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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
PubMed1995

Progenitor cells in the blood and marrow of patients with chronic phase chronic myeloid leukaemia respond differently to macrophage inflammatory protein-1 alpha.

Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a negative regulator of normal haemopoietic stem cell proliferation. Insensitivity to MIP-1 alpha of progenitor cells in chronic myeloid leukaemia (CML) could, therefore, explain myeloid expansion in this disease. We compared the effects of MIP-1 alpha on progenitor cells in normal marrow and in the blood and marrow of patients with chronic phase CML. Plastic-adherent precursors of granulocyte-macrophage colony-forming cells (P delta progenitors) are very primitive progenitor cells and are detected by incubating them for 1 week in liquid culture and assaying the CFU-GM released into the supernatant. Direct CFU-GM assays were also used in this study. Daily addition of 300 ng/ml/day of MIP-1 alpha to P delta progenitor assays of normal marrow cells suppressed CFU-GM production by 50% and in CML bone marrow P delta cultures by 20-30%. The response of CML blood P delta progenitors was heterogeneous. In five of nine cases, CFU-GM production was doubled in the presence of MIP-1 alpha and in four of nine cases, it was reduced. Addition of 100-500 ng MIP-1 alpha to direct assays of CFU-GM stimulated colony formation by normal marrow and CML blood cells to a similar extent.

Bone Marrow Cells↗
PubMed1995

Probability of progenitor renewal (PPR) and production of clonogenic progeny (CFU-GM) by primitive adherent progenitor cells in adult human bone marrow and umbilical cord blood.

Human haemopoietic tissues contain primitive plastic-adherent progenitor cells (P delta cells) that can be detected by measurement of their granulocyte-macrophage colony-forming cell (CFU-GM) progeny. Limiting dilution analysis and Poisson statistics are necessary for determining the frequency of P delta cells because each of them produces several CFU-GM. Limiting dilution also permits measurement of the abilities of individual P delta progenitors to produce CFU-GM. Here we report that the frequencies of P delta progenitors in cord blood and adult marrow are similar (5.6 and 7.8/10(5) mononuclear cells respectively) and individual cord blood P delta progenitors produce fewer CFU-GM than adult P delta progenitors. To test the possibility that the lower production of differentiated progeny by cord blood cells was the result of a higher rate of self-renewal, we devised a two-stage limiting dilution assay relying on the relative production of CFU-GM after two consecutive weeks of incubation. The probability of progenitor renewal (PPR) was derived from the number of wells (progenitors) that produced CFU-GM on both occasions compared with the number that produced CFU-GM on the first occasion only. The total number of CFU-GM produced on the second occasion compared with the number produced on the first occasion provided an index of the overall change in the size of the P delta cell population. The data indicate that P delta cells in cord blood have a higher PPR (0.59) than those in adult marrow (0.36). Also, the relative numbers of CFU-GM produced in the second and first weeks were greater for cord blood (1.2) than for adult marrow (0.36). Therefore P delta cells in cord blood have a greater capacity for self-maintenance and possibly for expansion than P delta cells in adult marrow.

Adult↗
PubMed1995

Evidence for a mechanism that can provide both short-term and long-term haemopoietic repopulation by a seemingly uniform population of primitive human haemopoietic precursor cells.

One of the controversies surrounding the repopulating capacities of haemopoietic stem cells is whether or not the same or different populations are responsible for short-term and long-term repopulation after transplantation. To address this question, we analysed results obtained from an in vitro model for the clonal production of granulocyte-macrophage colony-forming cells (CFU-GM) by individual primitive multilineage precursors in adult human bone marrow. The primitive precursors adhere to plastic and produce CFU-GM in a 1-week long 'delta' type culture. The clones that form are classified as having short maturation pathways (clones containing predominantly day 7 CFU-GM) or long maturation pathways (clones containing predominantly day 21 CFU-GM). The results indicate that individual primitive (P delta) cells produce clones that reach full maturity after different periods of time so that cells corresponding to a range of maturational stages can become available simultaneously. Consequently, transplanted stem cells may be able to provide both rapid and long-term mature cell recovery whilst at the same time reconstituting the stem cell pool. These results suggest that it might be possible to use highly purified stem cell populations, devoid of committed progenitors, for clinical transplantation.

Bone Marrow Cells↗
PubMed1995

Some factors determining the minimum number of cells required for successful clinical engraftment.

Theoretically, a single pluripotent haemopoietic stem cell should be able to reconstitute haemopoiesis following transplantation. However, clinical and experimental observations demonstrate that it is necessary to transplant numerous stem cells to obtain engraftment. Here we discuss some of the reasons for this apparent discrepancy and provide some quantitative estimates of the influence of kinetic factors in determining the number of stem cells required for clinical engraftment.

Cell Count↗
PubMed1994

SLe(x) expression of normal CD34 positive bone marrow haemopoietic progenitor cells.

We investigated sialylated Lewis x (sLe(x)) antigen expression on CD34 positive (CD34+) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD34+ bone marrow progenitors strongly expressed the sLe(x) antigen. This contradicts previous publications which reported sLe(x) expression on malignant blast cells but not on normal CD34+ progenitor cells.

Antigens, CD↗
PubMed1994

The kinetics of colony formation by CFU-GM in vitro.

Video-recordings of whole normal bone marrow granulocyte-macrophage colony (CFU-GM) cultures were made after 7, 14 and 21 d. Retrospective viewing of the tapes allowed the relationships to each other of the colonies scored on the three different occasions to be documented. The results show that, according to our scoring criteria, there is very little overlap between the numbers of colonies scored on days 7, 14 and 21. Moreover, only about 10% of the progenitors in a sample form day 21 colonies. The remaining progenitors form colonies earlier in the culture period and either disappear before day 21 or remain small.

Bone Marrow Cells↗
PubMed1994

The binding of acute myeloid leukemia blast cells to human endothelium.

AML blast cell adhesion to endothelium is in all likelihood a prerequisite for blast cell migration across the vascular wall in the periphery and the subsequent establishment of leukemic extravascular disease. A general feature of malignant cells is their acquisition of altered or aberrant adhesive capabilities which appear to be associated with their ability to metastasize. Aberrant expression of integrin adhesion molecules and of membrane oligosaccharide structures is found in AML and various solid tumors. With respect to AML, these alterations in adhesive phenotype may confer a proliferative advantage on the malignant cells in the marrow, may facilitate egress from the bone marrow into the peripheral vasculature and may enable AML blast cells to traverse the vessel wall and so establish extravascular disease. Oncogenes may be directly involved in the acquisition of such aberrant adhesive phenotypes. Neutrophil extravasation is described as a model for leukocyte migration across the vessel wall and brief summaries of experimental work involving aspects of AML blast cell and normal CD34+ bone marrow cell adhesion to endothelium in vitro are described.

Acute Disease↗
PubMed1994

Plastic-adherent cells in human bone marrow generate long-term hematopoiesis in vitro.

This study demonstrates that the human bone marrow mononuclear cell fraction that adheres to plastic during a 2-h incubation period includes stromal precursors and hematopoietic progenitor cells that can sustain hematopoiesis for at least 5 weeks in a long-term culture (LTC) system. Delta (delta) assays were performed on the 2-h plastic-adherent fraction of mononuclear cells, by assaying the numbers of CFU-GM released into the culture supernatant during 1 week of incubation, to investigate the properties of the original progenitor cells. The plastic-adherent delta (P delta) progenitor cells all express CD34, and a variable proportion express Thy-1. The numbers of CFU-GM produced are directly related to the numbers of mononuclear cells used to initiate the cultures, and limiting dilution experiments indicate a frequency for P delta cells of 2.5-10/10(5) mononuclear cells. The numbers of CFU-GM produced by individual P delta cells at limiting dilution vary considerably within experiments but the distributions of CFU-GM per P delta progenitor show little variation between samples. On average, P delta cells generate 6-9 early CFU-GM scored on day 21 of culture; 10-23 CFU-GM scored on day 14, and 18-40 mature CFU-GM scored on day 7. These results show that plastic-adherent cells in human bone marrow are capable of sustained hematopoiesis in vitro and of producing a spectrum of clonogenic progeny. They form the basis of an assay for investigating the kinetics of early human hematopoietic progenitor cells.

Antigens, CD↗
PubMed1994

In vitro proliferation by cells mobilized into the peripheral blood for collection and autologous transplantation.

We have investigated the properties of mobilized, cryopreserved, peripheral blood stem cells (PBSC), collected by leukapheresis over a period of 5 days, from eight myeloma patients in clinical remission. Cells were mobilized by treatment with cyclophosphamide and granulocyte colony-stimulating factor (G-CSF), and each day's collection was evaluated for its content of CD34+ cells, colony-forming units granulocyte/macrophage (CFU-GM), and plastic-adherent pre-CFU-GM. Peak values for these three parameters were observed at different times in different patients. There was no correlation between CD34+ content and CFU-GM, but there was some (r = 0.65) between CD34 numbers and colonies generated from a delta assay initiated using plastic-adherent pre-CFU-GM. In suspension cultures, the cells grew exponentially for 50 days. Thereafter, they did not divide, although they remained viable in culture for up to 1 month longer. Suspension cultures of PBSC grown with interleukin-3 (IL-3) displayed a predominantly myelomonocytic phenotype, but some megakaryocytes and erythroid cells were observed consistently. These results indicate that pre-CFU-GM in PBSC collections are capable of generating large numbers of clonogenic progeny in liquid culture and are capable of producing multiple lineages of differentiation.

Antigens, CD↗
PubMed1993

Acute myeloid leukaemia blast cells bind to human endothelium in vitro utilizing E-selectin and vascular cell adhesion molecule-1 (VCAM-1).

The adhesion of acute myeloid leukaemia (AML) blast cells to human umbilical vein endothelial cells (HUVECs) was investigated in vitro. Adhesion of blast cells from 10 cases of AML to unstimulated and interleukin-1 beta (IL-1) stimulated HUVECs was similar to or greater than that of control neutrophils. The extent to which endothelial E-selectin and vascular cell adhesion molecule-1 (VCAM-1) were involved in this adhesive process was investigated using blocking monoclonal antibodies to these proteins. In the majority of cases studied (7/8), anti-E-selectin significantly inhibited adhesion to IL-1 stimulated endothelium (26-65% inhibition) and in 5/8 cases so did anti-VCAM-1 (maximum of 31% inhibition). All cases were found to express the sialylated Lewis x antigen and very late activation antigen-4, ligands for E-selectin and VCAM-1 respectively. Our results indicate that leukaemic blast cells adhere to human endothelium and that there are E-selectin and, to a lesser extent, VCAM-1-dependent components to this process. Such adhesive interactions are likely to confer on AML blast cells the ability to migrate across the vascular wall and so to establish extravascular disease.

Acute Disease↗
PubMed1993

Rapid positive selection of CD34+ cells using magnetic microspheres coated with monoclonal antibody QBEND/10 linked via a cleavable disulphide bond.

Positive selection of CD34+ cells has applications in diagnostic pathology, in peripheral blood and bone marrow transplantation, and in studies on the function and regulation of primitive haemopoietic stem cells. Antibody-coated magnetic microspheres (dynabeads) can be used to isolate these cells by positive selection procedures. However, the advantages of using dynabeads in some positive selection protocols are compromised by the retention of the beads on the cells. We present a protocol which allows the rapid chemical release of the beads from positively sorted cells. The murine immunoglobulin (Ig) G1 CD34 antibody, QBEND/10, was immobilised onto dynabeads as part of a three-layered immune complex: QBEND/10 was attached to F(ab')2 anti-mouse immunoglobulin antibody fragments, which were immunologically bound to a mouse IgG1 myeloma protein. The myeloma protein covalently bonded the triplex to the beads. Thus, disulphide bonds in the hinge region of the F(ab')2 could be reduced with 10 microM dithiothreitol and CD34+ cells released within 20 min. Purified cells can be re-phenotyped by multiple markers and subsets identified. Purity of 97%, recovery of > 50%, and viability over 90% of the CD34+ cells was readily achieved. Furthermore, granulocyte-macrophage colony-forming cells were retained in the positive fraction. This methodology can be used to purify other cell types, including T and B lymphocytes.

Antibodies, Monoclonal↗
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↗