Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.     

Analysis of myogenesis by somatic cell hybridization. II. Retention of myogenic competence and suppression of transformed properties in hybrids between differentiation competent and incompetent rat L6 myoblasts

This study describes the characteristics of hybrids between two closely related rat myoblast lines, which differ both in the ability to express their program of differentiation and in the expression of neoplastic properties. Myogenic, nonneoplastic L6J1-S cells were hybridized with nonmyogenic, neoplastic L6J1-N1 cells. Six hybrid clones were isolated and expanded for analysis of myogenic competence, and four of these clones were also evaluated for parameters of transformation, including tumorigenicity, ability to clone in agar, and surface fibronectin. In addition to our analysis of isolated clones, we also assessed myogenic differentiation in colonies representing 226 early hybrid clones. Results of all these analyses demonstrate that the myogenic phenotype is retained and that the tumorigenic/transformed phenotype is suppressed in the hybrids. Furthermore, our results indicate that when the programs for myogenesis and neoplastic transformation are confronted within a single cell, they are expressed as mutually exclusive alternatives. In contrast to these results on myogenic X nonmyogenic L6 hybrids, it has been reported that isolated clones of A9 X L6 exhibited extinction of myogenic competence and retention of transformed properties. We have evaluated myotube formation in over 300 early hybrid clones between A9 and either diploid or subtetraploid L8 rat myoblasts. Our results demonstrate that all of these hybrid clones exhibit extinction regardless of the ploidy of the myoblast parent, and they further indicate that extinction is not a consequence of chromosome loss. These results support the conclusion that in A9 X L6 hybrids, the nonmyogenic, transformed phenotype is dominant.     

Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L- lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.     

Migratory behavior of cells on embryonic retina basal lamina

In order to study cell translocation in vitro on a physiological substrate a novel cell migration assay was developed using the inner limiting membrane of the avian embryonic retina. The matrix sheet consists of a laminin-rich basal lamina covered by a dense layer of neuroepithelial endfeet. The retina basal lamina does not contain fibronectin. Cells translocating on this substrate displace the neuroepithelial endfeet, leaving behind tracks in the endfeet monolayer. Motility of cells and the relative forward to lateral migration can be quantitated by measuring lengths, widths, and areas of the tracks. Using this assay system, the conditions and patterns of cell migration for a variety of cells have been examined. In the absence of serum all cell types show only minor migratory activity and addition of serum to the culture medium always enhances the rate of cell migration in a saturable, dose-response manner. The serum cannot be replaced by fibronectin or vitronectin (serum spreading factor). For maximum cell migration, serum has to be constantly present in the medium; however, 58% cell migration is obtained in serum-free medium when the matrix is preincubated with serum. According to the area and linearity of the tracks, the migratory behavior of the different cells can be classified into three groups: (i) fibroblasts and the nonpigmented Bowes melanoma cells form straight and long tracks; (ii) glioma, sarcoma, and carcinoma cells from straight but short tracks, and (iii) neuronal tumor cells, epithelial cells, and pigmented B16 melanoma cells form wide and short tracks. Comparative studies with low and high metastatic clones of tumorgenic cell lines show that migratory activity and metastatic potential of cells do not necessarily correlate. Finally, we show that fibroblasts deposit fibronectin fibrils on their paths as they migrate on the basal lamina. Fibronectin trails are also seen when fibroblasts are cultured on plain basal laminae that are pretreated with detergent to remove the endfeet monolayer. Likewise, when fibroblasts are cultured in the presence of antifibronectin antibodies, the fibronectin secreted by cells is detectable. Due to antibody treatment the cellular fibronectin is precipitated and its normal fibril formation is inhibited; however, the translocation of fibroblasts is not impaired.     

Variant endothelial cells. Fibronectin as a transducer of signals for migration and neovascularisation

A morphologic and growth control variant of bovine aortal endothelial cells has been isolated and shown to synthesise factor VIII antigen (McAuslan and Reilly '79). The variant also possesses the endothelial surface markers angiotensin converting enzyme and alpha 2-microglobulin. The normal cell synthesises fibronectin and deposits it underneath the cells; the variant also synthesises fibronectin. At least three times more fibronectin is distributed over the upper cell surface of variants. This correlates with the three-fold increased binding of the replication inhibitor Con A and suggests a role of fibronectin in endothelial cell growth control. When stimulated to migrate by CuII ions, the variant leaves deposits of fibronectin in its trail; in contrast, migrating normal cells do not, but they do redistribute their surface fibronectin. As revealed by scanning electron microscopy, variant cells are unusual in that they grow over or under cultured normal endothelial cells. It is proposed that during the process of neovascularisation, variant cells have a special function as lead cells that lay down fibronectin on which an endothelium can become established.     

Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro

We have previously reported the establishment of preadipocyte cell lines, termed dedifferentiated fat (DFAT) cells, from mature adipocytes of various animals. DFAT cells possess long-term viability and can redifferentiate into adipocytes both in vivo and in vitro. Furthermore, DFAT cells can transdifferentiate into osteoblasts and chondrocytes under appropriate culture conditions. However, it is unclear whether DFAT cells are capable of transdifferentiating into skeletal myocytes, which is common in the mesodermal lineage. Here, we show that DFAT cells can be induced to transdifferentiate into skeletal myocytes in vitro. Myogenic induction of DFAT cells resulted in the expression of MyoD and myogenin, followed by cell fusion and formation of multinucleated cells expressing sarcomeric myosin heavy chain. These results indicate that DFAT cells derived from mature adipocytes can transdifferentiate into skeletal myocytes in vitro.     

Alkaline phosphatase-positive reticular cells of chicken bone marrow--in vivo and in vitro studies

We examined alkaline phosphatase (ALPase)-positive reticular cells from chicken bone marrow in vitro in relation to other varieties of adherent cells. ALPase activity was found in both reticular and adipose cells which formed epithelioid cell colonies and were negative for fibronectin. We observed transition between two cell types. ALPase-negative macrophages as small round cells in culture revealed positive fibronectin and transformed into ALPase-negative spindle cells in long-term culture. Thus, we suggest two cell lineages, each with distinct cell characteristics.     

Establishment and characterization of partially differentiated chicken enterocyte cell clones

Three enterocyte cell clones were established in vitro from the intestine of a PA12 hen embryo. These cells exhibited epithelioid morphology and grew as monolayers. The cells were continuously propagated in culture up to 250 passages. Gradual increase in growth rate with time and in anchorage-independent growth in both agar and agarose showed that the three cell clones spontaneously transformed in vitro. The clones were heteroploid with one marker chromosome. Interestingly, they had features of partly differentiated enterocytes, especially microvilli, junctions connecting adjacent cells (tight junctions, desmosomes, hemidesmosomes, gap junctions), villin and cytokeratins. In addition, cells expressed brush border enzyme activity and transepithelial resistance. The fact that the levels of dipeptidyl peptidase IV (DPP-IV) and alkaline phosphatase activities fluctuated according to culture time and that MHC class II was induced by activation of cells with interferon suggested that the state of differentiation of the 3 cell clones could be modified in vitro. These clones are the first established avian enterocyte cell clones to be described. Because each cell clone exhibited differences in the level of differentiation and sensitivity to Salmonella infection, their use will allow comparative investigations concerning markers of differentiation of avian enterocytes and infection by host-adapted bacteria and parasites.     

Laminin B1 expression is required for laminin deposition into the extracellular matrix of PC12 cells.

The extracellular matrix of rat pheochromocytoma PC12 cells was shown by indirect immunofluorescence to consist of a network of fibronectin. The matrix did not contain laminin. The cells synthesized messenger RNA for fibronectin, laminin B2, and s-laminin but not for entactin or the B1 and A chains of laminin. Laminin B2 but not laminin B1 was detectable in the culture medium and in cell lysates. A full-length cDNA clone for the B1 chain of laminin was constructed in the plasmid p-444, which contains the neomycin-resistance marker and human beta-actin promoter. PC12 cells were transfected with this recombinant plasmid, and stable neomycin-resistant clones were isolated and characterized. Clones that synthesized laminin B1 messenger RNA were found to deposit a laminin-containing matrix. In many of these clones the deposition of the fibronectin matrix was greatly diminished. The laminin matrix was predominantly localized in the intercellular spaces forming a honeycomb pattern. The morphology of the laminin-synthesizing transfected cells was markedly different from the parental cells. The cells grew in tight clusters that were resistant to dissociating agents. It is concluded that the B1 chain of laminin contains information that is required for the formation of a stable laminin-containing extracellular matrix network either by interaction with cell surface receptors or other extracellular matrix components. Furthermore, expression of the laminin B1 gene may be a central regulatory point in determining extracellular matrix composition during embryogenesis.     

Analysis of the myogenic lineage in chick embryos

Abstract. Probabilistic and programmed lineage models for the generation of terminally differentiated skeletal muscle cells were tested in a clonal culture assay. Myogenic cells from the breast muscles of 10-day chick embryos were plated at an initial density of 250–1000 cells per 60 mm dish. Well-isolated individual cells were marked with a ring on the underside of the dishes, and clones arising from only these cells were followed. The presence of post-mitotic myoblasts in clones was assayed by peroxidase-antiperoxidase (PAP) and fluorescence immunocytochemical staining for both M-type creatine kinase (MCK) and skeletal muscle myosin heavy chain (MHC). Clones were fixed at intervals up to 76 h and were scored for the number of cells per clone and the number of MCK+ and MHC+ cells per clone. Quantitative and kinetic data were obtained indicating that post-mitotic myoblasts occurred overwhelmingly in homogeneous clones (all cells MCK⁺ and MHC⁺) which contained 2ⁿ cells ( n =0, 1, 2, 3, 4). This result does not support either probabilistic models of myogenesis or the existence of 'proliferative' mitoses at the end stages of differentiation. Rather, it indicates that myogenic precursor cells are a heterogeneous population, within which individual cells are predetermined to undergo a set number of symmetrical mitoses prior to yielding terminally differentiated progeny. These findings are strong evidence for a programmed, cell cycle-dependent lineage in the end stages of muscle differentiation.     

Autonomous proliferation of HTLV-CD4+ T cell clones derived from human T cell leukemia virus type I (HTLV-I)-associated myelopathy patients

That HTLV-I infects CD4(+) T cells and enhances their cell growth has been shown as successful long-term in vitro proliferation in the presence of IL-2. It is known that T cells isolated from HAM patients possess strong ability for cell proliferation in vitro and mRNA of various cytokines are abundantly expressed in CNS tissues of HAM patients. Hence, the cytokine-induced proliferation could have an important role in pathogenesis and immune responses of HAM. In this study, we examined the relationship between cell proliferation and ability of in vitro cytokine production of CD4(+) T cell clones isolated from HAM patients. We started a culture from a single cell to isolate cell clones immediately after drawing blood from the patients using limiting dilution method, which could allow the cell to avoid in vitro HTLV-I infection after initiation of culture. Many cell clones were obtained and the rate of proliferation efficiency from a single cell was as high as 80%, especially in the 4 weeks' culture cells from HAM patients. These cells were classified as mainly Th0 phenotype that produce both IFN-gamma and IL-4 after CD3-stimulation. However, the frequency of proviral DNA in these cloned cells was significantly low. Our results indicate that the ability of cell proliferation in HAM patients is not restricted in HTLV-I-infected T cells. HTLV-Iuninfected CD4(+) T cells, mainly Th0 cells, also have a strong ability to respond to IL-2-stimulation, showing that unusual immune activation on T cells has been observed in HAM patients.     

The adhesion modulating properties of tenascin-W

Tenascins are extracellular matrix glycoproteins associated with cell motility, proliferation and differentiation. Tenascin-C inhibits cell spreading by binding to fibronectin; tenascin-R and tenascin-X also have anti-adhesive properties in vitro. Here we have studied the adhesion modulating properties of the most recently characterized tenascin, tenascin-W. C2C12 cells, a murine myoblast cell line, will form broad lamellipodia with stress fibers and focal adhesion complexes after culture on fibronectin. In contrast, C2C12 cells cultured on tenascin-W fail to spread and form stress fibers or focal adhesion complexes, and instead acquire a multipolar shape with short, actin-tipped pseudopodia. The same stellate morphology is observed when C2C12 cells are cultured on a mixture of fibronectin and tenascin-W, or on fibronectin in the presence of soluble tenascin-W. Tenascin-W combined with fibronectin also inhibits the spreading of mouse embryo fibroblasts when compared with cells cultured on fibronectin alone. The similarity between the adhesion modulating effects of tenascin-W and tenascin-C in vitro led us to study the possibility of tenascin-W compensating for tenascin-C in tenascin-C knockout mice, especially during epidermal wound healing. Dermal fibroblasts harvested from a tenascin-C knockout mouse express tenascin-W, but dermal fibroblasts taken from a wild type mouse do not. However, there is no upregulation of tenascin-W in the dermis of tenascin-C knockout mice, or in the granulation tissue of skin wounds in tenascin-C knockout animals. Similarly, tenascin-X is not upregulated in early wound granulation tissue in the tenascin-C knockout mice. Thus, tenascin-W is able to inhibit cell spreading in vitro and it is upregulated in dermal fibroblasts taken from the tenascin-C knockout mouse, but neither it nor tenascin-X are likely to compensate for missing tenascin-C during wound healing.     

Concurrent modulation of cell surface fibronectin and adhesion to fibronectin in hepatoma cells

Rat hepatoma cells grown in vitro were poorly adhesive to plastic surfaces coated with fibronectin and lacked cell surface fibronectin matrix. They synthesized soluble fibronectin into the medium. The cell surface fibronectin matrix and the ability to attach to fibronectin-coated surface were restored in the 7777 cells upon passage as a tumor in rats and by coculturing these cells with normal liver-derived cells in vitro. Fibronectin matrix and the ability of cells to attach to fibronectin were thus modulated in a coordinated fashion, suggesting that the formation of a cell surface fibronectin matrix is dependent on the cell surface property that enables cells to interact with fibronectin.     

Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow

There is great interest in the therapeutic potential of non-hematopoietic stem cells obtained from bone marrow called mesenchymal stem cells (MSCs). Rare myogenic progenitor cells in MSC cultures have been shown to convert into skeletal muscle cells in vitro and also in vivo after transplantation of bone marrow into mice. To be clinically useful, however, isolation and expansion of myogenic progenitor cells is important to improve the efficacy of cell transplantation in generating normal skeletal muscle cells. We introduced into MSCs obtained from mouse bone marrow, a plasmid vector in which an antibiotic (Zeocin) resistance gene is driven by MyoD and Myf5 enhancer elements, which are selectively active in skeletal muscle progenitor cells. Myogenic precursor cells were then isolated by antibiotic selection, expanded in culture, and shown to differentiate appropriately into multinucleate myotubes in vitro. Our results show that using a genetic selection strategy, an enriched population of myogenic progenitor cells, which will be useful for cell transplantation therapies, can be isolated from MSCs.     

Different propensity for spontaneous differentiation of cell clones isolated from the human ovarian surface epithelial cell line HOC-7

Abstract. Limiting dilution culture of cell fractions obtained by discontinuous density gradient centrifugation was used to establish six different cell clones from HOC-7 ovarian adenocarcinoma cells (D1-D3, N1-N3). Clones D1-D3 revealed a phenotype similar to that seen in parental cells exposed to differentiation inducers such as dimethyl sulfoxide (DMSO, 0.8% [v/v]). They were flattened, slowly growing cells (doubling times: 42–46 h). The cells developed long cytoplasmic extensions and adopted a complicated growth pattern. Fixed-cell enzyme-linked immunosorbent assay (ELISA) and Western blotting demonstrated that these cells contained high levels of epidermal growth factor-receptor (EGF-R), carbohydrate antigen 125 (CA 125), fibronectin and desmoplakin, but low levels of myc oncoproteins. However, untreated parental cells and clones N1–N3 were fastgrowing (doubling times: 23–28 h), regularly shaped, polygonal cells ("cobblestone'monolayer) with low levels of EGF-R, CA 125, fibronectin and desmoplakin, but relatively higher amounts of myc oncoproteins. The similarity of the sublines to either untreated or inducertreated parental cells indicated that clones D1–D3 represented spontaneously differentiated HOC-7 cells, whereas clones N1–N3 originated from less-differentiated cells. The features examined in this model cell system proved to be closely related to ovarian cancer cell proliferation and differentiation. The observation of a tumorinherent propensity for spontaneous differentiation suggests that exogenous stimulation of existing differentiation pathways may represent an alternative approach for tackling the problem of growth control and differentiation in malignant tissues.     

Characterization of factor(s) in culture supernatants affecting cell social behavior.

Treatment of embryonic chick heart fibroblast cultures with 0.2 M urea reversibly increases cellular overlap. The increase in cellular overlapping over that in control cultures may be quantitated by the overlap ratio (R), the ratio of the number of superimposed nuclei observed, to the number expected to occur when cells are assumed to be distributed randomly over the culture substratum (R = observed/expected overlaps). Reversal of the urea-induced increase in R is blocked by 0.2 micrograms/ml cycloheximide. In the presence of cycloheximide, normal (low) overlap ratios are restored to urea-treated cultures by adding non-dialyzable material recovered by washing fibroblast monolayers with serum-free medium. The overlap ratio assay revealed no effect of supernatant material added either to urea-treated cultures in the continued presence of urea, or to untreated cultures. Although unfiltered supernatants were shown by SDS-polyacrylamide gel electrophoresis to contain fibronectin (CSP; LETS; MWappar. = 220,000 d) and smaller proteins, the ability to reverse the urea-induced increase in overlap ratio was present in Diaflo and Millipore filtrates of culture supernatants in which fibronectin was greatly depleted or absent. In contrast, purified fibronectin preparations failed to lower urea-induced increases in overlap-ratio. Partially purified, biologically active supernatants, prepared from 14C-leucine or 125I-labeled cultures, contained several macromolecules smaller than fibronectin that were labeled by both radioisotopes. In particular, one band (MWappar. = 58--60,000 d) was present in polyacrylamide gels of active supernatant and also depleted in gels of homogenates from urea-treated cultures. These results indicate that external macromolecules other than fibronectin are synthesized by cultured fibroblasts and can affect cell social behavior or culture morphology.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Clonal analysis of vertebrate myogenesis. VI. Acetylcholinesterase and acetylcholine receptor in myogenic and nonmyogenic clones from chick embryo leg cells.

Myogenic clones grown in vitro from cells of 4-, 6-, and 12-day chick embryo leg buds demonstrate reproducible stage-specific characteristics of morphology, extent of myotube formation, and culture medium requirements for differentiation, suggesting heterogeneity in the myogenic cell populations of the developing limb. To determine whether there is heterogeneity in the cytodifferentiation of different muscle colony types, clones have been examined for the appearance of two muscle-specific gene products—acetylcholinesterase (AChE) and acetylcholine receptor (AChR). AChE (detected by cytochemical reaction) and AChR (detected by autoradiography of [¹²⁵I]α-bungarotoxin binding) appeared in myotubes of all muscle colony types, and also appeared in about 5% of the mononucleated cells of all muscle colonies; but neither were detectable in cells of nonfused clones (colonies containing no myotubes). The results suggest that all muscle colony-forming cell types have equivalent capacities to elaborate muscle-specific gene products once the process of differentiation is initiated. However, when putative muscle colony-forming cells are grown under certain conditions that do not permit cell fusion (e.g., conditioned medium-requiring clones grown in fresh medium), mononucleated cells do not accumulate AChE or AChR. Conditioned medium-dependent differentiation thus differs from the fusion-specific processes affected by Ca²⁺ deprivation and phospholipase C treatment, since in these cases mononucleated cells exhibit differentiated functions. The apparent cytodifferentiation (without fusion) of some mononucleated cells within muscle colonies in which most mononucleated cells continue to proliferate raises questions concerning the control of myoblast differentiation and its relationship to the cell cycle and to fusion.