Proliferative potential of larval cells of the mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> and their capacity to differentiate into myogenic cells in culture

Cell differentiation and proliferation can be regulated by the extracellular matrix. To compare cell proliferation and myogenic differentiation in cultivated Mytilus larval cells on different substrates (collagen I and fibronectin), a double immunostaining with subsequent confocal microscopy were used for simultaneous detection of dividing and muscle cells. The proliferative activity was monitored using two markers, proliferating cell nuclear antigen (PCNA) and phospho-histone H3. The maximum number of mitotic (phosphohistone H3-positive) cells was observed after 4 h of cultivation (approximately 3%), but later, after 48 h, it decreased to 0.5%. Most of these cells formed small aggregates on all substrates tested. After 24 h of cultivation, the number of mitotic cells was approximately 5–7 times lower than that of S-phase (PCNA-positive) cells. The decrease in cell proliferation was accompanied by intensification of myogenic differentiation. First muscle cells were detected after 6 h of cultivation on fibronectin; numerous contracting muscle cells were observed after 24 h of cultivation. In contrast, the cells cultivated on collagen had mostly a rounded shape, did not spread, and showed a contracting activity only in rare cases. A small number of muscle cells with PCNA-positive nuclei were detected after 3-day-cultivation on fibronectin. We suggest that at early stages of cultivation, muscle precursor cells, or myoblasts, are able to synthesize DNA but lose this ability later.     

Real-time monitoring of extracellular matrix-mediated PC12 cell attachment and proliferation using an electronic biosensing device

The attachment and proliferation of a well-established, neuron-like cell line, rat pheochromocytoma (PC12) cells, on different extracellular matrices (ECMs) was monitored using cellular impedance sensing (CIS). Commonly used ECMs, including fibronectin, laminin, poly-l-lysine, collagen and poly-l-lysine followed by laminin, in addition to DMEM cell culture media alone as a control, were studied: CIS identified the dynamic progress of the adhesion and proliferation of the cells on different ECMs. Among these modified ECM surfaces, the laminin- and poly-l-lysine/laminin-modified surfaces were the best suited for the neuron-to-electrode surface attachment and proliferation, which was confirmed by MTT assays and a scanning electron microscopy analysis. This work provides a simple method to study neuron cell/ECM interactions in a real-time, label-free, and quantitative manner.     

Muscle and neuronal differentiation in primary cell culture of larval Mytilus trossulus (Mollusca: Bivalvia)

Molluscan in vitro technology allows the study of the differentiation of isolated cells undergoing experimental manipulations. We have used the immunofluorescence technique and laser scanning microscopy to investigate the organization of muscle proteins (actin, myosin, paramyosin, and twitchin) and the localization of neurotransmitters (serotonin and FMRFamide) in cultured mussel larval cells. Differentiation into muscle and neuron-like cells occurs during the cultivation of mussel cells from premyogenic and prenervous larval stages. Muscle proteins are colocalized in contractile cells through all stages of cultivation. The cultivation of mussel cells on various substrates and the application of integrin receptor blockers suggest that an integrin-dependent mechanism is involved in cell adhesion and differentiation. Dissociated mussel cells aggregate and become self-organized in culture. After 20 days of cultivation, they form colonies in which serotonin- and FMRFamide-immunoreactive cells are located centrally, whereas muscle cells form a contractile network at the periphery. The pattern of thick and thin filaments in cultivated mussel cells changes according to the scenario of muscle arrangement in vivo: initially, a striated pattern of muscle filaments forms but is then replaced by a smooth muscle pattern with a diffuse distribution of muscle proteins, typical of muscles of adult molluscs. Myogenesis in molluscs thus seems to be a highly dynamic and potentially variable process. Such a “flexible” developmental program can be regarded as a prerequisite for the evolution of the wide variety of striated and smooth muscles in larval and adult molluscs.     

Isolation and partial characterization of myogenic cells from mussel larvae in vitro

The main finding of the present study is the discovery of the possibility of a morphofunctional myogenic differentiation of larval mussel cells in vitro. The shape and extensive cytoskeletal network of the cultured contracting cells mimic largely those of smooth muscle cells in vivo. However, the behavior and protein composition of these cells are not completely identical with those of smooth muscle cells. Contracting mussel cells in vitro, as well as differentiated smooth muscles, demonstrate both phasic and tonic contractions. The paramyosin to myosin ratio in the cultured mussel cells is far less than that in the muscles of veliger larvae and adult mussels. We have found the protein carpets with various adhesive characteristics determine different development pathways. Myogenic differentiation is only observed in spreading cells. Non-spreading adherent cells plated on collagen carpet show high synthetic activity but the commitment of contractile phenotype is inhibited. Our results confirm that the myogenic program established in early embryogenesis of molluscs can be realized during the cultivation of cells from premyogenic larval stages.     

Comparative characteristic of Mytilus muscle cells developed in vitro and in vivo

The mussel cells from premyogenic larval stages are capable of differentiation into smooth muscle cells in vitro. However, the behavior and protein composition of these cells are not completely identical to those of smooth muscle cells of adult mussels. In this study we compared some properties of mussel muscle cells forming from cells of trochophore (premyogenic larval stage) in vitro with those of muscle cells of veliger and adult mussel. We found a substantial difference between the contractile apparatus protein composition of veliger muscle and cultivated cells. Myorod, one of the molecular markers of the phenotype of mollusc smooth muscle cells (Shelud'ko et al., 1999, Comp Biochem Physiol 122:277-285), is not a constituent of the contractile apparatus of veliger muscle. At the same time the protein composition of contractile apparatus in cultivated cells was similar to that of adult Mytilus muscles. There were only few quantitative differences between them. The contractile activity of cultivated cells was changing in time. The kinetic parameters of first spontaneous contractions were similar to those of phasic contractions, while their period was close to that of tonic contractions. After 50-55 hrs cultivation the cells produced both phasic and tonic contractions, but the character of contractile activity of cultivated cells was regulated after six days of cultivation only. However, there were no muscle cells in vitro, whose contractile activity was similar to that of veliger muscle cells. So, we concluded that properties of muscle cells forming from premyogenic larval mussel cells in culture are similar to those of muscle cells of the adult mussel, but not of veliger.     

Preference toward a polylysine enantiomer in inhibiting prions

Differential anti-prion activity of polylysine enantiomers was studied. Based on our recent discovery that poly-l-lysine (PLK) is a potent anti-prion agent, we investigated suppression of prions in cultured cells using poly-d-lysine (PDK). The results showed that PDK was more efficacious than PLK to inhibit prions. Protein misfolding cyclic amplification assay demonstrated improved efficacy of PDK in inhibiting plasminogen-mediated prion propagation, corresponding to the enantio-preference of PDK observed in cultured cells. Furthermore, our study demonstrated that polylysines formed a complex with plasminogen. These results propose to hypothesize a plausible mechanism that elicits prion inhibition by polylysine enantiomers.     

Primordial germ cell differentiation of nuclear transfer embryonic stem cells using surface modified electroconductive scaffolds

A combination of nanotopographical cues and surface modification of collagen and fibronectin is a potential platform in primordial germ cells (PGCs) differentiation. In the present study, the synergistic effect of nanotopography and surface modification on differentiation of nuclear transfer embryonic stem cells (nt-ESCs) toward PGC lineage was investigated. In order to achieve this goal, poly-anyline (PANi) was mix within poly-l-lactic acid (PLLA). Afterward, the random composite mats were fabricated using PLLA and PANi mix solution. The nanofiber topography notably upregulated the expressions of prdm14, mvh and c-kit compared with tissue culture polystyrene (TCP). Moreover, the combination of nanofiber topography and surface modification resulted in more enhancement of PGCs differentiation compared with non-modified nanofibrous scaffold. Additionally, gene expression results showed that mvh and c-kit were expressed at higher intensity in cells exposed to collagen and fibronectin rather than collagen or fibronectin solitary. These results demonstrated the importance of combined effect of collagen and fibronectin in order to develop a functional extracellular matrix (ECM) mimic in directing stem cell fate and the potential of such biofunctional scaffolds for treatment of infertility.     

Localization of αvβ3-like integrin in cultivated larval cells of the mussel Mytilus trossulus during neuronal and muscle differentiation

Using immunofluorescence phenotyping, the expression of αvβ3-like integrin was examined during neuronal and muscle differentiation in cell cultures derived from trochophore larvae of the mussel Mytilus trossulus. We have demonstrated that some mussel cells grown on fibronectin in vitro express the extracellular matrix (ECM) αvβ3 integrin-like receptor. At the same time, the distribution of αvβ3-like integrin is not ubiquitous, i.e. it depends on the cell type and the time of cultivation. Using immunohistochemical staining, we have found that only in some cells this integrin is co-localized with molluscan neuronal markers, neurotransmitters serotonin (5-HT) or Phe-Met-Arg-Phe-NH(2) neuropeptide (FMRFamide), and also with filament actin but not with paramyosin. Although we have previously shown that an integrin-dependent mechanism is involved in cell adhesion and differentiation of muscle cells of Mytilus, in this study, αvβ3-like integrin has not been found to participate in fibronectin adhesion of muscle cells but may be a linking agent between the ECM and the neuron-like cells.     

Detection of glycosaminoglycans on the surface of human umbilical vein endothelial cells using gold-conjugated poly-l -lysine with silver enhancement

Summary Endothelial glycosaminoglycans are important in a diverse range of vascular functions. In the course of a biochemical and histological study exploring the role of glycosaminoglycans in inflammation, we have investigated the use of gold-conjugated poly-l-lysine with silver enhancement to establish the nature and physical location of glycosaminoglycans on the surface of cultured human umbilical vein endothelial cells. Cationic gold was effective in locating anionic sites in both cultured endothelial cells and in paraffin-embedded renal tissue. By manipulating pH, and by using enzymes specific for degrading glycosaminoglycans, it was found that, at pH 1.2, staining was directed primarily at glycosaminoglycans. The surface of human umbilical vein endothelial cells was found to be extensively covered in heparan sulphate, the histological appearance of which was dependent upon the fixation procedure employed. Heparan sulphate was also seen to co-distribute with the extracellular matrix protein, fibronectin, when endothelial cultures were simultaneously stained with cationic gold and an antibody to cellular fibronectin.     

Enhancement of pancreatic islet cell monolayer growth by endothelial cell matrix and insulin

Summary The roles of glucose and insulin in the promotion of DNA synthesis in pancreatic islet cell monolayers were assessed using a variety of in vitro conditions. Several substrates including collagen, poly-l-lysine, Matrigel, and the extracellular matrix produced by cultured bovine endothelial cells (BCEM) were compared for their ability to promote monolayer growth. Islets grown on BCEM in combination with medium RPMI 1640 supplemented with 22.2 mM glucose or 10 μg/ml insulin gave the best results as determined by new DNA synthesis. The new-form monolayers were free of contaminating, fibroblasts. These results suggest that insulin is critical to pancreatic islet growth when the cells are attached to biocompatible matrices.     

BMP2 cross-linked by transglutaminase 2 to collagen-plla scaffold promotes osteogenic differentiation in mesenchymal stem cells

Transglutaminase 2 (TG2) was used to attach biologically-active BMP2 to collagen type I-coated poly-l-lactic acid (PLLA) nanofibrous scaffolds. Irreversibly cross-linked BMP2 retained its activity and induced Smad-dependent gene expression in cells seeded on PLLA–BMP2 scaffolds. These modified scaffolds promote osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) cultured in low-serum and growth factor free medium and support deposition of the calcified matrix and induction of the molecular osteogenic markers Runx2, osteopontin, osteonectin and bone sialoprotein. Importantly, the PLLA–BMP2 scaffolds did not support chondrogenic differentiation in hBMSCs as there was no expression of chondrogenic markers aggrecan, Sox 9, and collagen type II, and no deposition of cartilaginous glycosaminoglycan-rich matrix. Thus, TG2-mediated cross-linking of BMP2 to a scaffold is a novel approach to induce osteoblast-specific programming of hBMSCs in a spatially controlled manner.     

Effects of l-lysine and d-lysine on ɛ-Poly-l-lysine biosynthesis and their metabolites by Streptomyces ahygroscopicus GIM8

?-Poly-l-lysine (?-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of l-lysine, which is used as a safe food preservative. In this study, the effects of l-lysine and its isomer, d-lysine, on ?-PL biosynthesis and their metabolites by the ?-PL-producing strain Streptomyces ahygroscopicus GIM8 were determined. The results indicated that l-lysine added into the fermentation medium in the production phase mainly served as a precursor for ?-PL biosynthesis during the flask culture phase, leading to greater ?-PL production. At an optimum level of 3 mM l-lysine, a ?-PL yield of 1.16 g/L was attained, with a 41.4% increment relative to the control of 0.78 g/L. Regarding d-lysine, the production of ?-PL increased by increasing its concentrations up to 6 mM in the initial fermentation medium. Interestingly, ?-PL production (1.20 g/L) with the addition of 3 mM d-lysine into the initial fermentation medium in flasks was higher than that of the initial addition of 3 mM L-lysine (1.06 g/L). The mechanism by which d-lysine improves ?-PL biosynthesis involves its utilization that leads to greater biomass. After S. ahygroscopicus GIM8 was cultivated in the defined medium with L-lysine, several key metabolites, including 5-aminovalerate, pipecolate, and l-2-aminoadipate formed in the cells, whereas only l-2-aminoadipate was observed after d-lysine metabolism. This result indicates that l-lysine and d-lysine undergo different metabolic pathways in the cells. Undoubtedly, the results of this study are expected to aid the understanding of ?-PL biosynthesis and serve as reference for the formulation of an alternative approach to improve ?-PL productivity using l-lysine as an additional substrate in the fermentation medium.     

Extracellular matrix components direct porcine muscle stem cell behavior

In muscle tissue, extracellular matrix proteins, together with the vasculature system, muscle-residence cells and muscle fibers, create the niche for muscle stem cells. The niche is important in controlling proliferation and directing differentiation of muscle stem cells to sustain muscle tissue. Mimicking the extracellular muscle environment improves tools exploring the behavior of primary muscle cells. Optimizing cell culture conditions to maintain muscle commitment is important in stem cell-based studies concerning toxicology screening, ex vivo skeletal muscle tissue engineering and in the enhancement of clinical efficiency. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble the muscle extracellular matrix. Several important factors that determine myogenic commitment of the primary muscle cells were characterized by quantitative real-time RT-PCR and immunofluorescence. Adhesion of high PAX7 expressing satellite cells was improved if the cells were cultured on fibronectin or laminin coatings. Cells cultured on Matrigel and laminin coatings showed dominant integrin expression levels and exhibited an activated Wnt pathway. Under these conditions both stem cell proliferation and myogenic differentiation capacity were superior if compared to cells cultured on collagen type I, fibronectin and gelatin. In conclusion, Matrigel and laminin are the preferred coatings to sustain the proliferation and myogenic differentiation capacity of the primary porcine muscle stem cells, when cells are removed from their natural environment for in vitro culture.     

Analysis of heterogeneity of human keratinocytes interacting with immobilized fibronectin and collagenes I and IV types

The concept that stem cells form an independent subpopulations of somatic cells assumes the heterogeneity of cellular populations in adult tissues. As skin keratinocytes have natural affinity to extracellular matrix proteins, we made an attempt to reveal subpopulations of these cells depending on the time of their adhesion to substrates of collagen I and IV types and fibronectin. After selection for 10, 20 and 30 min the keratinocytes were cultivated for 24 h. The area of cell projection on a substrate and the spreading coefficient were measured (Kuzminykh, Petrov, 2004; Petrov et al., 2007). In 24 h statistically reliable morphological differences between the cells depending on the substratum were found. The size of the cells growing on collagen I type was twice as large as that of the cells cultivated on collagen IV type or in fibronectin. Irrespective of the substratum, up to 60-65% of the cells had a rounded form. The cultivation on collagens revealed the heterogeneity of keratinocytes both in the control cultures and under selection by adhesion time, while the cells grown on fibronectin behaved as a homogeneous population. These results suggest that, contrary to fibronectin, collagens stabilize some physiological states of keratinocytes corresponding to their interaction with extracellular matrix proteins in the organism.     

Laccase-catalyzed cross-linking of amino acids and peptides with dihydroxylated aromatic compounds

In order to design potential biomaterials, we investigated the laccase-catalyzed cross-linking between l-lysine or lysine-containing peptides and dihydroxylated aromatics. l-Lysine is one of the major components of naturally occurring mussel adhesive proteins (MAPs). Dihydroxylated aromatics are structurally related to 3,4-dihydroxyphenyl-l-alanine, another main component of MAPs. Mass spectrometry and nuclear magnetic resonance analyses show that the ε-amino group of l-lysine is able to cross-link dihydroxylated aromatics. Additional oligomer and polymer cross-linked products were obtained from di- and oligopeptides containing l-lysine. Potential applications in medicine or industry for biomaterials synthesised via the three component system consisting of the oligopeptide [Tyr-Lys]₁₀, dihydroxylated aromatics and laccase are discussed.     

Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocytes for Construction of Three-dimensional Cartilage Tissue

A differentiation method of human bone marrow mesenchymal stem cells (MSCs) to chondrocytes was developed for the construction of a three-dimensional (3D) cartilage tissue. The adhesive cells, which were isolated from a human bone marrow aspirate were embedded in type I collagen in a poly-l-lactate-glycolic acid copolymer (PLGA) mesh and cultivated for 4 week together with growth factors. The degree of cellular differentiation was estimated by quantitative RT-PCR of aggrecan and type II collagen mRNAs and by staining with Safranin O. The 3D culture showed a higher degree of differentiation even without growth factors than the conventional pellet culture with growth factors, namely, dexamethasone and transforming growth factor (TGF)-β 3. The 3D culture for 2 week with the combined addition of dexamethasone, TGF-β 3, and insulin-like growth factor (IGF)-I reached a 30% expression of aggrecan mRNA compared with that in primary human chondrocytes, while the aggrecan mRNA expression in the conventional pellet culture was less than 2%. The sequential two-step differentiation cultivation, during which the cells were cultivated in 3D for 1 week after the conventional two-dimensional (2D) culture for 1 week, could markedly accelerate the expression of aggrecan mRNA compared with the 3D cultivation for 2 week.     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

Enhanced neurite outgrowth of rat neural cortical cells on surface-modified films of poly(lactic-<Emphasis Type="Italic">co</Emphasis>-glycolic acid)

Neural cortical cells, isolated from prenatal rat cerebra, were grown on surface-modified poly(lactic-co-glycolic acid, 65:35) (PLGA) films coated with poly-D-lysine (PDL) with either laminin (LN), fibronectin (FN) or collagen (CN). Immunocytochemistry showed that the isolated cells were highly immunopositive for both neurofilament and MAP-2 with well-organized neurites and somatodendritic localization. The presence of PDL with LN or FN on the PLGA films was essential for increased neural cell growth. Also, PLGA films coated with either PDL/LN or PDL/FN mixtures had higher neurite outgrowth and regular differentiation.Revisions requested 30 September 2004; Revisions received 10 November 2004     

Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation.