan was sparsely deposited. The arrangement of the extracellular matrix may have important consequences for myelo- and lymphopoiesis.

Animals↗
PubMed1992

Physical, phenotypic and cytochemical characterisation of stroma-adherent blast colony-forming cells.

Primitive cells defined as long-term culture initiating cells (LTCIC) and blast colony-forming cells (Bl-CFC) bind to cultured stromal layers, but cells at later stages of maturation [granulocyte-erythroid-macrophage-monocyte colony-forming cells (GEMM-CFC) granulocyte-macrophage (CM-CFC) and erythroid burst-forming units (BFU-E)] do not. The precise relationship between the LTCIC and Bl-CFC is not known and this study was undertaken to determine their relative positions in the haemopoietic hierarchy. We have defined the Bl-CFC population in terms of its density profile and antigenic phenotype and compared these characteristics with GM-CFC and BFU-E. The progenitor cell populations did not differ in density. The major phenotypic difference was seen using the myeloid monoclonal antibody S17-25 which reacted with fewer Bl-CFC than GM-CFC. Also, we have cytochemically analysed the cells in colonies derived from Bl-CFC. Our studies indicate that the Bl-CFC precede BFU-E and GM-CFC but not the LTCIC.

Antigens, Differentiation↗
PubMed1991

The effects of interferon-alpha on the proliferation of CML progenitor cells in vitro are not related to the precise position of the M-BCR breakpoint.

We investigated the effects of brief (2 h) and continuous exposure to recombinant interferon-alpha (2a) (rIFN-alpha) on the proliferation of primitive (blast colony-forming cells, Bl-CFC) and committed myeloid progenitor cells (BFU-E and GM-CFC) derived from blood and bone marrow of patients with chronic myeloid leukaemia (CML) and normal subjects. In all three clonogenic assays, rIFN-alpha suppressed colony formation in a dose-dependent manner. No differences were detected in the proliferation of CML or normal Bl-CFC and GM-CFC exposed to rIFN-alpha. Erythroid colony formation by normal, but not by CML BFU-E, was inhibited by relatively low concentrations (100 U/ml) of rIFN-alpha. However, in patients whose blood or marrow contained a mixture of Philadelphia chromosome (Ph)-positive and Ph-negative BFU-E, cytogenetic analysis of individual erythroid colonies showed no differential inhibition by rIFN-alpha. We found no difference in the sensitivity to rIFN-alpha of GM-CFC from patients whose leukaemic cells expressed BCR/ABL mRNA with the b2a2 junction and that of GM-CFC from patients with the b3a2 mRNA. We conclude that (1) rIFN-alpha does not have a significant leukaemia-specific effect on the progenitor cells detected in these assays, and (2) the sensitivity of CML GM-CFC to rIFN-alpha is independent of the type of BCR/ABL message present in the cells. The clinical efficacy of rIFN-alpha could be due to selective toxicity to cells not assayed in this study, to effects on accessory cells or to alterations induced in progenitor cell/stromal cell interactions.

Blotting, Southern↗
PubMed1991

Production of heparan sulphate proteoglycans by human bone marrow stromal cells.

Haemopoietic progenitors from human bone marrow bind strongly to human marrow stromal cell cultures but the interaction only occurs if the stromal cells are maintained in methyl prednisolone. Heparan sulphate has been implicated in this interaction and in the binding of haemopoietic cell growth factors. In the present study we have compared the molecular structures of the heparan sulphate proteoglycans, metabolically labelled with [35S]sulphate, produced by methyl prednisolone-treated and untreated human marrow stromal cells in vitro. [35S]proteoglycans were examined in the cell layers (extracted with 1% (v/v) Triton X-100 in 6 M urea) and in the culture medium. Fractionation of proteoglycans by ion-exchange chromatography indicated that the heparan sulphate produced by the treated cultures eluted at a higher NaCl concentration than the counterpart from untreated cells. The heparan sulphate appeared to be mainly expressed on the cell surface, since it was efficiently extracted by treatment with dilute trypsin (50 micrograms ml-1 for 10 min). All cultures contained two heparan sulphate proteoglycan species, the major component eluted from a Sepharose CL-4B column with a median Kav of 0.33 and apparently contained an average of only one heparan sulphate chain. Small quantities of a larger proteoglycan, which was eluted in the void volume from the CL-4B column, was also detected, mainly in the cell layer extracts. The molecular structure of the heparan sulphate chains was analysed by oligosaccharide mapping, following specific enzymic depolymerisation, and separation of breakdown products by gradient PAGE. The maps revealed significant differences in overall enzyme susceptibilities and sulphation patterns of polysaccharides produced by methyl prednisolone-treated and untreated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow↗
PubMed1991

Deficiency of a phosphatidylinositol-anchored cell adhesion molecule influences haemopoietic progenitor binding to marrow stroma in chronic myeloid leukaemia.

The interactions between haemopoietic progenitor cells and marrow stromal cells that are essential for the regulation of normal haemopoiesis are defective in chronic phase chronic myeloid leukaemia (CML). The presence of primitive progenitor cells (blast colony-forming cells, Bl-CFC) in the blood of patients with CML is reflected by their reduced capacity to bind to marrow derived stromal layers in vitro. Whereas normal bone marrow Bl-CFC bind irreversibly to cultured stromal layers (and none are found in normal blood), the Bl-CFC in CML bind transiently and then detach. The normal cell adhesion mechanism is partially sensitive to treatment with phosphatidylinositol-specific phospholipase C (Pl-PLC), indicating the participation of a phosphatidylinositol (Pl)-linked structure; however, when CML cells were treated with Pl-PLC it had no effect on progenitor binding. Two other Pl-linked structures, decay-accelerating factor (DAF) and lymphocyte function associated antigen-3 (LFA-3) were normally expressed on CD34 positive CML cells and normally susceptible to Pl-PLC treatment. The treatment of normal cells with Pl-PLC, to mimic the situation in CML, resulted in the indiscriminate and inefficient binding of Bl-CFC to stroma. Moreover, treatment of the normal cells with 5637 conditioned medium (CM), which contains haemopoietic growth factors, also reduced the binding capacity of normal Bl-CFC; 5637CM treatment did not alter the expression of DAF. It is proposed that a Pl-linked cell adhesion molecule (CAM) is deficient in CML as a consequence of the constitutive activation of ABL kinase whilst, in normal cells, CAMs attached in this manner are responsible for efficient adhesion to stroma and are regulated by growth factors.

Antigens, CD↗
PubMed1991

Hemopoietic growth factors and receptors: bound and free.

Hemopoietic growth factors regulate blood cell production by interacting with specific receptors on hemopoietic progenitor cells. These factors are regarded conventionally as soluble molecules that are freely available to their target cells. There is growing evidence, however, that several of the factors can also exist as biologically active membrane- or matrix-bound entities. Conversely, the receptors for several of the factors, conventionally regarded as membrane-bound molecules, have been shown to exist as soluble entities that retain ligand binding activity. In principle, such soluble receptors could regulate the accessibility of the growth factors to target cells by performing carrier and/or blocking functions. Consideration of these new types of interactions should help us to understand the ways in which target cell responses to multifunctional growth factors can be controlled in vivo.

Animals↗
PubMed1990

Cell interactions and gene expression in early hematopoiesis.

As part of an investigation of the mechanisms controlling gene expression during lineage commitment, we have investigated the transcriptional status of hematopoietic lineage-specific genes and the interactions of early hematopoietic progenitor cells with stromal cells of the marrow microenvironment. The results indicate that a subset of otherwise lineage-restricted genes are transcriptionally active and/or DNAse I hypersensitive (i.e., "primed" for transcription) in multipotent, interleukin 3-dependent hematopoietic cells, and that they may become inaccessible and transcriptionally silent when cells are induced to adopt a single lineage during commitment. The external influences regulating gene expression in hematopoietic cells include binding interactions with stromal cells and exposure to locally presented growth factors. These interactions are thought to be essential for hematopoietic cell development and may be dysregulated in chronic myeloid leukemia.

Animals↗
PubMed1990

Haemopoietic stem cell subpopulations in mouse and man: discrimination by differential adherence and marrow repopulating ability.

Based on the properties of differential cell adherence, we have devised two assays for early progenitor cells in human bone marrow. One progenitor cell population binds to plastic and to pre-formed bone marrow derived stromal layers (P+S+) and gives rise to non-adherent granulocyte-macrophage colony-forming cells (GM-CFC); the other binds to stromal layers but not to plastic (P-S+); both are separable from GM-CFC which are P-S-. We have evaluated the relevance of differential binding properties to marrow repopulation in a murine model. Murine stem cells (spleen colony-forming cells--CFU-S) can be separated into P+S+, P-S+ and P-S- subpopulations by differential adhesion, thus paralleling the progenitor cell subpopulations in human marrow. Post irradiation (850 cGy X-rays) studies have shown that the P+S+ cells are essential for survival and recovery of marrow, spleen and blood cell populations. Also, in a model for purging autografts, we have demonstrated that the leukaemic cells can be separated from P+S+ repopulating cells by exploiting their different binding properties.

Animals↗
PubMed1990

Detection of the hybrid BCR/ABL messenger RNA in single CFU-GM colonies using the polymerase chain reaction.

In order to study which hemopoietic precursor cells express the hybrid BCR/ABL fusion mRNA we have developed a technique based on the polymerase chain reaction (PCR) for the examination of single hemopoietic colonies grown on semi-solid agar. The technique was developed by examining single CFU-GM colonies grown from newly diagnosed patients with chronic myeloid leukaemia (CML). RNA was isolated from individual 14 day colonies and reverse transcribed to a complementary DNA (cDNA) copy which formed the substrate for a PCR. We have studied 3 cases of CML using this method and have found that 5 out of 5, 9 out of 10 and 20 out of 23 colonies examined were positive. Thus we describe a simple and useful technique for the study of gene expression in a limited number of hemopoietic precursor cells.

Bone Marrow↗
PubMed1990

Hemopoietic progenitor cell binding to the stromal microenvironment in vitro.

Primitive clonogenic progenitor cells in human bone marrow bind to preformed marrow-derived stromal layers in vitro and generate colonies of blast cells. The binding interaction does not require calcium or magnesium ions and occurs equally well in serum-free and serum-supplemented culture medium. It does not appear to involve known cell adhesion molecules (CAMs) for which monoclonal antibodies are available (integrins, N-CAM, LFA-1, and ICAM-1), and we were unable to demonstrate a role for the progenitor cell antigen CD34 in progenitor cell adhesion to cultured stroma. The CAM expressed by the blast colony-forming cells may exist in transmembrane or phosphatidylinositol (PI)-linked forms because it is only partially degraded by exposure to trypsin or to PI-specific phospholipase C. However, binding of these cells to stroma is not prevented in the presence of monoclonal antibodies reacting with known PI-linked structures (Thy-1, CD14, and CD16). It is either masked by neuraminidase-sensitive residues or is no longer expressed as cells mature, respectively, along the granulocytic or erythroid lineages. The properties of the hemopoietic progenitor CAM are discussed with reference to the properties of other CAMs and of hemopoietic progenitor cell markers.

Antibodies, Monoclonal↗
PubMed1989

An in vitro model for the production of committed haemopoietic progenitor cells stimulated by exposure to single and combined recombinant growth factors.

A plastic-adherent mononuclear cell population in human bone marrow produces non-plastic-adherent nucleated cells in liquid cultures. These cells can be harvested from the culture medium and a proportion of them can be identified as granulocyte-macrophage colony-forming cells (GM-CFC) by plating them in semi-solid cultures with granulocyte-macrophage colony-stimulating factor (GM-CSF). The generation of GM-CFC from their plastic-adherent precursors can be amplified considerably by adding 5637 conditioned medium (CM) to the liquid phase of the adherent cell cultures. This effect of 5637 CM cannot be reproduced by recombinant (r) GM-CSF or interleukins (ILs) 1, 3 or 6 if they are added singly to the culture medium. In contrast, the combination of GM-CSF + IL-1 equalled or surpassed the activity of 5637 CM. The combinations of rGM-CSF + rIL-3 and rGM-CSF + IL-6 also mimicked the activity of 5637 CM but less effectively than GM-CSF + IL-1.

Bone Marrow↗
PubMed1989

Adhesive properties distinguish sub-populations of haemopoietic stem cells with different spleen colony-forming and marrow repopulating capacities.

The adhesive properties of clonogenic haemopoietic progenitor cells have been assessed by allowing them to bind to cultured marrow-derived stromal layers, stromal cell lines or bare plastic surfaces. We have compared the adhesive characteristics of murine marrow repopulating cells, spleen colony-forming cells (CFU-S) and granulocyte-macrophage colony-forming cells (GM-CFC) and of human blast colony-forming cells (Bl-CFC). The data indicate, first, that murine marrow repopulating cells bind to plastic and to stromal layers. Second, the CFU-S compartment contains three sub-populations: (a) CFU-S that bind to plastic and to stroma; (b) CFU-S that bind to stroma but not to plastic; (c) CFU-S that do not bind to stroma or plastic. Third, that more mature GM-CFC are not adhesive in these tests; and fourth, that recognitive events between stem cells and marrow stroma can operate across species barriers. Adhesion to both marrow-derived stroma and plastic appears to be a characteristic of haemopoietic stem cells that is lost or modulated as cells differentiate.

Animals↗