The growth of large megakaryocyte colonies from human bone marrow

Messner, Hans A.; Jamal, N; Izaguirre, Carlos A.

doi:10.1002/jcp.1041130410

Cited by 195 publications

(105 citation statements)

References 17 publications

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“…Another possibility would be that fibrinogen slows down differentiation processes, allowing more cell divisions before terminal differentiation. Recently Messner et al (25) have obtained improved results in mixed colony assays by replacing part of the serum with plasma. The presence of fibrinogen in plasma could explain the positive response obtained.…”

Section: Resultsmentioning

confidence: 99%

Fibrinogen and its fragment D stimulate proliferation of human hemopoietic cells in vitro.

Hatzfeld¹,

Hatzfeld²,

Maigne³

1982

Proc. Natl. Acad. Sci. U.S.A.

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Purified fibrinogen at concentrations of3-30 nM has been found to stimulate continuous growth ofhuman lymphoid and myeloid cell lines under serum-free conditions. A strong proliferative response resulted from the synergism elicited by the addition of fibrinogen to transferrin-supplemented medium. This effect was observed with the pre-B-cell line Raji, the T lymphomaderived JM, and the monocytic cell line U 937, either at high or low cell densities. With the promyelocytic cell line HL 60, fibrinogen did not shorten the doubling time of cultures seeded at high cell densities (2 x 105 cells per ml). However, at cell densities lower by 2 orders of magnitude and in the same medium, it promoted growth with a doubling time similar to that obtained at high cell concentrations. Fibrinogen also was found to increase the plating efficiency and colony size when human bone marrow cells were cultured in semisolid medium containing serum. In long-term bone marrow liquid cultures without fibrinogen, colony-forming cells were no longer detected after 6 weeks. In those cultured with fibrinogen, -50 granulocyte-macrophage colonies per 105 cells were obtained after 6 weeks, and 10, after 12 weeks. Purified fibrinogen fragment D possessed a stimulating activity similar to that of the intact fibrinogen molecule. This fragment cannot form fibrin, thus eliminating fibrin as a source of the mitogenic effect.The use of serum in tissue culture hinders the study of the regulation of cellular proliferation (1)(2)(3)(4)(5)(6) because the roles and interactions amongst various growth factors are difficult to analyze in nondefined media. Sato and his group (for a review see ref. 7) have contributed to the development of the concept of hormonally defined media and have shown how useful these media can be for analyzing the controls of proliferation and differentiation at the molecular level. Iscove and co-workers (8-10) have applied this concept to the culture ofmurine hemopoietic cells.A minimal medium is composed of defined factors that are all necessary in a particular combination for the growth of a particular cell type; removal ofeach factor individually prevents growth. These media facilitate the study of each growth requirement and its effects. By designing minimal media containing purified plasma components, we have analyzed their possible roles in the control of human hemopoietic cell proliferation (11).Fibrinogen is a major component of plasma, where its concentration is about 3 mg/ml. Blood (19), was provided by R. Gallo (National Cancer Institute, Bethesda, MD). Cell lines were maintained in suspension in RPMI 1640 medium with 2 g of sodium bicarbonate per liter, 1% penicillin/streptomycin and 1% L-glutamine stock solutions, and 10% heat-inactivated fetal calf serum in Coming TM 25 flasks. Defined media were prepared as described (11). Doubling times were estimated only when cultures contained more than 98% viable cells as determined by trypan blue exclusion.Long-Term Human Bone Marrow Culture and ColonyForming Cell Ass...

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Section: Resultsmentioning

confidence: 99%

Fibrinogen and its fragment D stimulate proliferation of human hemopoietic cells in vitro.

Hatzfeld¹,

Hatzfeld²,

Maigne³

1982

Proc. Natl. Acad. Sci. U.S.A.

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“…Megakaryocytes were cultured as described by Messner et al [24] and modified by Debili et al [20]. In short, 10-20 ml bone marrow from patients undergoing heart surgery was collected with informed consent in 5 ml RPMI containing 13 U/ml heparin.…”

Section: Megakaryocyte Culturesmentioning

confidence: 99%

Rapid alterations in lateral mobility of lipids in the plasma membrane of activated human megakaryocytes

Schootemeijer¹,

Beekhuizen²,

Gorter³

et al. 1994

European Journal of Biochemistry

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In the present study we measured membrane fluidity as the lateral mobility of the lipid probe l,l'-ditetradecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate by fluorescence recovery after photobleaching in the plasma membrane of a single megakaryocyte, the progenitor cell of platelets. Megakaryocytes after 13 days in culture (maturation stage 111) had a lateral diffusion coefficient (D) of (4.56-+0.10)X10-9 cm2/s and a mobile fraction of 6 5 2 2 % (means ? SEM, n = 140).Megakaryocytes isolated from rib had a similar D and mobile fraction. Stimulation with a-thrombin (1-10 U/ml) induced a dose-dependent decrease in D to (3.40 ?0.22)X10-9 cmz/s between 1 -5 min after stimulation ( P < 0.001). The mobile fraction did not change. A similar decrease in D was found following stimulation with ADP (20 kM) and ionomycin (100 nM). Modulation of calpain I activity with calpain I inhibitor or tetracain had no effect. Pretreatment with cytochalasin B or colchicine decreased D to (3.64 Ifr 0.29)X em% ( P < 0.013) respectively. After stimulation D decreased further in cytochalasin-treated cells (3.37 2 0.16)X lo-' cm2/s ( P < 0.020) but remained at the same level in colchicine-treated cells. Both treatments increased the mobile fraction to 73 -75% in stimulated megakaryocytes ( P < 0.03).These data indicate that the diffusion velocity of lipids in megakaryocytes is low and decreases further after stimulation. These changes are independent of calpain I. Treatments that decrease the cytoskeletal mass and thereby increase the mobility of proteins in the plasma membrane increase the number of lipids that participate in this process.cm2/s ( P < 0.003) and (3.96 ? 0.1 8)XOne of the factors that determines the sensitivity of blood platelets to activating stimuli is the fluidity of the plasma membrane. Platelets enriched in cholesterol, as seen in hyperlipoproteinemic patients or after prolonged incubation with liposomes, have a decreased membrane fluidity and respond to thrombin and ADP with increased aggregation and secretion [l-31. On the other hand, when the fluidity is increased by incubation with alkyl alcohols, benzyl alcohol, and phenolic compounds, a lower aggregation tendency is found [4].There is little insight into the mobility of lipids after platelets have been stimulated, partly because different approaches have led to conflicting results. Studies based on fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, which detects the angular movement of a lipophilic molecule around a perpendicular axis in the plane of the membrane, showed a rigidification of the membrane after stimulation ionomycin had no effect [6]. 1 -(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene binds more specifically to the outer layer of the plasma membrane [6, 71. Such labeled platelets again showed a decrease in membrane fluidity at low doses of thrombin (G0.2 U/ml) [8, 91, but higher doses made the membrane more fluid [9]. A similar fall was found after stimulation with ADP (0.2-5 pM) [9] and a low concentration of ionomycin (100 n...

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“…One interesting postulation would be that GP160 is a marker of pluripotent stem cells or a marker of stem cells common to lymphoid and myeloid cells, and these stem cells bearing GP160 are the targets for leukemogenesis of non-T ALL and myelo-monocytic leukemia. The presence of a common progenitor of myeloid and lymphoid cells in normal and disturbed hemopoiesis has been reported (36)(37)(38). However, little is known about the target cells for leukemogenesis of human lymphoid and myeloid leukemias.…”

Section: Discussionmentioning

confidence: 99%

Distinct human leukemia-associated cell surface glycoprotein GP160 defined by monoclonal antibody SN6.

Haruta¹,

Seon²

1986

Proc. Natl. Acad. Sci. U.S.A.

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Distinct human leukemia-associated cell surface glycoprotein GP160 defined by monoclonal antibody SN6(human non-T-cell and myelo-monocytic leukemia antigen/cell surface glycoprotein/immunodiagnosis) YURO Communicated by David Harker, July 10, 1986 ABSTRACT In this study, a monoclonal antibody (mAb) termed SN6 was generated by immunizing a mouse with a non-T-cell leukemia antigen preparation isolated from cell membranes of leukemia cells derived from a patient (FJ) with non-T/non-B-cell-type acute lymphoblastic leukemia (ALL). SN6 was tested against a variety of cultured and uncultured human cell specimens by using a sensitive cellular radioimmunoassay. Among the 26 cultured malignant and nonmalignant cell lines tested, SN6 reacted with all of the 6 leukemic non-T/non-B (including pre-B)-cell lines tested-i.e., KM-3, NALM-16, REH, NALL-1, NALM-1, and NALM-6. Of these cell lines, 5 were derived from individual patients with ALL; the remaining 1 was from a patient with chronic myelocytic leukemia in blast crisis. In addition, SN6 reacted with 3 of 3 leukemic myelo-monocytic cell lines tested-i.e., ML-2, HL-60, and U937. SN6 did not react with any other cell lines. A consistent result was obtained with 42 fresh (uncultured) cell specimens derived from individual patients with several different types of leukemias. SN6 reacted with 11 of 16 non-T/non-B (including pre-B)-cell ALL specimens. In addition, it reacted with various myelo-monocytic leukemia cell specimens to various degrees. SN6 did not show a significant reaction with normal peripheral blood cells tested, which included B cells, T cells, granulocytes, monocytes, and erythrocytes. However, it reacted with a small population (-1 % as determined by immunofluorescence staining) of normal bone marrow cells. The approximate molecular mass of the glycoprotein antigen defined by SN6 was determined to be 160,000 by radioimmunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Only one component of 80,000 daltons was formed upon reduction of the 160,000 molecular mass antigen. Therefore, this antigen is apparently a homodimer of a 80,000-dalton subunit. This conclusion was further corroborated by two-dimensional gel analysis, which showed a single well-defined spot for the reduced antigen. We designate this distinct human leukemiaassociated cell surface antigen "GP160."

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The growth of large megakaryocyte colonies from human bone marrow

Cited by 195 publications

References 17 publications

Fibrinogen and its fragment D stimulate proliferation of human hemopoietic cells in vitro.

Fibrinogen and its fragment D stimulate proliferation of human hemopoietic cells in vitro.

Rapid alterations in lateral mobility of lipids in the plasma membrane of activated human megakaryocytes

Distinct human leukemia-associated cell surface glycoprotein GP160 defined by monoclonal antibody SN6.

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