Fibronectin promotes rat Schwann cell growth and motility

Techniques are now available for culturing well characterized and purified Schwann cells. Therefore, we investigated the role of fibronectin in the adhesion, growth, and migration of cultured rat Schwann cells. Double-immunolabeling shows that, in primary cultures of rat sciatic nerve, Schwann cells (90%) rarely express fibronectin, whereas fibroblasts (10%) exhibit a granular cytoplasmic and fibrillar surface-associated fibronectin. Secondary cultures of purified Schwann cells do not express fibronectin. Exogenous fibronectin has a small effect on promoting the adhesion of Schwann cells to the substrate and does not significantly affect cell morphology, but it produced a surface fibrillar network on fibronectin on the secondary Schwann cells. Tritiated thymidine autoradiography revealed that addition of fibronectin to the medium, even at low concentrations, markedly stimulates Schwann cell proliferation, in both primary and secondary cultures. In addition, when cell migration was measured in a Boyden chamber assay, fibronectin was found to moderately, but clearly, stimulate directed migration or chemotaxis.     

Tumor necrosis factor-alpha as a proliferative signal for an IL-2-dependent T cell line: strict species specificity of action

The ability of TNF-alpha to stimulate T cell proliferation was examined. We demonstrate that murine rTNF-alpha induces the proliferation of CT6, a murine T cell line previously thought to be responsive only to IL-2. This activity appears to be the result of the direct action of murine rTNF-alpha on the CT6 cells because neither 1) murine IL-2 or murine IL-4, lymphokines also capable of inducing CT6 proliferation, were detected in culture supernatants from murine rTNF-alpha-treated CT6 cells nor 2) did antibodies specific for IL-2 or IL-4 inhibit murine rTNF-alpha-induced CT6 proliferation. Unlike many of the activities displayed by TNF-alpha, its ability to induce CT6 cell proliferation shows strict species specificity as indicated by the failure of human rTNF-alpha to stimulate these cells. Flow cytometric analysis and binding of radiolabeled TNF-alpha have indicated that receptors for TNF-alpha on these cells are specific for murine TNF-alpha. The ability of murine rTNF-alpha to induce the proliferation of certain T cell lines further indicates that this molecule plays an important role in regulation of T cell-mediated immune responses.     

Transmission and regulation of biochemical stimulus via a nanoshell directly adsorbed on the cell membrane to enhance chondrogenic differentiation of mesenchymal stem cell

A nanoscale artificial extracellular matrix (nanoshell) formed by layer-by-layer adsorption can enhance and modulate the function of stem cells by transferring biochemical stimulus to the cell directly. Here, the nanoshell composed of fibronectin (FN) and chondroitin sulfate (CS) is demonstrated to promote chondrogenic differentiation of mesenchymal stem cells (MSCs). The multilayer structure of nanoshell is formed by repeating self-assembly of FN and CS, and its thickness can be controlled through the number of layers. The expression of chondrogenic markers in MSCs coated with the FN/CS nanoshell was increased as the number of bilayers in the nanoshell increased until four, but when it exceeds five bilayers, the effect began to decrease. Finally, the MSCs coated with optimized four bilayers of FN/CS nanoshell have high chondrogenic differentiation efficiency and showed the potential to increase formation of cartilage tissue when it is transplanted into mouse kidney. So, the precise regulation of stem cell fate at single cell level can be possible through the cellular surface modification by self-assembled polymeric film.     

Opposing effects of collagen I and vitronectin on fibronectin fibril structure and function

Extracellular matrix fibronectin fibrils serve as passive structural supports for the organization of cells into tissues, yet can also actively stimulate a variety of cell and tissue functions, including cell proliferation. Factors that control and coordinate the functional activities of fibronectin fibrils are not known. Here, we compared effects of cell adhesion to vitronectin versus type I collagen on the assembly of and response to, extracellular matrix fibronectin fibrils. The amount of insoluble fibronectin matrix fibrils assembled by fibronectin-null mouse embryonic fibroblasts adherent to collagen- or vitronectin-coated substrates was not significantly different 20 h after fibronectin addition. However, the fibronectin matrix produced by vitronectin-adherent cells was ~ 10-fold less effective at enhancing cell proliferation than that of collagen-adherent cells. Increasing insoluble fibronectin levels with the fibronectin fragment, anastellin did not increase cell proliferation. Rather, native fibronectin fibrils polymerized by collagen- and vitronectin-adherent cells exhibited conformational differences in the growth-promoting, III-1 region of fibronectin, with collagen-adherent cells producing fibronectin fibrils in a more extended conformation. Fibronectin matrix assembly on either substrate was mediated by α5β1 integrins. However, on vitronectin-adherent cells, α5β1 integrins functioned in a lower activation state, characterized by reduced 9EG7 binding and decreased talin association. The inhibitory effect of vitronectin on fibronectin-mediated cell proliferation was localized to the cell-binding domain, but was not a general property of αvβ3 integrin-binding substrates. These data suggest that adhesion to vitronectin allows for the uncoupling of fibronectin fibril formation from downstream signaling events by reducing α5β1 integrin activation and fibronectin fibril extension.     

The primary effects of clinorotation on cultured human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are specific cells capable of long-term proliferation and differentiation into various stromal tissue cell types. The state of MSCs depends on the cellular microenvironment and several soluble factors. We proposed that gravity could, in addition, influence MSCs features. To prove this hypothesis, we studied the effects of prolonged clinorotation on cultured human MSC morphology, proliferation rate and expression of specific cellular markers. Human bone marrow-derived MSCs were isolated by Histopaque-1.077 density centrifugation and cultured in DMEM-LG with 10% FBS. MSC cultures were composed of fibroblastoid cells negative for hemopoietic cell markers and positive for ASMA, collagen-1, fibronectin, CD54, CD105 and CD106. Cells were exposed to clinorotation from 1 hour to 10 days. It was shown that the proliferative rate was decreased in experimental cultures as compared to cells growing in normal conditions. Clinorotated MSCs appeared more flattened and reached confluence at a lower cell density. The obtained results suggest that cultured human mesenchymal stem cells sense the changes in gravity vector and may respond to microgravity by altered functional activity.     

Stimulation of integrin-mediated cell contractility by fibronectin polymerization

Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function.     

Different culture method changing CD105 expression in amniotic fluid MSCs without affecting differentiation ability or immune function

MSCs are kind of cultured cells that reside in different tissues as inducers or regulators of physiological and pathological processes. Here, we derived MSCs from amniotic fluid and compared their differentiation ability and immunosuppression effect on PHA-activated PBMC with those of MSCs isolated from umbilical cords. Amniotic fluid MSCs were isolated and cultured on commercial AFC medium and classic MSC medium, and the number and size of colonies were used to evaluate differences in primary and passaged culture. Rate of proliferation, population doubling time, cell morphology, cell surface markers and mRNA expression were measured in subcultured cells. Furthermore, a comparative study was performed with umbilical cord MSCs to assess the ability of differentiation and immunosuppressive effect of PHA-stimulated PBMCs. Amniotic fluid MSCs were isolated and expanded by three methods, and exhibited nearly all the characteristics of umbilical cord MSCs. Compared with umbilical cord MSCs, amniotic fluid MSCs had an enhanced osteogenic and chrondrogenic differentiation capability, and stronger immunosuppression effect of inhibition of PHA-activated PBMC division. Culture with commercial AFCs medium yielded the highest percentage of CD105 expression and showed some advantages in primary cell isolation, cell source-specific marker retention and cell proliferation. We demonstrated that amniotic fluid MSCs exhibited some advantages over umbilical cord MSCs, and different culture media caused cell proliferation, cell surface marker and cell morphology change, but were not associated with varying differentiation capability and immune effects.     

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.     

Comparison between Culture Conditions Improving Growth and Differentiation of Blood and Bone Marrow Cells Committed to the Endothelial Cell Lineage

The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.     

Thrombin stimulation of matrix fibronectin

Trypsin, thrombin, and peptide analogues of the new amino terminus of the proteolyzed thrombin receptor, SFLLRN and SFLLRNPNDKYEPF, stimulated embryonic fibroblasts cultured as 3-dimensional tissue-like aggregates to elaborate a fibronectin-rich extracellular matrix. Enzymatically inactive thrombin and the control peptide FLLRN failed to stimulate matrix production. The induction of cell proliferation correlated with production of the fibronectin matrix. The regions of active cell proliferation in the fibroblast aggregates co-localized with the matrix and peptide analogues of the RGD cell-adhesion site of fibronectin reversibly inhibited the accumulation of the fibronectin matrix and the stimulation of cell proliferation by SFLLRN. Two different preparations of the fibronectin matrix stimulated cell proliferation in aggregates cultured in growth factor-free medium. We suggest that the stimulation of matrix production is a necessary event for mitogenic signaling in mesenchymal tissue. The tight coupling between the matrigenic and mitogenic activities of growth factors was absent in monolayer cultures of chick embryonic fibroblasts since thrombin and trypsin induced proliferation of monolayer-cultured cells without inducing the production of a fibronectin matrix. © 1996 Wiley-Liss, Inc.     

Mesenchymal stromal cells promote tumor growth through the enhancement of neovascularization

Mesenchymal stromal cells (MSCs), also called mesenchymal stem cells, migrate and function as stromal cells in tumor tissues. The effects of MSCs on tumor growth are controversial. In this study, we showed that MSCs increase proliferation of tumor cells in vitro and promote tumor growth in vivo. We also further analyzed the mechanisms that underlie these effects. For use in in vitro and in vivo experiments, we established a bone marrow-derived mesenchymal stromal cell line from cells isolated in C57BL/6 mice. Effects of murine MSCs on tumor cell proliferation in vitro were analyzed in a coculture model with B16-LacZ cells. Both coculture with MSCs and treatment with MSC-conditioned media led to enhanced growth of B16-LacZ cells, although the magnitude of growth stimulation in cocultured cells was greater than that of cells treated with conditioned media. Co-injection of B16-LacZ cells and MSCs into syngeneic mice led to increased tumor size compared with injection of B16-LacZ cells alone. Identical experiments using Lewis lung carcinoma (LLC) cells instead of B16-LacZ cells yielded similar results. Consistent with a role for neovascularization in MSC-mediated tumor growth, tumor vessel area was greater in tumors resulting from co-injection of B16-LacZ cells or LLCs with MSCs than in tumors induced by injection of cancer cells alone. Co-injected MSCs directly supported the tumor vasculature by localizing close to vascular walls and by expressing an endothelial marker. Furthermore, secretion of leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2 and vascular endothelial growth factor was increased in cocultures of MSCs and B16-LacZ cells compared with B16-LacZ cells alone. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis.     

Collagen type I together with fibronectin provide a better support for endothelialization

Endothelialization of vascular implants is limited by the inability of cells to retain adhesion when exposed to flow. Extracellular matrix proteins, including fibronectin and collagen, enhance cell adherence on materials. This study investigated the behaviour of Human Umbilical Vein Endothelial Cells (HUVEC) on extracellular matrix coated polystyrene. Collagen and fibronectin were coated as single and double layers to analyse differences in cell proliferation, morphology, and cell-protein interactions. Significantly higher endothelial cell proliferation and migration rates were observed on the collagen and collagen+fibronectin coating compared to the uncoated or fibronectin-coated sample. Immmunofluorescent microscopy showed evidence of extracellular matrix remodelling in the double, collagen+fibronectin coating. These results strongly suggest that a double coating of collagen+fibronectin provides a better support structure for endothelial cell growth and contributes to improve the ability of vascular implants to become and remain endothelialized.     

Dental pulp stem cells (DPSCs) increase prostate cancer cell proliferation and migration under in vitro conditions

Cancer as a multistep and complicated disease is regulated by several molecular and cellular events. Cancer treatment could be managed at the early stages when the tumor is confined in the tissue. However, disseminated cancer cells metastasize to other body parts and generate new tumors resulting in mortality. Mesenchymal stem cells (MSCs) are found in different body parts and helps adult tissue regeneration. The role of MSCs in cancer progression has emerged as one of the important aspects in cancer biology and is the aim of interest in recent years. In the current study, effects of Dental Pulp Stem Cells (DPSCs) on PC-3 prostate cancer cell proliferation and migration were conducted by cell proliferation, apoptosis, gene expression and cell migration analysis in vitro. Condition medium (CM) obtained from DPSCs increased cell proliferation of PC-3 cells and decreased apoptosis. Either administration of CM or trans well co-culture of DPSCs increased cell migration in scratch assay, confirmed by gene expression analysis of migratory genes including fibronectin, laminin and collagen type I (Col I). Furthermore, DPSCs participated in a self-organized structure with PC-3 cells in co-culture conditions. Overall, results indicated that DPSCs could promote PC-3 cancer cell proliferation and metastasis in co-culture conditions in vitro.     

The influence of mesenchymal stromal cells on B-cell line growth and immunoglobulin synthesis

There are conflicting data on the influence of mesenchymal stromal cells (MSCs) on B-lymphocyte growth, differentiation, and immunoglobulin (Ig) production. The aim of this study was to investigate the influence of MSCs derived from the adipose tissue of healthy donors and cancer patients on the proliferation and Ig synthesis of the lymphoblastoid cell line Namalva and myeloma cell line U266. Cocultivation of Namalva cells with MSCs stimulated their proliferation and decreased the doubling time and minimal inoculation dose necessary for cloning of these lymphoblastoid cells. The presence of MSCs supported the survival and proliferation of Namalva cells cultivated in growth factor-deficient medium. MSCs also stimulated the proliferation of U266 myeloma cells. MSCs derived from adipose tissue of both healthy donors and patients with breast cancer effectively stimulated cell proliferation of B-cell lines. The presence of MSCs in mixed cultures had no influence on the production of IgM or IgE by Namalva or U266 cells, respectively. Cocultivation of Namalva or U266 cells with MSCs provided tight contacts between cells of both types.     

Investigation of the mesenchymal stem cell compartment by means of a lentiviral barcode library

The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic “barcodes” has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F-derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.     

Mesenchymal stem cells inhibit generation and function of both CD34+-derived and monocyte-derived dendritic cells

Mesenchymal stem cells (MSCs) are not only able to evade the immune system, but they have also been demonstrated to exert profound immunosuppressive properties on T cell proliferation. However, their effect on the initiators of the immune response, the dendritic cells (DCs), are relatively unknown. In the present study, the effects of human MSCs on the differentiation and function of both CD34+ -derived DCs and monocyte-derived DCs were investigated. The presence of MSCs during differentiation blocked the differentiation of CD14+CD1a- precursors into dermal/interstitial DCs, without affecting the generation of CD1a+ Langerhans cells. In line with these observations, MSCs also completely prevented the generation of immature DCs from monocytes. The inhibitory effect of MSCs on DC differentiation was dose dependent and resulted in both phenotypical and functional modifications, as demonstrated by a reduced expression of costimulatory molecules and hampered capacity to stimulate naive T cell proliferation. The inhibitory effect of MSCs was mediated via soluble factors. Taken together, these data demonstrate that MSCs, next to the antiproliferative effect on T cells, have a profound inhibitory effect on the generation and function of both CD34+ -derived and monocyte-derived DCs, indicating that MSCs are able to modulate immune responses at multiple levels.