Cultured Schwann cells assemble normal-appearing basal lamina only when they ensheathe axons

Previous work demonstrated that Schwann cells (SCs) must interact with nerve cells (NCs) in order to generate their basal lamina (BL) in culture (M. B. Bunge, A. K. Williams, and P. M. Wood, 1982, Dev. Biol. 92, 449-460). The present study was undertaken to determine if this interaction requires proximity of NCs to SCs. Coverslips carrying isolated SCs were placed into culture dishes containing normally contacting SCs + NCs, NCs alone, or SCs alone and were maintained in these dishes for 3-4 weeks in medium known to foster the differentiation of axon-related SCs (BL formation, myelination). The SCs on the coverslip were not allowed to contact the cells in the culture dish. In other experiments, SCs isolated on coverslips were simply cultured in medium conditioned by contacting SCs + NCs, NCs alone, or SCs alone. The accumulation of BL components was monitored by light microscopic immunocytochemistry and the assembly of BL structure assessed by electron microscopy. When SCs were cocultured with but not contacted by neurons, immunostaining for BL constituents revealed a patchy deposition of material in sharp contrast to the linear deposition observed on axon-related SCs. Electron microscopy of these isolated SCs revealed short segments of BL, strands or clumps of BL-like material extending away from the cell surface, and accumulation of this material between cells. A greater number of isolated SCs were immunostained when grown with contacting SCs + NCs than with NCs or SCs. The conditioned medium experiments yielded similar results; only patchy BL was observed and more immunostaining was detected on isolated SCs when the medium had been conditioned by contacting SCs + NCs than by NCs alone or SCs alone. Immunostaining was less overall in the conditioned medium experiments than in the cell coculture work. In addition, standard SC + NC cultures grown in differentiation-supporting medium were studied by electron microscopy. SCs that were not contacted by axons but were positioned between fascicles of normally contacting SCs + NCs were identified under phase microscopy and then examined for the presence of BL. These SCs exhibited only occasional segments of BL or detached BL-like material. Lastly, within differentiated fascicles, nonensheathing SCs were compared with neighboring myelinating SCs that were in substantial contact with axons. BL-deficient nonensheathing SCs were found directly adjacent to axons and BL-coated myelinating SCs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anti-β1 integrin antibody inhibits schwann cell meylination

Schwann cells (SCs) co-cultured with sensory neurons require ascorbate supplementation for basal lamina assembly and differentiation into myelinating cells. The ascorbate requirement can be bypased by adding a purifed basal lamina component, laminin, to SC/neuron cocultures. We have examined the role of laminin receptors, Namely, the β1 subfamily of integrins, in the process of myelination. We demonstrate by immunostaining or immunoprecipitation that undifferentiated SCs in contact with axons express large amounts of the β1 subunit in association with the α1 or α6 subunit. In co-cultures of myelinating SCs, α1β1 is no longer present, α6β1 is still present but at reduced levels, and α6β4 is expressed at much higher levels than in co-cultures of undifferentiated SCs. Immunogold labelling at the electron microscope level suggested that β1 integrins are randomly distributed on undifferentiated SCs, become localized to the SC surface contacting basal lamina in differentiating SCs before the onset of myelination, and are not detected on myelinating SCs. Fab fragments of β1 function-blocking antibody block both attachment of isolated SCs to laminin and formation of myelin sheaths by SCs co-cultured with neurons in ascorbate-supplemented medium. SCs unable to myelinate in the presence of the anti-β1 antibody assemble patchy basal lamina that is only loosely attached to the cell surface and in some cases appears to be detaching from the membrane. In contrast, an α1β1 function-blocking antibody only partially blocks attachment of isolated SCs to laminin but has no inhibitory effect on SC myelination. These results are consistent with the hypothesis that a member of the β1 subfamily of integrins other than α1β1 binds laminin present in basal lamina to the SC surface and transduces signals that are critical for initiation of SC differentiation into a myelinating cell. 1994 John Wiley & Sons, Inc.     

Developmental changes in the ultrastructure of the lamprey lateral line nerve during metamorphosis

The ultrastructure of the trunk lateral line nerve of larval and adult lampreys was studied with transmission electron microscopy. We confirmed that lampreys' lateral line nerve lacks myelin. Nevertheless, all axons were wrapped by Schwann cell processes. In the larval nerve, gaps between Schwann cells were observed, where the axolemma was covered only by a basal lamina, indicating an earlier developmental stage. In the adult nerve, glial (Schwann cell) ensheathment was mostly complete. Additionally, we observed variable ratios of axons to Schwann cells in larval and adult preparations. In the larval nerve, smaller axons were wrapped by one Schwann cell. Occasionally, a single Schwann cell surrounded two axons. Larger axons were associated with two to five Schwann cells. In the adult nerve, smaller axons were surrounded by one, but larger axons by three to eight Schwann cells. The larval epineurium contained large adipose cells, separated from each other by single fibroblast processes. This layer of adipose tissue was reduced in adult preparation. The larval perineurium was thin, and the fibroblasts, containing large amounts of glycogen granules, were arranged loosely. The adult perineurium was thicker, consisting of at least three layers of fibroblasts separated by collagen fibrils. The larval and adult endoneurium contained collagen fibrils oriented orthogonally to each other. Both larval and adult lateral line nerves possessed a number of putative fascicles weakly defined by a thin layer of perineurial fibroblasts. These results indicate that after a prolonged larval stage, the lamprey lateral line nerve is subjected to additional maturation processes during metamorphosis. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Effects of cis-4-hydroxy- -proline, an inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells

The proline analog cis-4-hydroxy- -proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. We confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen, cultures grown in the presence or absence of CHP were metabolically labeled with [³H]leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. CHP had no significant effect on either total protein synthesis (medium plus cell layer) or cell number. We conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. We discuss the evidence for and against specificity of CHP action in other systems.     

Localization of the nerve growth factor receptor on fetal human schwann cells in culture

Previous studies have established that Schwann cells (SC) in culture express an NGF receptor. In this study, cultures of fetal human SC were established from fetal nerves and various light microscopic (LM) and electron microscopic (EM) techniques were used to localize the NGF receptor on the SC. Results indicate that NGF receptor is localized to the plasma membrane of the SC. Quantitative digital analysis determined that the distal portion of the SC process had high concentrations of NGF receptor. The possible functional significance of this latter observation is discussed in terms of SC migration and ensheathment of axons.     

Effects of cis-4-hydroxy-L-proline, and inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells

The proline analog cis-4-hydroxy-L-proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. We confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen, cultures grown in the presence or absence of CHP were metabolically labeled with [3H]leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. CHP had no significant effect on either total protein synthesis (medium plus cell layer) or cell number. We conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. We discuss the evidence for and against specificity of CHP action in other systems.     

Schwann cell extracellular matrix molecules and their receptors

The major cellular constituents of the mammalian peripheral nervous system are neurons (axons) and Schwann cells. During peripheral nerve development Schwann cells actively deposit extracellular matrix (ECM), comprised of basal lamina sheets that surround individual axon-Schwann cell units and collagen fibrils. These ECM structures are formed from a diverse set of macromolecules, consisting of glyco-proteins, collagens and proteoglycans. To interact with ECM, Schwann cells express a number of integrin and non-integrin cell surface receptors. The expression of many Schwann cell ECM proteins and their receptors is developmentally regulated and, in some cases, dependent on axonal contact. Schwann cell ECM acts as an organizer of peripheral nerve tissue and strongly influences Schwann cell adhesion, growth and differentiation and regulates axonal growth during development and regeneration.     

Neuron-schwann cell interaction in basal lamina formation

The availability of tissue culture systems that allow the growth of nerve cells, Schwann cells, and fibroblasts separately or in various combinations now makes possible investigation of the role of cell interactions in the development of the peripheral nervous system. Using these systems it was earlier found that basal lamina is formed on the Schwann cell surface in cultures of sensory ganglion cells and Schwann cells without fibroblasts. It is here reported that the presence of nerve cells is required for the generation of basal lamina on the Schwann cell plasmalemma. Utilizing nerve cell-Schwann cell preparations devoid of fibroblasts, this was found in the following ways. (1) When nerve cells are removed from 3- to 5-week-old cultures, the basal lamina disappears from Schwann cells. (2) If nerve cells are added back to such Schwann cell populations, Schwann cell basal lamina reappears. (3) Removal of nerve cells from older (3–4 months) cultures does not lead to basal lamina loss; areas presumed not to have been coated with lamina before neurite degeneration remain so, suggesting that the lamina persists but is not reformed. (4) If basal lamina is removed with trypsin, it is reformed in neuron plus Schwann cell cultures but not in Schwann cell populations alone. Thus, the formation but not the persistence of Schwann cell basal lamina requires the presence of nerve cells.     

Ensheathment of the olfactory nerves in the adult rat

The ensheathment of the olfactory nerve fibres is achieved by cooperation of two cell types. The olfactory ensheathing cells have a rounded outer surface enclosed in a continuous single basal lamina, and enclose an inner compartment from which overlapping processes of the same and adjacent cells enwrap interweaving territories of tightly apposed aligned axons. The olfactory nerve fibroblasts are highly flattened, dense cells generating multiple layers of very thin processes encircling individual or groups of olfactory ensheathing cells. This paper illustrates the unique ultrastructural features of this ensheathment.     

Movements of the Schwann cell nucleus implicate progression of the inner (axon-related) Schwann cell process during myelination

Although it has been known for several decades that peripheral myelin is formed from an extended, spiraled, and compacted sheet of Schwann cell (SC) plasma membrane, the mechanism by which this unique spiraling is accomplished remains unknown. We have studied the movements of SC nuclei before, during, and subsequent to myelin formation (over periods of 24-72 h) to determine if this nuclear motion (noted in earlier reports) would provide useful insights into the mechanism of myelinogenesis. We used rodent sensory neuron and SC cultures in which initiation of myelinogenesis is relatively synchronized and bright field conditions that allowed resolution of the axon, compact myelin, and position of the SC nucleus. Observed areas were subsequently examined by electron microscopy (EM); eight myelinating SCs with known nuclear movement history were subjected to detailed EM analysis. We observed that, prefatory to myelination, SCs extended along the length of larger axons, apparently competing with adjacent SCs for axonal surface contact. This lengthening preceded the deposition of compact myelin. SC nuclear circumnavigation of the axon was found to attend early myelin sheath formation. This movement was rarely greater than 0.25 turns per 3 h; on the average, more nuclear motion was seen in relation to internodes that formed during observation (0.8 +/- 0.1 turns/24 h) than in relation to those that had begun to form before observation (0.3 +/- 0.1 turns/24 h). Nuclear circumnavigation generally proceeded in one direction, could be in similar or opposite direction in neighboring myelinating SCs on the same axon, and was not proportional to the number of major dense lines within the myelin sheath. A critical finding was that, in all eight cases examined, the overall direction of nuclear movement was the same as that of the inner end of the spiraling SC process, and thus opposite the direction of the outer end of the spiral. We conclude that the correspondence of the direction of nuclear rotation and inner end of the spiraling cytoplasmic lip implicates active progression of the inner lip over the axonal surface to form the membranous spiral of myelin, the nuclear motion resulting from towing by the advancing adaxonal lip. This interpretation fits with finding basal lamina and macular adhering junctions associated with the external lip of SC cytoplasm; these attributes would imply anchorage rather than movement of this region of the SC.     

A morphologic and histochemical study of the mesentery in the guinea pig

The guinea pig mesentery is a uniform, continuous, thin (18 micron) sheet of connective tissue covered by a single layer of flattened mesothelial cells on both surfaces. Tight and gap junctions provide for cell-to-cell adhesion among mesothelial cells. These cells possess numerous micropinocytotic vesicles; a conspicuous basal lamina separates the mesothelium from the underlying connective tissue. Most of the material found between the two serous coverings consisted of a three-dimensional meshwork of abundant collagenous fibers intermingled with a sparse net of very thin (0.4 micron) elastic fibers. Two distinct populations of collagen fibrils are segregated into different compartments of the mesentery. One population is formed of thick (56 nm) fibrils which associate to form closely packed fibers. The second population, composed of loosely arranged thin (38 nm) fibrils which do not become assembled into fibers, is found underlying the basal lamina that separates the mesothelium from the connective tissue. These observations strongly suggest that the mesentery contains both collagens type I and type III. The guinea pig mesentery contains 6.8 mg of sulfated glycosaminoglycans/g dry weight. Most of these glycosaminoglycans (78%) were identified as dermatan sulfate, whilst the rest (22%) corresponded to heparan sulfate.     

The Cysteine-rich Region of Type VII Collagen Is a Cystine Knot with a New Topology

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX₃CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.     

Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).     

Laminin 5 can promote assembly of the lamina densa in the skin equivalent model

Skin equivalents were prepared by culturing human keratinocytes on the surface of type I collagen gel contracted by human skin fibroblasts (dermal equivalents) and by raising the gel to an air-liquid interface. A stratified squamous epithelium was formed with a well-differentiated cornified layer at the top of keratinocyte layers within 7 days after plating of the keratinocytes on the dermal equivalents. Although major basement membrane components such as collagens IV and VII and laminin 5 were detected immunohistochemically at the dermal-epidermal junction, a lamina densa was rarely observed by electron microscopy even in 14-day skin equivalents. When laminin 5 (1, 5 or 20 μg/ml) was added to the culture medium on day 7 through day 14, types IV and VII collagens at the dermal-epidermal junction stained more strongly by immunohistochemistry compared with the control. Patches of lamina densa were present along the epidermal-dermal junction, and vesicles containing electron-opaque sheets approximately 0.6 μm in diameter that reacted with anti-collagen IV antibody were also observed in basal keratinocytes in 14-day skin equivalents by electron microscopy. Morphometric analysis showed that the total length of lamina densa along the dermal-epidermal junction as well as in the vesicles increased up to 180%, 230% or 520% of control cultures by the addition of laminin 5 (1, 5 or 20 μg/ml, respectively). These results suggest that laminin 5 accelerates formation of the lamina densa along dermal-epidermal junction of the skin equivalents, depending on the concentration of laminin 5 supplemented exogenously.     

Evidence that contact with connective tissue matrix is required for normal interaction between Schwann cells and nerve fibers

Explants of fetal rat sensory ganglia, cultured under conditions allowing axon and Schwann cell outgrowth in the absence of fibroblasts, occasionally develop nerve fascicles that are partially suspended in culture medium above the collagen substrate. In these suspended regions, fascicles are abnormal in that Schwann cells are decreased in number, are confined to occasional clusters along the fascicle, provide ensheathment for only a few axons at the fascicle periphery, and do not form myelin. When these fascicles are presented with a substrate of reconstituted rat-tail collagen, Schwann cell numbers increase, ensheathment of small nerve fibers occurs normally, and larger axons are myelinated. We conclude that, for normal development, Schwann cells require contact with extracellular matrix as well as axons. The Schwann cell abnormalities in suspended fascicles are similar to those observed in nerve roots of dystrophic mice.     

New multiple labelling method for improved satellite cell identification in human muscle: application to a cohort of power-lifters and sedentary men

Presently applied methods to identify and quantify human satellite cells (SCs) give discrepant results. We introduce a new immunofluorescence method that simultaneously monitors two SC markers (NCAM and Pax7), the basal lamina and nuclei. Biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects were re-examined. Significantly different results from those with staining for NCAM and nuclei were observed. There were three subtypes of SCs; NCAM⁺/Pax7⁺ (94%), NCAM⁺/Pax7⁻ (4%) and NCAM⁻/Pax7⁺ (1%) but large individual variability existed. The proportion of SCs per nuclei within the basal lamina of myofibres (SC/N) was similar for all groups reflecting a balance between the number of SCs and myonuclei to maintain homeostasis. We emphasise that it is important to quantify both SC/N and the number of SCs per fibre. Our multiple marker method is more reliable for SC identification and quantification and can be used to evaluate other markers of muscle progenitor cells.