Extracellular Matrix Proteins and Transcription of Matrix-Associated Genes in Mesenchymal Stromal Cells during Modeling of the Effects of Microgravity

Abstract We have studied the deposition of extracellular matrix proteins in the adherent stroma of long-term murine bone marrow cultures. Stable hematopoiesis was maintained for greater than 12 wk. At selected intervals, culture dishes were sacrificed by removing all nonadherent cells and air drying the dishes. The adherent stromal layer was analyzed for the presence of intracellular and extracellular collagen, fibronectin, and laminin using double immunofluorescent staining with specific antisera against these matrix components. In cultures examined during the first 2 wk, large numbers of stromal cells contained collagen, fibronectin, and laminin. Over the next 2 wk, an extensive extracellular network of fibronectin, laminin, and collagen was deposited on the dishes, which persisted throughout the life of the cultures. In contrast to a previous report, we detected substantial numbers of endothelial cells by means of immunofluorescent staining of stromal cells with antisera to type IV collagen, laminin, and factor VIII antigen. Although deposition of these extracellular matrix proteins coincides with onset of active hematopoietic cell production, the relative roles of the stromal cells and the extracellular matrix in supporting hematopoiesis in murine bone marrow cell cultures remain to be determined.

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Extracellular matrix production by the adherent cells of long-term murine bone marrow cultures

Blood ◽

10.1182/blood.v61.3.540.bloodjournal613540 ◽

1983 ◽

Vol 61 (3) ◽

pp. 540-547 ◽

Cited By ~ 4

Author(s):

KS Zuckerman ◽

MS Wicha

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Stromal Cells ◽

Extracellular Matrix Proteins ◽

Matrix Proteins ◽

Immunofluorescent Staining ◽

Murine Bone Marrow ◽

Bone Marrow Cultures ◽

Marrow Cultures

We have studied the deposition of extracellular matrix proteins in the adherent stroma of long-term murine bone marrow cultures. Stable hematopoiesis was maintained for greater than 12 wk. At selected intervals, culture dishes were sacrificed by removing all nonadherent cells and air drying the dishes. The adherent stromal layer was analyzed for the presence of intracellular and extracellular collagen, fibronectin, and laminin using double immunofluorescent staining with specific antisera against these matrix components. In cultures examined during the first 2 wk, large numbers of stromal cells contained collagen, fibronectin, and laminin. Over the next 2 wk, an extensive extracellular network of fibronectin, laminin, and collagen was deposited on the dishes, which persisted throughout the life of the cultures. In contrast to a previous report, we detected substantial numbers of endothelial cells by means of immunofluorescent staining of stromal cells with antisera to type IV collagen, laminin, and factor VIII antigen. Although deposition of these extracellular matrix proteins coincides with onset of active hematopoietic cell production, the relative roles of the stromal cells and the extracellular matrix in supporting hematopoiesis in murine bone marrow cell cultures remain to be determined.

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Spreading and actin cytoskeleton organization of cartilage and bone marrow stromal cells cocultured on various extracellular matrix proteins

Cell and Tissue Biology ◽

10.1134/s1990519x15010083 ◽

2015 ◽

Vol 9 (1) ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

E. I. Sachenberg ◽

N. N. Nikolaenko ◽

G. P. Pinaev

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Actin Cytoskeleton ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Extracellular Matrix Proteins ◽

Marrow Stromal Cells ◽

Matrix Proteins ◽

Cytoskeleton Organization ◽

Actin Cytoskeleton Organization

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Beta7 Integrins Regulate Podia Formation in Multiple Myeloma (MM) Cells for the Interaction with the Cellular and Non-Cellular Bone Marrow (BM) Stroma

Blood ◽

10.1182/blood.v120.21.3979.3979 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3979-3979

Author(s):

Karthik Ramasamy ◽

Henry Lekgetho ◽

Jose Luis Orgaz ◽

Amna Anwar ◽

Victoria Sanz-Moreno ◽

...

Keyword(s):

Extracellular Matrix ◽

Cell Adhesion ◽

Cell Lines ◽

Stromal Cells ◽

Conflicts Of Interest ◽

3D Culture ◽

Extracellular Matrix Proteins ◽

Cytoskeletal Proteins ◽

Matrix Proteins ◽

2D And 3D

Abstract Abstract 3979 Adhesion of MM cells to the BM microenvironment can lead to cell adhesion mediated drug resistance (CAM-DR). We have previously shown that treatment with Dexamethasone (a drug commonly used against MM alone or in combination with new therapeutic agents) induces in MM cells increased assembly of CD138, and F-actin containing membrane extensions named podia. Using this 2D co-culture model we found that podia from MM cells from patients or MM cell lines elongate on the surface of BM stromal cells and also interconnect distant MM cells correlating with CAM-DR. Formation of podia was observed after at least 6 days of co-culture when many soluble factors and extracellular matrix proteins may have been secreted and remodelled. We also showed that preventing SDF1-alpha binding to the CXCR4 receptor in MM cells inhibits podia formation. In order to investigate the possible role of ECM proteins and their ligands in podia assembly we have now tested the formation of podia in 3D co-cultures of MM and stromal cells embedded in gels composed of polymerised extracellular matrix proteins found in the BM including collagen I and collagen IV. We now found that MM cells incubated overnight in 3D matrix lattices assembled podia that elongated along fibres of polymerised extracellular matrix that put them in contact with stromal cells or other distant MM cells. We also found that culture of MM cells in 3D matrices in the absence of stromal cells did not result in podia formation. These results suggest that stromal cells promote podia formation in contact with the extracellular matrix. It has been shown by Neri et al1that beta7 integrins are involved in MM cell adhesion to BM cells and extracellular matrix during recruitment to the BM and they are involved in CAM-DR. We found that integrin beta7 KD MM cells failed to form podia to the same extent as the parental cell lines in 2D and 3D cultures. Podia assembled by beta7 KD cells were shorter and highly irregular in shape in comparison to long and straight podia formed by parental MM cells. Using immunostaining, we found that podia in beta7 KD cells lacked internal F-actin as well as vimentin filaments that we identified in podia of parental MM cells. However, total levels of these cytoskeletal proteins remained unchanged as detected by western blot. Dexamethasone-induced increased formation of podia was also dependent on the expression of beta7 integrins by MM cells and the presence of BM stromal cells in 2D and 3D culture conditions. We conclude that podia promote the formation of cellular networks of distant MM cells as well as the contact with BM stromal cells in 3D and this process is involved in CAM-DR against Dexamethasone. Podia assemble in contact with stromal cells and along extracellular matrix cables and require polymerisation of actin and vimentin cytoskeletal filaments downstream of beta7 integrins. Disclosures: No relevant conflicts of interest to declare.

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