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E Spooncer

Publications and source records attributed to E Spooncer.

At least 55 records · Page 3Linked to original sources
PubMed1988

Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis.

The proliferation and development of haemopoietic stem cells takes place in close association with marrow stromal cells. This intimate cell contact presumably enables the stem cells and their progeny to respond to stimuli present on the stromal cell surface. While the nature of these stimuli has not been determined, it is likely that growth factors play some role. Recently, it was demonstrated that the natural and the recombinant haemopoietic growth factor, granulocyte/macrophage colony stimulating factor (GM-CSF), could be adsorbed out of solution by an extract of human marrow stromal extracellular matrix (ECM) with retention of biological activity. However, the precise ECM molecules involved were not identified. Here, we clearly demonstrate that the major sulphated glycosaminoglycan of mouse marrow stroma, heparan sulphate, possesses the ability to adsorb both GM-CSF and the multilineage haemopoietic growth factor, Interleukin 3 (IL-3). Furthermore, these growth factors, once bound, can be presented in the biologically active form to haemopoietic cells.

Adsorption↗
PubMed1988

Interleukin-3-specific modification of cell membrane "fluidity" of haemopoietic cells.

The work reported here clearly demonstrates that a specific growth factor, interleukin-3 (IL-3), which acts on multipotent haemopoietic stem cells as well as on committed myeloid progenitor cells of different lineages (Schrader, 1988; Whetton and Dexter, 1986), specifically induces a modification of the physical state ("fluidity") of the cell membranes of two IL-3-responsive and apparently normal haemopoietic cell lines. Furthermore, in a derived IL-3 independent myeloid leukaemic cell line, no such physical response to IL-3 binding was observed. The rapidity of the "normal" response suggests further that it may be associated with, or even constitute per se a critical early effect elicited by IL-3 in sensitive cells, the necessity for which is abrogated in the malignant derivative.

Animals↗
PubMed1988

Restriction of expression of an integrated recombinant retrovirus in primary but not immortalized murine hematopoietic stem cells.

A recombinant retrovirus (DHFR*-SVADA) in which human adenosine deaminase (ADA) cDNA is transcribed from an internal SV40 promoter was used to infect murine hematopoietic stem and progenitor cells. Human ADA enzyme was not expressed in infected primary murine pluripotent stem cell-derived spleen or progenitor colonies (CFU-GM, CFU-Mix, BFU-E). In contrast, human ADA enzyme activity was readily detected in progenitor colonies derived from immortalized multipotent factor-dependent cells. The level of human enzyme was near endogenous murine enzyme levels and was equivalent in undifferentiated stem cells and differentiated myeloid, erythroid, and mixed colonies. These results indicate that cellular properties other than the stage of differentiation are important in determining the expression of foreign sequences introduced by retroviruses. Cell lines that are immortalized but still capable of induced differentiation may contain factors that abrogate blocks to expression that are manifested in primary hematopoietic stem cells.

Adenosine Deaminase↗
PubMed1987

Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells.

When highly enriched multipotential spleen colony forming cells (CFU-S) obtained following fluorescence activated cell sorting (FACS-CFU-S) are cultured on marrow stromal cells, they undergo proliferation and development to produce mature haemopoietic cells (Spooncer et al., Nature, 316:62-64, 1985). We now show that FACS-CFU-S behave in a similar way when cultured on monolayers of 3T3 cells, indicating that the 3T3 cells can supply at least part of the environment which is representative of marrow stromal cells and provide, therefore, a system for studying stromal cell: haemopoietic cell interactions. We also demonstrate that IL-3-dependent multipotential stem cell lines (FDCP-Mix), but not a variety of other "committed" IL-3-dependent cell lines, resemble FACS-CFU-S in terms of their ability to proliferate and differentiate when cultured on 3T3 cells in the absence of IL-3. In this system, attachment of the FDCP-Mix to the 3T3 cells is critical for the subsequent maintenance of viability and stimulation of development of the cells. When the FDCP-Mix cells are physically separated from the 3T3 cells, they die and their death cannot be prevented by using 3T3-cell-conditioned medium. The extracellular matrix generated by 3T3 cells is not sufficient for promoting attachment or viability of the FDCP-Mix cells, indicating the importance of integral membrane components. However, attachment and development of FDCP-Mix cells occurs on 3T3 cells that have been lightly fixed with glutaraldehyde indicating that active metabolism is not essential for the effects promoted by the 3T3 cells. We suggest that the ability of FACS-CFU-S and FDCP-Mix cells to respond to 3T3 cells involves specific ligand/receptor interactions.

Animals↗
PubMed1987

Growth and differentiation in the hemopoietic system.

Hemopoiesis is regulated by a complex series of interactions, including interactions among hemopoietic cells themselves, hemopoietic cells and the extracellular matrix, hemopoietic cells and marrow stromal cells, and hemopoietic cells and growth factors. In vitro culture systems have allowed a reductionist approach to the solution of these various problems and have facilitated experiments at the mechanistic level. The hemopoietic system is organized hierarchically with multipotential self-renewing stem cells, committed progenitor cells, and mature cells. The various stimuli necessary for growth and development of these cells are rapidly being elucidated. The nature of commitment (or differentiation) remains an enigma, but model systems have been developed in which various aspects of this problem can be investigated. In this respect, growth and differentiation factors obviously have a major role to play. Now that many of these factors have been molecularly cloned (and pure target cell populations are available) their role in vivo and their mode of action can be examined.

Animals↗
PubMed1987

Erythropoietic repopulating ability of stem cells from long-term marrow culture.

Hemopoietic precursors are heterogeneous with respect to their capacity for self-renewal and long-term repopulating ability. Bone marrow cultures produce a variety of precursors over many weeks, including CFU-S; however, it is important to determine whether these populations retain the functional ability shown by fresh marrow. The most primitive precursor or stem cells have the most long-term repopulating ability. We here describe direct measurements of this ability in cells from marrow cultures by using competitive repopulation assays. Cultured adherent cells repeatedly showed less capacity than fresh marrow cells to repopulate erythropoiesis in irradiated recipients, whereas cultured suspension cells consistently had less capacity than adherent cells. Concentrations of macroscopic CFU-S measured at nine or 12 days were similar in cultured adherent and suspension cells and generally lower than those in fresh marrow. In every experiment, the long-term repopulating ability of the marrow cells used was substantially reduced after transfer into tissue culture. Thus, primitive stem cells may not proliferate in such cultures despite extensive production of CFU-S and more differentiated cell types.

Animals↗
PubMed1986

Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors.

Interleukin-3 (IL-3)-dependent cell lines (FDCP-mix) were cloned and isolated from long-term bone-marrow cultures infected with src-MoMuLV. These cell lines have many of the characteristics of hematopoietic stem cells. Early isolates of the FDCP-mix cells form spleen colonies in irradiated mice and establish long-term hematopoiesis on irradiated marrow stroma in vitro in the absence of IL-3. These two properties of the cells are lost within 15 weeks of establishing the cell lines, but the cell lines retain their ability to differentiate in a multilineage response to hematopoietic growth factors and to hematopoietic stromal cells, as well as to self-renew in the presence of IL-3. The choice between differentiation and self-renewal in FDCP-mix cells can clearly be modified by culture conditions: in particular, cultures containing horse serum preferentially promote self-renewal, whereas cultures containing fetal calf serum preferentially promote differentiation. The FDCP-mix cell lines are not leukemic, nor do they contain the src oncogene. Their ability to respond to hematopoietic growth factors and stroma in a similar manner to normal hematopoietic cells makes them a valuable model for studying the regulation of hemopoietic cell self-renewal and differentiation.

Animals↗
PubMed1986

Perturbed hemopoiesis and the generation of multipotential stem cell clones in src-infected bone marrow cultures is an indirect or transient effect of the oncogene.

Multipotential stem cell lines, derived specifically from long-term bone marrow cultures infected with a recombinant retrovirus carrying v-src, lack v-src. Stable consequences thus result from transient actions or indirect effects of v-src on other cells, with the latter possibility being favored by its mosaic expression in marrow cultures.

Animals↗
PubMed1986

The kinetic response of haemopoietic precursor cells, in vivo, to highly purified, recombinant interleukin-3.

Interleukin-3 is a murine haemopoietic cell growth factor which has now been prepared in recombinant form, rIL-3. This purified material has been shown to act, in vitro, in a comparable manner to the native material and has recently been shown to have an in vivo effect on the committed, in vitro colony-forming cells. We have examined the effects, in vivo, of low acute and chronic infusion doses on the pluripotent haemopoietic spleen colony-forming cells, CFU-S, (stem cells). The proliferation rate of CFU-S is rapidly increased after administration of rIL-3. This is followed by a migration of the more mature CFU-S to the spleen where, particularly under chronic rIL-3 treatment there is a large increase in CFU-S numbers. The increased proliferation of CFU-S is accompanied by increased differentiation in the form of a large increase in in vitro IL-3 responsive cells. The changes observed were not the result of endotoxin contamination in the rIL-3 preparation. Significantly, however, larger doses of lipopolysaccharide, did mimic the effects of rIL-3 in vivo. IL-3 was not detected in the blood following LPs treatment but it is suggested that LPS acts indirectly on haemopoietic precursor cells by eliciting local production of IL-3, perhaps from adjacent T cells or stromal cells.

Animals↗
PubMed1986

Isolation of haemopoietic spleen colony forming cells.

A simplified and more widely applicable modification of the recently developed methodology for sorting and collecting purified populations of the pluripotent haemopoietic spleen colony-forming cell, CFU-S, is described. Based on their relatively low density and high affinity for wheat germ agglutinin, CFU-S are collected using a fluorescence activated cell sorter. Normal bone marrow cells are labelled with flurorescein labelled wheat germ agglutinin (WGA - FITC) and then subjected to a density cut on metrizamide. Cells with density less than 1.080 gm. ml-1 are sorted on a FACS-IV instrument. Highly fluorescent cells with medium forward and low perpendicular light scatters are collected. These cells are then re-sorted to the same criteria as for the first sort. This double sorting procedure gives a population of cells which, corrected for spleen seeding efficiency, contains about 90% CFC-S. Mixed CFC, in vitro, are also enriched but from the ratios of mixed to spleen CFC, and of 11 to 8 day spleen colony forming ability, there is a clear selection for the earlier, more primitive and/or non-cycling Go-phase CFC-S. This is confirmed by direct observation of autoradiographic labelling indices.

Animals↗
PubMed1985

The role of stromal cells and growth factors in haemopoiesis and modulation of their effects by the src oncogene.

In the haemopoietic system the mature blood cells have only a finite lifetime. For example, a circulating granulocyte in the peripheral blood has an approximate half-life of 7 h (Cartwright, Athens & Wintrobe, 1964; Dancey, Dubelbeiss, Harker & Finch, 1976) whilst the lifetime of an erythrocyte is approximately 120 days (Wickramasinghe & Weatherall, 1982). This constant 'death' of mature functional haemopoietic cells means that new blood cells must replace those that are removed. The process of haemopoiesis provides the mature functional blood cells to replace those lost as a consequence of performing their biological functions (e.g. lymphocytes and macrophages in the immune response) or through apparent old age and breakdown (e.g. erythrocytes that are 110-120 days old). The major questions that we are required to answer about this process are 'where do all these new cells come from?', 'what regulates their production?' and 'how is this mechanism of control lost in haemopoietic disorders such as leukaemia, hyperproliferative diseases and anaemias?'. Recent work in the field of haemopoiesis has given some clues to the answers to the questions, which provide an intriguing insight into not only haemopoiesis itself but the possible lesions associated with the various blood disorders.

Animals↗
PubMed1984

Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes.

Sialylated fucosyl lactosaminoglycan was isolated from human neutrophilic granulocytes and its structure was elucidated. The lactosaminoglycan glycopeptides were digested by endo-beta-galactosidase and "the core portion" and released oligosaccharides were analyzed by permethylation, fast atom bombardment mass spectrometry, and exoglycosidases. In addition, lactosaminoglycan saccharides were obtained by hydrazinolysis and the structures of fractionated sialyl oligosaccharides were analyzed by fast atom bombardment mass spectrometry and permethylation coupled with exoglycosidase treatment. The structure of one of the major components was found to be: (Formula: see text). This structure is unique in that 1) four linear polylactosaminyl side chains are attached to the core portion, 2) the side chain arising from position 4 of 2,4-linked mannose contains one or more alpha 1----3 fucosyl residues, 3) the side chain arising from position 6 of 2,6-linked mannose is terminated with NeuNAc alpha 2----3Gal(Fuc alpha 1----3)GlcNAc, sialyl Lex, and 4) the side chain arising from position 2 of 2,4-linked mannose is terminated with sialic acid through alpha 2----6 linkage.

Amino Sugars↗
PubMed1984

Isolation and characterization of polyfucosylated lactosaminoglycan from human granulocytes.

Lactosaminoglycan was isolated from human granulocytes and the structure of neutral lactosaminoglycan was elucidated. The lactosaminoglycan glycopeptides and lactosaminoglycan saccharides obtained by hydrazinolysis were analyzed by permethylation. In addition, the lactosaminoglycan was digested by endo-beta-galactosidase and the "core" portion and released oligosaccharides were analyzed by specific glycosidases, permethylation, and fast atom bombardment mass spectrometry. The structure of the major component in the neutral lactosaminoglycan was found to be: sequence in text where m + n + o + p greater than 6, and the mean value of fucose content = 4.5. This structure is unique in that 1) four linear polylactosaminyl chains are attached to the core portions, 2) N-acetylglucosamine residues in the polylactosaminyl side chains are substituted with fucose through an alpha 1----3 linkage and at least 1 mol of Gal beta 1----4(Fuc alpha 1----3)GlcNAc terminal structure is present, and 3) the tetraantennary core is a major component. Since this polyfucosylated lactosaminoglycan is abundantly present in human granulocytes, we propose that this lactosaminoglycan is a major carrier for the granulocyte-specific antigen, Gal beta 1----4(Fuc alpha 1----3)GlcNAc, which is recognized by the My-1 monoclonal antibody (Huang, L. C., Civin, C. I., Magnani, J. L., Shaper, J. H., and Ginsburg, V. (1983) Blood 61, 1020-1023).

Adult↗