scholarly journals Evidence for direct action of human biosynthetic (recombinant) GM-CSF on erythroid progenitors in serum-free culture

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1867-1871 ◽  
Author(s):  
AR Migliaccio ◽  
M Bruno ◽  
G Migliaccio
Keyword(s):  
Direct Action ◽  
Erythroid Progenitors ◽  
Serum Free ◽  
Gm Csf ◽  
Plastic Surface ◽  
Blast Cells ◽  
Fetal Bovine ◽  
Serum Free Conditions ◽  
Stimulating Factor

Abstract The biologic activity of human biosynthetic granulocyte-monocyte colony stimulating factor (GM-CSF) was investigated in serum-free culture of erythroid progenitors derived from adult peripheral blood. The morphology of erythroid bursts and the cloning efficiency of BFU-E under serum-free conditions were similar to those observed in dishes with fetal bovine serum (FBS). For these experiments, progenitor cells were partially purified by Ficoll-Paque density centrifugation, adherence to a plastic surface, and complement-mediated cytotoxicity of Leu-1+ elements. For some studies, blastlike cells were harvested directly from 6-day-old semisolid cultures. In serum-free culture of the light-density cell fraction, biosynthetic erythropoietin (Ep) was sufficient for formation of pure and mixed erythroid colonies whereas GM-CSF was required for granulocyte-monocytic colonies. When adherent and Leu-1+ cells were removed, or when in vitro differentiated blast cells were used as a source of progenitors, neither Ep or GM-CSF alone induced colony formation. In dishes supplemented with both growth factors, erythroid bursts were detected. Although the presence of GM- CSF alone did not induce formation of any colony or clusters, BFU-E were recorded when Ep was added 8 days later, suggesting that BFU-E could be maintained. Terminal maturation of the resulting erythroid bursts was delayed by 8 days. These results provide evidence that GM- CSF acts directly on early erythroid progenitors. Furthermore, they suggest that both Ep and GM-CSF are necessary to start the differentiation process.

scholarly journals Evidence for direct action of human biosynthetic (recombinant) GM-CSF on erythroid progenitors in serum-free culture

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1867-1871 ◽  
Author(s):  
AR Migliaccio ◽  
M Bruno ◽  
G Migliaccio
Keyword(s):  
Direct Action ◽  
Erythroid Progenitors ◽  
Serum Free ◽  
Gm Csf ◽  
Plastic Surface ◽  
Blast Cells ◽  
Fetal Bovine ◽  
Serum Free Conditions ◽  
Stimulating Factor

The biologic activity of human biosynthetic granulocyte-monocyte colony stimulating factor (GM-CSF) was investigated in serum-free culture of erythroid progenitors derived from adult peripheral blood. The morphology of erythroid bursts and the cloning efficiency of BFU-E under serum-free conditions were similar to those observed in dishes with fetal bovine serum (FBS). For these experiments, progenitor cells were partially purified by Ficoll-Paque density centrifugation, adherence to a plastic surface, and complement-mediated cytotoxicity of Leu-1+ elements. For some studies, blastlike cells were harvested directly from 6-day-old semisolid cultures. In serum-free culture of the light-density cell fraction, biosynthetic erythropoietin (Ep) was sufficient for formation of pure and mixed erythroid colonies whereas GM-CSF was required for granulocyte-monocytic colonies. When adherent and Leu-1+ cells were removed, or when in vitro differentiated blast cells were used as a source of progenitors, neither Ep or GM-CSF alone induced colony formation. In dishes supplemented with both growth factors, erythroid bursts were detected. Although the presence of GM- CSF alone did not induce formation of any colony or clusters, BFU-E were recorded when Ep was added 8 days later, suggesting that BFU-E could be maintained. Terminal maturation of the resulting erythroid bursts was delayed by 8 days. These results provide evidence that GM- CSF acts directly on early erythroid progenitors. Furthermore, they suggest that both Ep and GM-CSF are necessary to start the differentiation process.

scholarly journals Effect of recombinant granulocyte-macrophage colony-stimulating factor on murine thrombocytopoiesis in vitro and in vivo

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1433-1438
Author(s):  
T Ishibashi ◽  
H Kimura ◽  
Y Shikama ◽  
T Uchida ◽  
S Kariyone ◽  
...  
Keyword(s):  
Tritiated Thymidine ◽  
In Vivo Studies ◽  
Colony Stimulating Factor ◽  
Serum Free ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

To investigate the effect of recombinant granulocyte-macrophage colony- stimulating factor (rGM-CSF) on murine megakaryocytopoiesis in vitro, the factor was added to both serum-free colony assays and liquid marrow cultures. GM-CSF had a significant megakaryocytic colony-stimulating activity. After 2 hours of preincubation with and without 10 ng/mL rGM- CSF, the percentage of megakaryocyte colony-forming cell (CFU-MK) in DNA synthesis was determined by tritiated-thymidine suicide using colony growth. The reduction of CFU-MK colony numbers in marrow culture was 47.5% +/- 9.9%, 20.9% +/- 5.2% (control), respectively, indicating that the factor affected cell cycle at CFU-MK levels. When acetylcholinesterase (AchE) production was measured fluorometrically after 4 days of liquid culture, rGM-CSF elicited an increase in AchE activity in a dose-dependent fashion. To determine if the hematopoietin acts directly on megakaryocytic differentiation, 2 ng/mL rGM-CSF was added to serum-free cultures of 295 single megakaryocytes isolated from CFU-MK colonies. An increase in size was observed in 65% of cells initially 10 to 20 microns in diameter, 71% of cells 20 to 30 microns, and 40% of cells greater than 30 microns. Conversely, in absence of GM- CSF, 17%, 31%, and 10% of cells in each group increased in diameter. These data suggest that rGM-CSF promotes murine megakaryocytopoiesis in vitro and that the response to the factor is direct. To determine if the factor influences megakaryocytic/thrombocytic lineage in vivo, 1 and 5 micrograms of rGM-CSF were administered intraperitoneally every 12 hours for 6 consecutive days. Although a two- to three-fold increase in peripheral granulocytes was observed, neither megakaryocytic progenitor cells or platelets changed. Histologic analysis of bone marrow megakaryocytes showed no increase in size and number. The in vivo studies demonstrated no effect of GM-CSF on thrombocytopoiesis. The discrepancies between the in vitro and in vivo effects of GM-CSF require additional investigations.

scholarly journals Effect of recombinant granulocyte-macrophage colony-stimulating factor on murine thrombocytopoiesis in vitro and in vivo

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1433-1438 ◽  
Author(s):  
T Ishibashi ◽  
H Kimura ◽  
Y Shikama ◽  
T Uchida ◽  
S Kariyone ◽  
...  
Keyword(s):  
Tritiated Thymidine ◽  
In Vivo Studies ◽  
Colony Stimulating Factor ◽  
Serum Free ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract To investigate the effect of recombinant granulocyte-macrophage colony- stimulating factor (rGM-CSF) on murine megakaryocytopoiesis in vitro, the factor was added to both serum-free colony assays and liquid marrow cultures. GM-CSF had a significant megakaryocytic colony-stimulating activity. After 2 hours of preincubation with and without 10 ng/mL rGM- CSF, the percentage of megakaryocyte colony-forming cell (CFU-MK) in DNA synthesis was determined by tritiated-thymidine suicide using colony growth. The reduction of CFU-MK colony numbers in marrow culture was 47.5% +/- 9.9%, 20.9% +/- 5.2% (control), respectively, indicating that the factor affected cell cycle at CFU-MK levels. When acetylcholinesterase (AchE) production was measured fluorometrically after 4 days of liquid culture, rGM-CSF elicited an increase in AchE activity in a dose-dependent fashion. To determine if the hematopoietin acts directly on megakaryocytic differentiation, 2 ng/mL rGM-CSF was added to serum-free cultures of 295 single megakaryocytes isolated from CFU-MK colonies. An increase in size was observed in 65% of cells initially 10 to 20 microns in diameter, 71% of cells 20 to 30 microns, and 40% of cells greater than 30 microns. Conversely, in absence of GM- CSF, 17%, 31%, and 10% of cells in each group increased in diameter. These data suggest that rGM-CSF promotes murine megakaryocytopoiesis in vitro and that the response to the factor is direct. To determine if the factor influences megakaryocytic/thrombocytic lineage in vivo, 1 and 5 micrograms of rGM-CSF were administered intraperitoneally every 12 hours for 6 consecutive days. Although a two- to three-fold increase in peripheral granulocytes was observed, neither megakaryocytic progenitor cells or platelets changed. Histologic analysis of bone marrow megakaryocytes showed no increase in size and number. The in vivo studies demonstrated no effect of GM-CSF on thrombocytopoiesis. The discrepancies between the in vitro and in vivo effects of GM-CSF require additional investigations.

scholarly journals Construction of Recombinant Human GM-CSF and GM-CSF-ApoA-I Fusion Protein and Evaluation of Their Biological Activity

2021 ◽  
Vol 14 (5) ◽  
pp. 459
Author(s):  
Mariya Pykhtina ◽  
Svetlana Miroshnichenko ◽  
Vladimir Romanov ◽  
Antonina Grazhdantseva ◽  
Galina Kochneva ◽  
...  
Keyword(s):  
Bone Marrow Cells ◽  
E Coli ◽  
Gm Csf ◽  
Human Apolipoprotein ◽  
Proliferative Effect ◽  
Erythroleukemia Cells ◽  
Blast Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

In this study, two strains of the yeast P. pastoris were constructed, one of which produced authentic recombinant human granulocyte-macrophage colony-stimulating factor (ryGM-CSF), and the other was a chimera consisting of ryGM-CSF genetically fused with mature human apolipoprotein A-I (ApoA-I) (ryGM-CSF-ApoA-I). Both forms of the cytokine were secreted into the culture medium. The proteins’ yield during cultivation in flasks was 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Both forms of recombinant GM-CSF stimulated the proliferation of human TF-1 erythroleukemia cells; however, the amount of chimera required was 10-fold that of authentic GM-CSF to induce a similar proliferative effect. RyGM-CSF exhibited a 2-fold proliferative effect on BFU-E (burst-forming units—erythroid) at a concentration 1.7 fold less than non-glycosylated E. coli-derived GM-CSF. The chimera together with authentic ryGM-CSF increased the number of both erythroid precursors and BMC granulocytes after 48 h of incubation of human bone marrow cells (BMCs). In addition, the chimeric form of ryGM-CSF was more effective at increasing the viability of the total amount of BMCs, decreasing apoptosis compared to the authentic form. ryGM-CSF-ApoA-I normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. The chimera form of GM-CSF exhibited the properties of a multilinear growth factor, modulating the activity of GM-CSF and, perhaps, it may be more suitable for the normalization of granulopoiesis.

scholarly journals A GM-colony-stimulating factor (CSF) activated ribonuclease system transregulates M-CSF receptor expression in the murine FDC-P1/MAC myeloid cell line.

1992 ◽  
Vol 3 (5) ◽  
pp. 535-544 ◽  
Author(s):  
B C Gliniak ◽  
L S Park ◽  
L R Rohrschneider
Keyword(s):  
Protein Synthesis ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Mrna Degradation ◽  
Receptor Expression ◽  
Metabolic Inhibitors ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Stimulating Factor

The murine myeloid precursor cell line FDC-P1/MAC simultaneously expresses receptors for multi-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF. Growth of FDC-P1/MAC cells in either multi-CSF or GM-CSF results in the posttranscriptional suppression of M-CSF receptor (c-fms proto-oncogene) expression. We use the term transregulation to describe this control of receptor expression and have further characterized this regulatory process. The removal of FDC-P1/MAC cells from GM-CSF stimulation resulted in the re-expression of c-fms mRNA independent of M-CSF stimulation and new protein synthesis. Switching FDC-P1/MAC cells from growth in M-CSF to GM-CSF caused the selective degradation of c-fms mRNA within 6 h after factor switching. Blocking protein synthesis or gene transcription with metabolic inhibitors effectively prevented GM-CSF stimulated degradation of c-fms mRNA. These results suggest that the transregulation of c-fms transcripts by GM-CSF requires the transcriptional activation of a selective mRNA degradation factor. In vitro analysis, the use of cytoplasmic cell extracts, provided evidence that a ribonuclease is preferentially active in GM-CSF stimulated cells, although the specificity for mRNA degradation in vitro is broader than seen in vivo. Together, these data suggest that GM-CSF can dominantly transregulate the level of c-fms transcript through the transcriptional activation of a ribonuclease degradation system.

scholarly journals Differentiation of blast cells from a Down's syndrome patient with transient myeloproliferative disorder

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 508-512
Author(s):  
J Suda ◽  
M Eguchi ◽  
Y Akiyama ◽  
Y Iwama ◽  
T Furukawa ◽  
...  
Keyword(s):  
Down's Syndrome ◽  
Down’S Syndrome ◽  
Colony Growth ◽  
Myeloproliferative Disorder ◽  
In Vitro Proliferation ◽  
Gm Csf ◽  
Blast Cells ◽  
Macrophage Colony ◽  
Male Neonate

A male neonate with Down's syndrome and congenital myeloproliferative disorder was studied. His blood picture showed the unique coexistence of leukocytosis with matured cells and a large number of blast cells. The in vitro proliferation and differentiation of blast cells into various lineages in the presence of phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LCM) was examined by using a liquid culture and a methylcellulose culture system. The differentiation of blast cells into myeloid cells was confirmed by specific cytochemical stainings, electron microscopy, and an immunologic study. No specific factors in the plasma of the patient promoted the proliferation or differentiation of blast cells. The cellular composition of colonies grown in methylcellulose culture from single blast cells was studied by a micromanipulation technique. High plating efficiency was observed. Of 136 cultures, 78 showed colony growth. Half of the blast cells were colony-forming cells that could proliferate and differentiate into basophils, neutrophils, eosinophils, macrophages, and erythrocytes in the presence of PHA-LCM. Using the blast cells with a high differentiation capacity to the basophil pathway, we studied the effect of recombinant granulocyte-macrophage colony-stimulating factor (GM- CSF). Recombinant GM-CSF support neutrophils, eosinophils, and macrophages but not typical basophils. These findings of the cell differentiation of blast cells into various kinds of cells in vitro were in agreement with the finding of neutrophilia, eosinophilia, basophilia, and thrombocythemia in this patient.

scholarly journals Interleukin-6 (IL-6) is an intermediate in IL-1-induced proliferation of leukemic human megakaryoblasts

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 1972-1979 ◽  
Author(s):  
MA Brach ◽  
B Lowenberg ◽  
L Mantovani ◽  
U Schwulera ◽  
R Mertelsmann ◽  
...  
Keyword(s):  
Thymidine Incorporation ◽  
Interleukin 1 ◽  
Leukemic Cells ◽  
Megakaryoblastic Leukemia ◽  
Gm Csf ◽  
Endogenous Production ◽  
Neutralizing Monoclonal Antibody ◽  
In Vitro Effects ◽  
Stimulating Factor

Abstract We have examined the in vitro effects of recombinant human (rh) interleukin-1 (IL-1) on the growth of purified megakaryoblasts obtained from patients with acute megakaryoblastic leukemia. We demonstrate that both IL-1 alpha and IL-1 beta treatment of these cells led to stimulation of DNA synthesis (as shown by increase of 3H-thymidine incorporation up to 35-fold) and also resulted in colony formation of leukemic megakaryoblasts. However, the stimulatory effect of IL-1 was dependent on endogenous production of IL-6, because addition of neutralizing monoclonal antibody (MoAb) to IL-6 abrogated the stimulatory activity of IL-1. In contrast, neutralizing MoAbs to granulocyte (G)-colony stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF failed to counteract the growth- enhancing effects of IL-1. Leukemic megakaryoblasts accumulated IL-6 mRNA and released IL-6 protein into their culture supernatant when exposed to rh IL-1 but failed to disclose transcripts for G-, GM-, and M-CSF under these conditions. Analysis of IL-6 receptor (IL-6R) transcript levels demonstrated that megakaryoblasts constitutively expressed IL-6R mRNA and that these transcripts are down-regulated to undetectable levels upon exposure to IL-1 and IL-6. Increase of 3H- thymidine incorporation by megakaryoblasts could be duplicated by exogenous IL-6 that could be blocked by neutralizing MoAb to IL-6. In conclusion, our results suggest that leukemic megakaryoblasts could produce and secrete IL-6, and express IL-6R, and that the growth- enhancing effect of IL-1 on these cells is indirect, via production of IL-6 by leukemic cells.

scholarly journals Treatment with monocyte-derived partially purified GM-CSF but not G-CSF aborts the development of transplanted chloroleukemia in rats

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 1077-1080 ◽  
Author(s):  
JJ Jimenez ◽  
AA Yunis
Keyword(s):  
Conditioned Medium ◽  
Molecular Size ◽  
Myelogenous Leukemia ◽  
Rat Lung ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

We have previously demonstrated that cultured rat chloroleukemia cells, MIA C51, will terminally differentiate to macrophages when treated with rat lung-conditioned medium in vitro and in vivo. In the present study we fractionated rat monocyte-conditioned medium by ultrafiltration according to molecular size. The fraction with molecular weight (mol wt) 30 to 50 Kd containing partially purified granulocyte-macrophage colony-stimulating factor (GM-CSF) activity caused the differentiation of C51 cells to macrophages in vitro and in diffusion chambers in vivo. Treatment of young rats with this fraction aborted the development of chloroleukemia from transplanted C51 cells. In contrast, the fraction with mol wt 10 to 30 Kd containing virtually all the G-CSF activity exhibited no differentiation activity either in vitro or in vivo. It is concluded that in this rat myelogenous leukemia model partially purified GM-CSF but not G-CSF contains the effector molecule(s) causing terminal differentiation of C51 cells and tumor cell rejection.

scholarly journals Effects of recombinant GM-CSF on the blast cells of acute myeloblastic leukemia

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 313-316 ◽  
Author(s):  
T Hoang ◽  
N Nara ◽  
G Wong ◽  
S Clark ◽  
MD Minden ◽  
...  
Keyword(s):  
Suspension Culture ◽  
Adherent Cells ◽  
Colony Stimulating Factor ◽  
Response Curves ◽  
Self Renewal ◽  
Gm Csf ◽  
Blast Cells ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

The effects of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) were compared to those of media conditioned by the continuous bladder carcinoma line, HTB9 (HTB9-CM), using three criteria. First, both GM-CSF and HTB9-CM stimulated blast colony formation in methylcellulose cultures, patient-to-patient variations were seen in the dose-response curves, and GM-CSF was effective, but less so that HTB9-CM. Second, GM-CSF also enhanced growth of blast progenitors in suspension culture, indicating its capacity to support self-renewal. GM-CSF was as effective as HTB9-CM in the production of adherent cells during the growth of blast cells in suspension, a finding that is interpreted to mean that GM-CSF also supports postdeterministic events in blast differentiation. Finally, colonies growing in the presence of GM-CSF were not phenotypically different than those stimulated by HTB9-CM.

scholarly journals Extracellular Vesicles Derived From Canine Mesenchymal Stromal Cells in Serum Free Culture Medium Have Anti-inflammatory Effect on Microglial Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Yukina Kuwahara ◽  
Karin Yoshizaki ◽  
Hidetaka Nishida ◽  
Hiroaki Kamishina ◽  
Sadatoshi Maeda ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Stromal Cells ◽  
Culture Medium ◽  
Serum Free ◽  
Naturally Occurring ◽  
Serum Free Condition ◽  
Cell Sources ◽  
Fetal Bovine ◽  
Inflammatory Effect

Mesenchymal stem/stromal cells (MSCs) have been used as cell sources for treating dogs with naturally-occurring diseases. Extracellular vesicles (EVs) derived from MSCs are now recognized as pivotal to modulating the immune response and supporting tissue repair. Manufacture of MSC-EVs for clinical application mandates removal of the xeno-proteins, including fetal bovine serum. The objective of this study was to examine whether canine MSCs survived and secreted EVs in serum-free medium (SFM) conditions and to assess the immunomodulatory effect of EVs in vitro. Canine MSCs were found to survive and secrete EVs under SFM conditions. The surface markers of MSCs in the SFM were similar to MSCs in complete culture medium. Canine MSC-EVs had a diameter of ~300 nm and were positive for EV markers. MSC-derived EVs from the serum-free condition reduced the levels of IL-1β by BV-2 cells in response to LPS stimulation. These results warrant further studies of the use of SFM for producing EVs derived from canine MSCs.

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