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M Y Gordon

Publications and source records attributed to M Y Gordon.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1988

The origin of stromal cells in patients treated by bone marrow transplantation.

The source of stromal cells following bone marrow transplantation has been investigated by culturing marrow cells obtained from patients who have a graft cells from a donor of the opposite sex. Two culture systems have been used for these studies. The first supports the proliferation of fibroblast colony-forming cells (F-CFC) and the formation of confluent layers of fibroblastoid cells; the second (the long-term bone marrow culture system) results in the formation of a complex and heterogeneous layer of adherent cells composed of fibroblasts, fat cells, endothelial cells, macrophages and 'blanket' cells. A survey of the work done using these culture systems shows that, although the issue of the transplantability of stromal cells remains controversial, it now seems reasonably certain that repopulation of the fibroblast component by donor cells is rare. In contrast, one study has shown that endothelial-like cells can engraft in transplant recipients. Another study has demonstrated a donor origin for the macrophages found in long-term cultures of marrow from transplanted patients. This finding might have been predicted because macrophages are derived from haemopoietic stem cells.

Bone Marrow↗
PubMed1988

Heparan sulfate is necessary for adhesive interactions between human early hemopoietic progenitor cells and the extracellular matrix of the marrow microenvironment.

Human hemopoietic blast colony-forming cells (BI-CFCs) recognize and adhere to the extracellular matrix (ECM) produced by marrow-derived stromal cells in vitro. We have investigated the requirements for this interaction by testing the capacity of BI-CFCs to adhere to ECM components under a variety of conditions. Binding was prevented completely by prior treatment of stromal ECM with nitrous acid, in large part by treatment with heparitinase or hyaluronidase, and slightly by treatment with chondroitinases. Whereas heparan sulfate isolated from marrow stromal cultures effectively blocked binding, heparan sulfate from bovine kidney did not. Chondroitin sulfate and hyaluronic acid did not have any effect in this test. In contrast, collagen was not sufficient for the interaction because dishes coated with collagen type I or IV did not act as adhesive surfaces for BI-CFCs. Ligands for integrin receptors (e.g., fibronectin) did not participate in BI-CFC binding because the synthetic pentapeptide glycine-arginine-glycine-asparagine-serine did not compete with stroma in binding BI-CFCs. These findings indicate that heparan sulfate in the bone marrow microenvironment is necessary for BI-CFC binding to ECM and may contribute to localizing hemopoietic stem cells in hemopoietic tissue.

Antigens, Surface↗
PubMed1988

Hierarchical organization of hematopoietic microenvironments: role of proteoglycans.

The orderly process of hematopoietic cell differentiation depends on the organization of specific microenvironments which function by assembling progenitor cells and appropriate concentrations of necessary growth factors. We propose that this organization involves a spectrum of microenvironmental complexity concomitant with changing cellular requirements, that it is achieved by the extracellular matrix produced by marrow stromal cells, and that its specificity is conferred by the heterogeneity intrinsic in the conformation of proteoglycans and their constituent glycosaminoglycans.

Animals↗
PubMed1988

Increased DR antigen expression by committed hemopoietic progenitor cells but not primitive progenitor cells in chronic myeloid leukemia.

We used a complement-dependent cytotoxic assay to study the expression of DR antigens by hemopoietic progenitor cells derived from normal marrow and from the blood of patients with chronic phase chronic myeloid leukemia (CML). Almost all normal and CML day-12 granulocyte-macrophage colony-forming cells (d12 GM-CFC) and erythroid burst-forming units (BFU-E) expressed DR antigens. A primitive progenitor cell, the "blast colony-forming cell" (Bl-CFC), also expressed DR antigens whether derived from normal marrow or CML blood. The expression of DR antigen by normal and CML Bl-CFC was similar but the density of DR antigens on CML d12 GM-CFC and BFU-E was much greater than that of their counterparts from normal marrow. The similarity in DR antigen density on normal and CML Bl-CFC means that purging of CML marrow using a DR-specific antibody would not select in favor of normal cells and thus would not be useful in an autograft setting.

Cell Survival↗