Biochemical characterization of heparan sulfate derived from murine hemopoietic stromal cell lines: A bone marrow-derived cell line S17 and a fetal liver-derived cell line AFT024

2002 ◽  
Vol 87 (2) ◽  
pp. 160-172 ◽  
Author(s):  
Katia Arcanjo ◽  
Gisele Belo ◽  
Cristiane Folco ◽  
Claudio C. Werneck ◽  
Radovan Borojevic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Heparan Sulfate ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cell ◽  
Biochemical Characterization ◽  
Fetal Liver

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2328-2328
Author(s):  
Katja C. Weisel ◽  
Ying Gao ◽  
Jae-Hung Shieh ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stromal Cell Line ◽  
Mes Cells

Abstract The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

Expression of the Smad3 Transgene Restores Radiosensitivity and Migratory Capacity to a Smad3−/− Clonal Bone Marrow Stromal Cell Line.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4307-4307
Author(s):  
Michael W. Epperly ◽  
Shaonan Cao ◽  
Xichen Zhang ◽  
Emily E. Greenberger ◽  
Julie Goff ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Gene Product ◽  
Cell Lines ◽  
Stromal Cell ◽  
Cell Tracking ◽  
Bone Marrow Stromal Cell ◽  
Saturation Density ◽  
Marrow Stromal Cell ◽  
Migratory Capacity

Abstract An intact Smad3 gene product is critical for a functioning signal transduction pathway following TGF binding to the TGF-β receptor. We have previously established Smad3−/− and Smad3+/+ long term bone marrow cultures (LTBMCs) and isolated clonal bone marrow stromal cell lines from each. The Smad3−/− cells were smaller in size but had a faster cell doubling time (24 hours compared to 48 hours) and increased saturation density compared to +/+ cells (15.3 ± 1.0 x 105 cells/25 mm2 flask compared to 3.8 ± 0.1 x 105, p = 0.003). The plating efficiency of the lines was similar (18.3 ± 2.7 compared to 15.5 ± 1.7, p = 0.417). We transfected the Smad3−/− cell line with a retrovirus containing the Smad3 transgene, and selected a subclone expressing the transgene mRNA, designated Smad3−/−(3). Smad3−/−(3) cells were increased in size to that of Smad3+/+ cells, and showed decreased cell saturation density. Using the Cytoworks computer controlled cell tracking Bioreactor, we measured the migration of each clonal line. Tissue culture wells of 100 cells per well were followed for 5 days tracking each cell in quadruplicate wells per cell line. Smad3+/+ cells migrated significantly faster over 5 days in culture compared to Smad3−/− cells. (The average velocities were 0.62 μm/min for Smad3+/+ and 0.36 μm/min for Smad3−/−, p<0.0001). Over 5 days, the average velocities for Smad3+/+ cells were 0.51, 0.51, 0.52, 0.72, 0.91 μm/min, and for Smad3−/− cells were 0.28, 0.38, 0.41, 0.37, 0.35 μm/min. The 5 p-values comparing these cell lines were all <0.0001. The 7 day clonagenic irradiation survival curve showed that Smad3+/+ and Smad3−/−(3) cells were significantly more sensitive (D0 = 1.75 ± 0.03 and 1.51 ± 0.07 Gy, respectively) compared to the Smad3−/− cell line (D0 = 2.43 ± 0.06 Gy, p=0.0016 and 0.0103). These results demonstrate a concordance of radioresistance and decreased migratory capacity in bone marrow stromal cells devoid of a functioning Smad3 gene product, and restoration of both properties following overexpression of the transgene product. These data may help explain the decreased radiation fibrosis observed in Smad3−/− mice.

Biological and Molecular Characterization by Integrative Genomics Approach of CMA-03/06, a Newly Established Interleukin-6 Independent Variant of the CMA-03 Human Myeloma Cell Line.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1814-1814
Author(s):  
Donata Verdelli ◽  
Lucia Nobili ◽  
Katia Todoerti ◽  
Laura Mosca ◽  
Sonia Fabris ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Molecular Characterization ◽  
Cell Lines ◽  
Stromal Cells ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
Growth And Survival ◽  
Genome Wide

Abstract Abstract 1814 Poster Board I-840 Background The growth and survival of multiple myeloma (MM) cells in the bone marrow microenvironment is regulated by functional complex interactions between the tumor cells and the surrounding bone marrow stromal cells mediated by adhesion molecules and the production of several cytokines of which interleukin-6 (IL-6) has been identified as the most important. Major advances in the investigation of MM biology were made possible by the availability of human myeloma cell lines (HMCLs). The IL-6-dependent CMA-03 cell line was established in our laboratory from a peritoneal effusion of a refractory relapsed MM patient. By gradually decreasing the IL-6 added to the culture, an IL-6-independent variant, CMA-03/06, could be obtained. Aims. To perform a biological and molecular characterization of this novel cell line, and to provide insights into the signaling pathways and target genes involved in the growth and survival of CMA-03/06. Methods. The growth, immunophenotypic, cytogenetic and fluorescence in situ hybridization (FISH) characterization of CMA-03/06 cell line was performed by means of standard procedures. IL-6 production into the culture media was determined using a high sensitivity IL-6 specific ELISA. Genome-wide profiling data were generated by means of Affymetrix GeneChip® Human Mapping 250K Nsp arrays; copy number (CN) alterations were calculated using the DNAcopy Bioconductor package, based on circular binary segmentation method. Global gene expression profiling (GEP) was performed by means of the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix); the supervised analyses were done using the SAM software version 3.0. Results Unlike CMA-03, the addition of IL-6 to the culture medium of CMA-03/06 cells or co-culture with multipotent mesenchymal stromal cells did not induce an increase in CMA-03/06 proliferation. IL-6 was not detected in the supernatants from either CMA-03 or CMA-03/06 cell lines within 48 h, suggesting that the IL-6 independence of CMA03/06 cells is not a result of the development of an autocrine IL-6 loop. Nevertheless, IL-6 induced the activation of STAT3 and STAT1 in both cell lines, even if a slight constitutive STAT3 phosphorylation was found in CMA-03/06. The immunophenotypic analysis showed a significant difference in the expression of three antigens in the 2 cell lines: CD45 was considerably reduced in CMA-03/06 cells, whereas they were found positive for both chains of IL-6 receptor, CD126 and CD130, almost undetectable in CMA-03. Conventional cytogenetic and FISH analyses did not reveal differences between the 2 HMCLs. The genome-wide analysis allowed the identification of about 100 altered chromosomal regions common to both HMCLs, mostly DNA gains. Comparison of CMA-03/06 and CMA-03 cells evidenced a different CN in only 15 small chromosomal regions, 8 of which did not contain any transcript, whereas few genes were located on the other ones. GEP analysis of CMA-03/06 compared with CMA-03 identified 21 upregulated and 47 downregulated genes, many of which particularly relevant for MM biology, mainly involved in cellular signaling, cell cycle, cell adhesion, cell development, regulation of transcription, immunologic, inflammatory or defense activity, apoptosis. None of the genes differentially expressed in CMA-03/06 compared with CMA-03 except 1 were positioned on the chromosomal regions showing a different CN. Finally, CMA-03/06 cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA-03 cells. Conclusions Our data show the IL-6 independence of CMA-03/06 cell line in the absence of an autocrine IL-6 loop; the cells, however, maintain the IL-6 signaling pathway responsiveness. A consistent number of genes particularly relevant for MM biology were found deregulated in CMA-03/06 cell line compared with CMA-03. Furthermore, CMA-03/06 cell line shows an increased resistance to apoptosis. The novel CMA03/06 cell line may thus represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3079-3089
Author(s):  
J Mladenovic ◽  
SM Anderson
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conditioned Media ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf

The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.

scholarly journals Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 447-455 ◽  
Author(s):  
D Zipori ◽  
J Toledo ◽  
K von der Mark
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cell ◽  
Leukemic Cells ◽  
Matrix Composition ◽  
Mouse Bone Marrow ◽  
Type I

Abstract Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

scholarly journals Effect of AGM and Fetal Liver-Derived Stromal Cell Lines on Globin Expression in Adult Baboon (P. anubis) Bone Marrow-Derived Erythroid Progenitors

PLoS ONE ◽  
2012 ◽  
Vol 7 (5) ◽  
pp. e36846 ◽  
Author(s):  
Donald Lavelle ◽  
Kestutis Vaitkus ◽  
Maria Armila Ruiz ◽  
Vinzon Ibanez ◽  
Tatiana Kouznetsova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Erythroid Progenitors

scholarly journals Cloning and Characterization of a Novel Oxidoreductase KDRF from a Human Bone Marrow-derived Stromal Cell Line KM-102

1997 ◽  
Vol 272 (4) ◽  
pp. 2570-2577 ◽  
Author(s):  
Ryuta Koishi ◽  
Ichiro Kawashima ◽  
Chigusa Yoshimura ◽  
Mie Sugawara ◽  
Nobufusa Serizawa
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Stromal Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Stromal Cell Line

scholarly journals Hemonectin mediates adhesion of engrafted murine progenitors to a clonal bone marrow stromal cell line from Sl/Sld mice

Blood ◽  
1991 ◽  
Vol 77 (8) ◽  
pp. 1691-1698 ◽  
Author(s):  
P Anklesaria ◽  
JS Greenberger ◽  
TJ Fitzgerald ◽  
B Sullenbarger ◽  
M Wicha ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cumulative Number ◽  
Specific Staining ◽  
Marrow Stromal Cell

Abstract Mutant Sl/Sld mice exhibit decreased marrow hematopoiesis. The defect is known to reside in the marrow microenvironment of these animals, which is reproduced in vitro by primary marrow explants as well as by cloned marrow stromal cell lines. Bone marrow progenitor cells are incapable of adhering to primary Sl/Sld stromal cells or cloned stromal cell lines derived from them to form cobblestone-islands and proliferate. The role of hemonectin, a marrow-specific adhesion protein in the defective hematopoiesis of the Sl/Sld mice, was studied. Indirect immunoperoxidase staining of marrow in situ from Sl/Sld mice showed little specific staining while specific staining was seen in a pericellular distribution in marrow from +/+ mice. Hemonectin expression in several cloned stromal cell lines from Sl/Sld mice was compared by immunoblotting with that in cloned stromal cell lines from normal +/+ littermates. Cell line Sld3, which has the least hematopoiesis supportive capacity in vitro, showed no detectable hemonectin by immunoblotting, while Sld1 and Sld2 showed detectable but greatly reduced amounts compared with normal +/+ 2.4, GBI/6, and D2XRII. Confluent cultures incubated with purified hemonectin and engrafted with enriched progenitors showed a significant increase in the cumulative number of cobbleston-islands and day 14 spleen colony- forming units (CFU-s) forming progenitors (39.15 +/- 3.6/dish; 16.3 +/- 3.1/dish, respectively), compared with untreated Sld3 cultures (cobblestone-islands 8.1 +/- 3.6/dish; CFU-s forming progenitors 8.8 +/- 0.05/dish). Hemonectin-mediated progenitor cell binding to the Sld3 stromal cells was specifically inhibited by antihemonectin but not by preimmune serum. These data support the role of hemonectin in early progenitor-stromal cell interactions.

scholarly journals Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3079-3089 ◽  
Author(s):  
J Mladenovic ◽  
SM Anderson
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conditioned Media ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gm Csf

Abstract The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.

scholarly journals Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 447-455
Author(s):  
D Zipori ◽  
J Toledo ◽  
K von der Mark
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cell ◽  
Leukemic Cells ◽  
Matrix Composition ◽  
Mouse Bone Marrow ◽  
Type I

Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

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