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Joel S Greenberger - One of the best experts on this subject based on the ideXlab platform.
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expression of the smad3 transgene restores radiosensitivity and migratory capacity to a smad3 clonal bone marrow Stromal Cell Line
Blood, 2005Co-Authors: Michael W Epperly, Shaonan Cao, Xichen Zhang, Emily E Greenberger, Julie P Goff, Hong Wang, Al Bahnson, Donna Shields, Darcy Franicola, Joel S GreenbergerAbstract: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 10 5 Cells/25 mm 2 flask compared to 3.8 ± 0.1 x 10 5 , 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 = 1.75 ± 0.03 and 1.51 ± 0.07 Gy, respectively) compared to the Smad3−/− Cell Line (D 0 = 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.
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establishment of a novel clonal murine bone marrow Stromal Cell Line for assessment of p53 responses to genotoxic stress
Toxicology, 2002Co-Authors: Nikolai V Gorbunov, Joel S Greenberger, James E Morris, Brian D ThrallAbstract:The p53 protein is widely regarded as an important sensor of genotoxic damage in Cells, and mutations in p53 are the most frequent observed in human cancers. Rapid assays for evaluating the potential of a chemical or physical agent to alter the transcriptional regulatory role of p53 may therefore serve as useful tools in toxicological research. In this study, the use of enhanced green fluorescent protein (EGFP) as a live Cell reporter to assess the transactivation response of p53 to chemical and physical agents was evaluated. A stable murine bone marrow Stromal Cell Line (D2XRIIGFP24) expressing EGFP under control of p53 response elements was established. D2XRIIGFP24 Cells displayed low constitutive background fluorescence which was significantly enhanced in response to exposure to agents that induced p53 protein levels. Increases in EGFP fluorescence in response to oxidative and nitrosative stress as well as UVC irradiation were dose-dependent, detectable within 3 h of exposure and correlated closely with the amount of p53 protein accumulated within the Cell. The results demonstrate the potential for rapid and early detection of p53 transactivation using the EGFP reporter approach and indicate this approach is adaptable to a variety of fluorescent assay techniques and a useful Cell model for molecular toxicology research.
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perfusion enhances functions of bone marrow Stromal Cells in three dimensional culture
Cell Transplantation, 1998Co-Authors: Shuichi Mizuno, Julie Glowacki, Joel S GreenbergerAbstract:Abstract Perfusion of medium through three-dimensional (3D) collagen sponges enhanced viability and function of cocultivated marrow Stromal and hematopoietic Cell Lines. Cells of the murine bone marrow Stromal Cell Line GPIa were cultured in novel 3D collagen sponges, made from pepsin-digested bovine skin. Static cultures of sponges were maintained in dishes with media changes every other day. Perfused sponges were contained in a glass column with medium flow set at 1.3 mL/min. In some sponges, the 32D cl3 c-fms m (CRX-1) hematopoietic progenitor Cell Line was added 7 days after GPIa Cells. At 7 and 16 days, light microscopic evaluation showed poor viability of Cells in static sponge cultures. In perfused sponge cultures, there was greater Cellularity throughout the sponge and abundant accumulation of metachromatic extraCellular matrix surrounding GPIa Cells. Chondroitin 6-sulfate and heparan sulfate were identified as components of the matrix by immunohistochemical methods. DNA synthesis was evaluated by 15-h exposure of cultures to bromodeoxyuridine (BrdU), with subsequent immunohistochemical localization with monoclonal anti-BrdU antibody. Cells positive for BrdU were identified at the outer surfaces of both static and perfused sponges; however, positive Cells were also seen throughout the internal areas of the sponges that were perfused. These results suggest that better nutrient exchange occurred in perfused sponges. In static cocultures of GPIa and CRX-1 Cells, there was no detectable viability of the IL-3–dependent CRX-1 Cells; however, under perfused conditions, CRX-1 Cells flourished within the sponges as documented by BrdU incorporation. Thus, medium perfusion enhanced GPIa Stromal Cell Line viability and function in 3D collagen sponge cultures, as demonstrated by BrdU incorporation, matrix production, and support of CRX-1 Cells. This novel culture system may be useful for examining the interactions of bone marrow Stromal Cells with extraCellular matrix molecules, soluble and matrix-bound factors, and with other Cell types.
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quantitation of murine hematopoietic stem Cells in vitro by limiting dilution analysis of cobblestone area formation on a clonal Stromal Cell Line
Experimental Hematology, 1993Co-Authors: S Neben, P Anklesaria, Joel S Greenberger, Peter MauchAbstract:Murine hematopoietic stem Cells with varying proliferative capacity can be assayed by limiting dilution analysis of "cobblestone area" (CA) formation on Stromal layers in microlong-term bone marrow cultures. Cobblestone area forming Cell (CAFC) frequency determined at early time points (day 7) correlates with mature stem Cells measured as day 8 CFU-S, whereas CAFC frequency determined at day 28 correlates more closely with long-term marrow repopulating ability. Here we report a modification of the CAFC assay in which a clonal bone marrow Stromal Cell Line, GB1/6, is substituted for fresh marrow-derived Stromal layers. This modification simplifies the initial culture setup, eliminates inhomogeneities in the Stromal layer and reduces the need for mice. Normal bone marrow CAFC frequencies were the same for both types of Stromal Cell underlayer, demonstrating the ability of a clonal Cell Line to completely replace the heterogeneous microenvironment of fresh stroma for in vitro stem Cell support.
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adipogenesis in a myeloid supporting bone marrow Stromal Cell Line
Journal of Cellular Biochemistry, 1992Co-Authors: Jeffery M Gimble, Kellee Youkhana, Xianxin Hua, Kay L Medina, Mary Sullivan, Helen B Bass, Chisun S Wang, Joel S GreenbergerAbstract:The bone marrow stroma contains pre-adipocyte Cells which are part of the hemopoietic microenvironment. Cloned Stromal Cell Lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine Stromal Cell Line and compared to other Stromal and pre-adipocyte Cell Lines. In long-term cultures, the +/+2.4 Stromal Cells support myeloid Cell growth, consistent with their expression of macrophage-colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 Cells failed to support Stromal Cell dependent B Lineage lymphoid Cells in vitro, suggesting that these Stromal Cells exhibit only a myelopoietic support function. The +/+2.4 Cells differentiate into adipocytes spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of agonists based on morphologic and gene marker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination, a time dependent increase in the steady state mRNA and enzyme activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis was accompanied by a slight decrease in the signal intensity of the macrophage-colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow Stromal Cell Lines. These data demonstrate that although the lympho-hematopoietic support function of pre-adipocyte bone marrow Stromal Cell Lines is heterogeneous, they share a common mechanism of adipogenesis.
Deborah E Banker - One of the best experts on this subject based on the ideXlab platform.
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acute myeloid leukemia Cells are protected from spontaneous and drug induced apoptosis by direct contact with a human bone marrow Stromal Cell Line hs 5
Experimental Hematology, 2001Co-Authors: Sara M Garrido, Frederick R Appelbaum, Cheryl L Willman, Deborah E BankerAbstract:In vitro culture systems that parallel in vivo growth conditions are needed to study leukemia biology and to accurately test therapeutic efficacies. We investigated the effects of the HS-5 human bone marrow Stromal Cell Line on cultured primary leukemia Cell survival and chemosensitivity.A total of 30 bone marrow (BM) samples from untreated acute myeloid leukemia (AML) patients were cultured for 96 hours with serum and growth factors, without HS-5, or in direct contact with HS-5 monolayers, or with HS-5 separated from AML Cells by transwell inserts. In some experiments, cytosine arabinoside or daunomycin was added for the last 18 hours of culture. Apoptosis frequencies, bcl-2 protein expression, proliferating Cell nuclear antigen expression, and Cell cycle distributions were determined in four-color flow cytometry analyses of CD45(+) leukemia Cells. In comparison to control growth conditions, direct contact with HS-5 significantly inhibited culture-induced and drug-induced apoptosis of AML Cells. Direct contact of AML Cells with HS-5 significantly increased short-term proliferation and viability, and colony formation of primary AML Cells. HS-5-mediated apoptosis inhibition was not consistently associated with increased bcl-2 protein in AML Cells. Noncontact conditions inhibited drug-induced apoptosis significantly less than direct contact with HS-5. Coculture of AML Cells on HS-5 monolayers improved in vitro leukemia Cell survival and attenuated chemotherapy-induced leukemia Cell killing. This has practical significance, increasing the fraction of primary AML samples that can be analyzed in vitro, and allows drug sensitivity testing in growth conditions more similar to the bone marrow microenvironment.
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acute myeloid leukemia Cells are protected from spontaneous and drug induced apoptosis by direct contact with a human bone marrow Stromal Cell Line hs 5
Experimental Hematology, 2001Co-Authors: Sara M Garrido, Frederick R Appelbaum, Cheryl L Willman, Deborah E BankerAbstract:Abstract Objective In vitro culture systems that parallel in vivo growth conditions are needed to study leukemia biology and to accurately test therapeutic efficacies. We investigated the effects of the HS-5 human bone marrow Stromal Cell Line on cultured primary leukemia Cell survival and chemosensitivity. Materials and Methods A total of 30 bone marrow (BM) samples from untreated acute myeloid leukemia (AML) patients were cultured for 96 hours with serum and growth factors, without HS-5, or in direct contact with HS-5 monolayers, or with HS-5 separated from AML Cells by transwell inserts. In some experiments, cytosine arabinoside or daunomycin was added for the last 18 hours of culture. Apoptosis frequencies, bcl -2 protein expression, proliferating Cell nuclear antigen expression, and Cell cycle distributions were determined in four-color flow cytometry analyses of CD45 + leukemia Cells. Results In comparison to control growth conditions, direct contact with HS-5 significantly inhibited culture-induced and drug-induced apoptosis of AML Cells. Direct contact of AML Cells with HS-5 significantly increased short-term proliferation and viability, and colony formation of primary AML Cells. HS-5–mediated apoptosis inhibition was not consistently associated with increased bcl -2 protein in AML Cells. Noncontact conditions inhibited drug-induced apoptosis significantly less than direct contact with HS-5. Conclusion Coculture of AML Cells on HS-5 monolayers improved in vitro leukemia Cell survival and attenuated chemotherapy-induced leukemia Cell killing. This has practical significance, increasing the fraction of primary AML samples that can be analyzed in vitro, and allows drug sensitivity testing in growth conditions more similar to the bone marrow microenvironment.
Sara M Garrido - One of the best experts on this subject based on the ideXlab platform.
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acute myeloid leukemia Cells are protected from spontaneous and drug induced apoptosis by direct contact with a human bone marrow Stromal Cell Line hs 5
Experimental Hematology, 2001Co-Authors: Sara M Garrido, Frederick R Appelbaum, Cheryl L Willman, Deborah E BankerAbstract:In vitro culture systems that parallel in vivo growth conditions are needed to study leukemia biology and to accurately test therapeutic efficacies. We investigated the effects of the HS-5 human bone marrow Stromal Cell Line on cultured primary leukemia Cell survival and chemosensitivity.A total of 30 bone marrow (BM) samples from untreated acute myeloid leukemia (AML) patients were cultured for 96 hours with serum and growth factors, without HS-5, or in direct contact with HS-5 monolayers, or with HS-5 separated from AML Cells by transwell inserts. In some experiments, cytosine arabinoside or daunomycin was added for the last 18 hours of culture. Apoptosis frequencies, bcl-2 protein expression, proliferating Cell nuclear antigen expression, and Cell cycle distributions were determined in four-color flow cytometry analyses of CD45(+) leukemia Cells. In comparison to control growth conditions, direct contact with HS-5 significantly inhibited culture-induced and drug-induced apoptosis of AML Cells. Direct contact of AML Cells with HS-5 significantly increased short-term proliferation and viability, and colony formation of primary AML Cells. HS-5-mediated apoptosis inhibition was not consistently associated with increased bcl-2 protein in AML Cells. Noncontact conditions inhibited drug-induced apoptosis significantly less than direct contact with HS-5. Coculture of AML Cells on HS-5 monolayers improved in vitro leukemia Cell survival and attenuated chemotherapy-induced leukemia Cell killing. This has practical significance, increasing the fraction of primary AML samples that can be analyzed in vitro, and allows drug sensitivity testing in growth conditions more similar to the bone marrow microenvironment.
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acute myeloid leukemia Cells are protected from spontaneous and drug induced apoptosis by direct contact with a human bone marrow Stromal Cell Line hs 5
Experimental Hematology, 2001Co-Authors: Sara M Garrido, Frederick R Appelbaum, Cheryl L Willman, Deborah E BankerAbstract:Abstract Objective In vitro culture systems that parallel in vivo growth conditions are needed to study leukemia biology and to accurately test therapeutic efficacies. We investigated the effects of the HS-5 human bone marrow Stromal Cell Line on cultured primary leukemia Cell survival and chemosensitivity. Materials and Methods A total of 30 bone marrow (BM) samples from untreated acute myeloid leukemia (AML) patients were cultured for 96 hours with serum and growth factors, without HS-5, or in direct contact with HS-5 monolayers, or with HS-5 separated from AML Cells by transwell inserts. In some experiments, cytosine arabinoside or daunomycin was added for the last 18 hours of culture. Apoptosis frequencies, bcl -2 protein expression, proliferating Cell nuclear antigen expression, and Cell cycle distributions were determined in four-color flow cytometry analyses of CD45 + leukemia Cells. Results In comparison to control growth conditions, direct contact with HS-5 significantly inhibited culture-induced and drug-induced apoptosis of AML Cells. Direct contact of AML Cells with HS-5 significantly increased short-term proliferation and viability, and colony formation of primary AML Cells. HS-5–mediated apoptosis inhibition was not consistently associated with increased bcl -2 protein in AML Cells. Noncontact conditions inhibited drug-induced apoptosis significantly less than direct contact with HS-5. Conclusion Coculture of AML Cells on HS-5 monolayers improved in vitro leukemia Cell survival and attenuated chemotherapy-induced leukemia Cell killing. This has practical significance, increasing the fraction of primary AML samples that can be analyzed in vitro, and allows drug sensitivity testing in growth conditions more similar to the bone marrow microenvironment.
Frederick R Appelbaum - One of the best experts on this subject based on the ideXlab platform.
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acute myeloid leukemia Cells are protected from spontaneous and drug induced apoptosis by direct contact with a human bone marrow Stromal Cell Line hs 5
Experimental Hematology, 2001Co-Authors: Sara M Garrido, Frederick R Appelbaum, Cheryl L Willman, Deborah E BankerAbstract:In vitro culture systems that parallel in vivo growth conditions are needed to study leukemia biology and to accurately test therapeutic efficacies. We investigated the effects of the HS-5 human bone marrow Stromal Cell Line on cultured primary leukemia Cell survival and chemosensitivity.A total of 30 bone marrow (BM) samples from untreated acute myeloid leukemia (AML) patients were cultured for 96 hours with serum and growth factors, without HS-5, or in direct contact with HS-5 monolayers, or with HS-5 separated from AML Cells by transwell inserts. In some experiments, cytosine arabinoside or daunomycin was added for the last 18 hours of culture. Apoptosis frequencies, bcl-2 protein expression, proliferating Cell nuclear antigen expression, and Cell cycle distributions were determined in four-color flow cytometry analyses of CD45(+) leukemia Cells. In comparison to control growth conditions, direct contact with HS-5 significantly inhibited culture-induced and drug-induced apoptosis of AML Cells. Direct contact of AML Cells with HS-5 significantly increased short-term proliferation and viability, and colony formation of primary AML Cells. HS-5-mediated apoptosis inhibition was not consistently associated with increased bcl-2 protein in AML Cells. Noncontact conditions inhibited drug-induced apoptosis significantly less than direct contact with HS-5. Coculture of AML Cells on HS-5 monolayers improved in vitro leukemia Cell survival and attenuated chemotherapy-induced leukemia Cell killing. This has practical significance, increasing the fraction of primary AML samples that can be analyzed in vitro, and allows drug sensitivity testing in growth conditions more similar to the bone marrow microenvironment.
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acute myeloid leukemia Cells are protected from spontaneous and drug induced apoptosis by direct contact with a human bone marrow Stromal Cell Line hs 5
Experimental Hematology, 2001Co-Authors: Sara M Garrido, Frederick R Appelbaum, Cheryl L Willman, Deborah E BankerAbstract:Abstract Objective In vitro culture systems that parallel in vivo growth conditions are needed to study leukemia biology and to accurately test therapeutic efficacies. We investigated the effects of the HS-5 human bone marrow Stromal Cell Line on cultured primary leukemia Cell survival and chemosensitivity. Materials and Methods A total of 30 bone marrow (BM) samples from untreated acute myeloid leukemia (AML) patients were cultured for 96 hours with serum and growth factors, without HS-5, or in direct contact with HS-5 monolayers, or with HS-5 separated from AML Cells by transwell inserts. In some experiments, cytosine arabinoside or daunomycin was added for the last 18 hours of culture. Apoptosis frequencies, bcl -2 protein expression, proliferating Cell nuclear antigen expression, and Cell cycle distributions were determined in four-color flow cytometry analyses of CD45 + leukemia Cells. Results In comparison to control growth conditions, direct contact with HS-5 significantly inhibited culture-induced and drug-induced apoptosis of AML Cells. Direct contact of AML Cells with HS-5 significantly increased short-term proliferation and viability, and colony formation of primary AML Cells. HS-5–mediated apoptosis inhibition was not consistently associated with increased bcl -2 protein in AML Cells. Noncontact conditions inhibited drug-induced apoptosis significantly less than direct contact with HS-5. Conclusion Coculture of AML Cells on HS-5 monolayers improved in vitro leukemia Cell survival and attenuated chemotherapy-induced leukemia Cell killing. This has practical significance, increasing the fraction of primary AML samples that can be analyzed in vitro, and allows drug sensitivity testing in growth conditions more similar to the bone marrow microenvironment.
Maria G Roubelakis - One of the best experts on this subject based on the ideXlab platform.
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a novel function for the haemopoietic supportive murine bone marrow ms 5 mesenchymal Stromal Cell Line in promoting human vasculogenesis and angiogenesis
British Journal of Haematology, 2012Co-Authors: Bob Zhou, Grigorios Tsaknakis, Kate E Coldwell, Cheen P Khoo, Maria G RoubelakisAbstract:Summary The bone marrow contains specific microenvironmental stem Cell niches that maintain haemopoiesis. CXCL12-expressing mesenchymal Stromal Cells are closely associated with the bone marrow sinusoidal endothelia, forming key elements of the haemopoietic stem Cell niche, yet their ability to regulate endothelial function is not clearly defined. Given that the murine nestin+ Cell Line, MS-5, provides a clonal surrogate bone marrow Stromal niche capable of regulating both murine and human primitive haemopoietic stem/progenitor Cell (HSC/HPC) fate in vitro, we hypothesized that MS-5 Cells might also support new blood vessel formation and function. Here, for the first time, we demonstrate that this is indeed the case. Using proteome arrays, we identified HSC/HPC active angiogenic factors that are preferentially secreted by haemopoietic supportive nestin+MS-5 Cells, including CXCL12 (SDF-1), NOV (CCN3), HGF, Angiopoietin-1 and CCL2 (MCP-1). Concentrating on CXCL12, we confirmed its presence in MS-5 conditioned media and demonstrated that its antagonist in receptor binding, AMD-3100, which mobilizes HSC/HPCs and endothelial progenitors from bone marrow, could significantly reduce MS-5 mediated human vasculogenesis in vitro, principally by regulating human endothelial Cell migration. Thus, the clonal nestin+MS-5 murine bone marrow Stromal Cell Line not only promotes human haemopoiesis but also induces human vasculogenesis, with CXCL12 playing important roles in both processes.
