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Sample records for autologous skeletal myoblasts

  1. New perspectives in the treatment of damaged myocardium using autologous skeletal myoblasts

    International Nuclear Information System (INIS)

    Rigatelli, Gianluca; Rossini, Katia; Vindigni, Vincenzo; Mazzoleni, Francesco; Rigatelli, Giorgio; Carraro, Ugo

    2004-01-01

    Autologous skeletal myoblast transplantation may be used to ameliorate the healing process following myocardium infarct and, hopefully, cardiomyopathies. Despite successful animal experimentation, several issues need to be addressed in clinical settings, i.e., the impact of the delivery route, the extent of short- and long-term survival, and differentiation of the injected skeletal myoblasts. The authors offer some new hypotheses resulting from basic research, i.e., where and when to inject the myogenic cells, whatever their source, how to decrease new myofiber atrophy and improve their regeneration. Although these new hypotheses still need to be tested in humans, they may be decisive for future experimental studies and will lead to making endovascular cell implantation a more effective way to treat ischemic heart disease and failure

  2. Skeletal muscle myoblasts possess a stretch-responsive local angiotensin signalling system.

    Science.gov (United States)

    Johnston, Adam P W; Baker, Jeff; De Lisio, Michael; Parise, Gianni

    2011-06-01

    A paucity of information exists regarding the presence of local renin-angiotensin systems (RASs) in skeletal muscle and associated muscle stem cells. Skeletal muscle and muscle stem cells were isolated from C57BL/6 mice and examined for the presence of a local RAS using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), Western blotting and liquid chromatography-mass spectrometry (LC-MS). Furthermore, the effect of mechanical stimulation on RAS member gene expression was analysed. Whole skeletal muscle, primary myoblasts and C2C12 derived myoblasts and myotubes differentially expressed members of the RAS including angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) type 1 (AT(1)) and type 2 (AT(2)). Renin transcripts were never detected, however, mRNA for the 'renin-like' enzyme cathepsin D was observed and Ang I and Ang II were identified in cell culture supernatants from proliferating myoblasts. AT(1) appeared to co-localise with polymerised actin filaments in proliferating myoblasts and was primarily found in the nucleus of terminally differentiated myotubes. Furthermore, mechanical stretch of proliferating and differentiating C2C12 cells differentially induced mRNA expression of angiotensinogen, AT(1) and AT(2). Proliferating and differentiated muscle stem cells possess a local stress-responsive RAS in vitro. The precise function of a local RAS in myoblasts remains unknown. However, evidence presented here suggests that Ang II may be a regulator of skeletal muscle myoblasts.

  3. One-Step Purification of Human Skeletal Muscle Myoblasts and Subsequent Expansion Using Laminin-Coated Surface.

    Science.gov (United States)

    Chowdhury, Shiplu Roy; binti Ismail, Annis; Chee, Sia Chye; bin Laupa, Mohd Suffian; binti Jaffri, Fadhlun; Saberi, Salfarina Ezrina Mohmad; Idrus, Ruszymah Bt Hj

    2015-11-01

    Skeletal myoblasts have been extensively used to study muscle growth and differentiation, and were recently tested for their application as cell therapy and as a gene delivery system to treat muscle and nonmuscle diseases. However, contamination of fibroblasts in isolated cells from skeletal muscle is one of the long-standing problems for routine expansion. This study aimed to establish a simple one-step process to purify myoblasts and maintain their purity during expansion. Mixed cells were preplated serially on laminin- and collagen type I-coated surfaces in a different array for 5, 10, and 15 min. Immunocytochemical staining with antibodies specific to myoblasts was performed to evaluate myoblast attachment efficiency, purity, and yield. It was found that laminin-coated surface favors the attachment of myoblasts. Highest myoblast purity of 78.9% ± 6.8% was achieved by 5 min of preplating only on the laminin-coated surface with a yield of 56.9% ± 3.3%. Primary cells, isolated from skeletal muscle (n = 4), confirm the enhancement of purity through preplating on laminin-coated surface for 5 min. Subsequent expansion after preplating enhanced myoblast purity due to an increase in myoblast growth than fibroblasts. Myoblast purity of ∼ 98% was achieved when another preplating was performed during passaging. In conclusion, myoblasts can be purified and efficiently expanded in one step by preplating on laminin-coated surface, which is a simple and robust technique.

  4. Intraurethral Injection of Autologous Minced Skeletal Muscle

    DEFF Research Database (Denmark)

    Gräs, Søren; Klarskov, Niels; Lose, Gunnar

    2014-01-01

    noted. CONCLUSIONS: Intraurethral injection of minced autologous muscle tissue is a simple surgical procedure that appears safe and moderately effective in women with uncomplicated stress urinary incontinence. It compares well to a more complicated regenerative strategy using in vitro expanded muscle......PURPOSE: Intraurethral injection of in vitro expanded autologous skeletal muscle derived cells is a new regenerative therapy for stress urinary incontinence. We examined the efficacy and safety of a simpler alternative strategy using freshly harvested, minced autologous skeletal muscle tissue...... with its inherent content of regenerative cells. MATERIALS AND METHODS: A total of 20 and 15 women with uncomplicated and complicated stress urinary incontinence, respectively, received intraurethral injections of minced autologous skeletal muscle tissue and were followed for 1 year. Efficacy was assessed...

  5. The Use of Platelet-Rich and Platelet-Poor Plasma to Enhance Differentiation of Skeletal Myoblasts: Implications for the Use of Autologous Blood Products for Muscle Regeneration.

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    Miroshnychenko, Olga; Chang, Wen-Teh; Dragoo, Jason L

    2017-03-01

    Platelet-rich plasma (PRP) has been used to augment tissue repair and regeneration after musculoskeletal injury. However, there is increasing clinical evidence that PRP does not show a consistent clinical effect. Purpose/Hypothesis: This study aimed to compare the effects of the following non-neutrophil-containing (leukocyte-poor) plasma fractions on human skeletal muscle myoblast (HSMM) differentiation: (1) PRP, (2) modified PRP (Mod-PRP), in which transforming growth factor β1 (TGF-β1) and myostatin (MSTN) were depleted, and (3) platelet-poor plasma (PPP). The hypothesis was that leukocyte-poor PRP would lead to myoblast proliferation (not differentiation), whereas certain modifications of PRP preparations would increase myoblast differentiation, which is necessary for skeletal muscle regeneration. Controlled laboratory study. Blood from 7 human donors was individually processed to simultaneously create leukocyte-poor fractions: PRP, Mod-PRP, PPP, and secondarily spun PRP and Mod-PRP (PRP ss and Mod-PRP ss , respectively). Mod-PRP was produced by removing TGF-β1 and MSTN from PRP using antibodies attached to sterile beads, while a second-stage centrifugal spin of PRP was performed to remove platelets. The biologics were individually added to cell culture groups. Analysis for induction into myoblast differentiation pathways included Western blot analysis, reverse-transcription polymerase chain reaction, and immunohistochemistry, as well as confocal microscopy to assess polynucleated myotubule formation. HSMMs cultured with PRP showed an increase in proliferation but no evidence of differentiation. Western blot analysis confirmed that MSTN and TGF-β1 could be decreased in Mod-PRP using antibody-coated beads, but this modification mildly improved myoblast differentiation. However, cell culture with PPP, PRP ss , and Mod-PRP ss led to a decreased proliferation rate but a significant induction of myoblast differentiation verified by increased multinucleated

  6. Developmental Programming in Response to Intrauterine Growth Restriction Impairs Myoblast Function and Skeletal Muscle Metabolism

    Science.gov (United States)

    Yates, D. T.; Macko, A. R.; Nearing, M.; Chen, X.; Rhoads, R. P.; Limesand, S. W.

    2012-01-01

    Fetal adaptations to placental insufficiency alter postnatal metabolic homeostasis in skeletal muscle by reducing glucose oxidation rates, impairing insulin action, and lowering the proportion of oxidative fibers. In animal models of intrauterine growth restriction (IUGR), skeletal muscle fibers have less myonuclei at birth. This means that myoblasts, the sole source for myonuclei accumulation in fibers, are compromised. Fetal hypoglycemia and hypoxemia are complications that result from placental insufficiency. Hypoxemia elevates circulating catecholamines, and chronic hypercatecholaminemia has been shown to reduce fetal muscle development and growth. We have found evidence for adaptations in adrenergic receptor expression profiles in myoblasts and skeletal muscle of IUGR sheep fetuses with placental insufficiency. The relationship of β-adrenergic receptors shifts in IUGR fetuses because Adrβ2 expression levels decline and Adrβ1 expression levels are unaffected in myofibers and increased in myoblasts. This adaptive response would suppress insulin signaling, myoblast incorporation, fiber hypertrophy, and glucose oxidation. Furthermore, this β-adrenergic receptor expression profile persists for at least the first month in IUGR lambs and lowers their fatty acid mobilization. Developmental programming of skeletal muscle adrenergic receptors partially explains metabolic and endocrine differences in IUGR offspring, and the impact on metabolism may result in differential nutrient utilization. PMID:22900186

  7. Developmental Programming in Response to Intrauterine Growth Restriction Impairs Myoblast Function and Skeletal Muscle Metabolism

    Directory of Open Access Journals (Sweden)

    D. T. Yates

    2012-01-01

    Full Text Available Fetal adaptations to placental insufficiency alter postnatal metabolic homeostasis in skeletal muscle by reducing glucose oxidation rates, impairing insulin action, and lowering the proportion of oxidative fibers. In animal models of intrauterine growth restriction (IUGR, skeletal muscle fibers have less myonuclei at birth. This means that myoblasts, the sole source for myonuclei accumulation in fibers, are compromised. Fetal hypoglycemia and hypoxemia are complications that result from placental insufficiency. Hypoxemia elevates circulating catecholamines, and chronic hypercatecholaminemia has been shown to reduce fetal muscle development and growth. We have found evidence for adaptations in adrenergic receptor expression profiles in myoblasts and skeletal muscle of IUGR sheep fetuses with placental insufficiency. The relationship of β-adrenergic receptors shifts in IUGR fetuses because Adrβ2 expression levels decline and Adrβ1 expression levels are unaffected in myofibers and increased in myoblasts. This adaptive response would suppress insulin signaling, myoblast incorporation, fiber hypertrophy, and glucose oxidation. Furthermore, this β-adrenergic receptor expression profile persists for at least the first month in IUGR lambs and lowers their fatty acid mobilization. Developmental programming of skeletal muscle adrenergic receptors partially explains metabolic and endocrine differences in IUGR offspring, and the impact on metabolism may result in differential nutrient utilization.

  8. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

    Science.gov (United States)

    Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

  9. MicroRNA-128 targets myostatin at coding domain sequence to regulate myoblasts in skeletal muscle development.

    Science.gov (United States)

    Shi, Lei; Zhou, Bo; Li, Pinghua; Schinckel, Allan P; Liang, Tingting; Wang, Han; Li, Huizhi; Fu, Lingling; Chu, Qingpo; Huang, Ruihua

    2015-09-01

    MicroRNAs (miRNAs or miRs) play a critical role in skeletal muscle development. In a previous study we observed that miR-128 was highly expressed in skeletal muscle. However, its function in regulating skeletal muscle development is not clear. Our hypothesis was that miR-128 is involved in the regulation of the proliferation and differentiation of skeletal myoblasts. In this study, through bioinformatics analyses, we demonstrate that miR-128 specifically targeted mRNA of myostatin (MSTN), a critical inhibitor of skeletal myogenesis, at coding domain sequence (CDS) region, resulting in down-regulating of myostatin post-transcription. Overexpression of miR-128 inhibited proliferation of mouse C2C12 myoblast cells but promoted myotube formation; whereas knockdown of miR-128 had completely opposite effects. In addition, ectopic miR-128 regulated the expression of myogenic factor 5 (Myf5), myogenin (MyoG), paired box (Pax) 3 and 7. Furthermore, an inverse relationship was found between the expression of miR-128 and MSTN protein expression in vivo and in vitro. Taken together, these results reveal that there is a novel pathway in skeletal muscle development in which miR-128 regulates myostatin at CDS region to inhibit proliferation but promote differentiation of myoblast cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Laminin-Coated Poly(Methyl Methacrylate) (PMMA) Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population.

    Science.gov (United States)

    Zahari, Nor Kamalia; Idrus, Ruszymah Binti Haji; Chowdhury, Shiplu Roy

    2017-10-30

    Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h -1 ) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h -1 ) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.

  11. Bone Marrow Mesenchymal Stromal Cells Stimulate Skeletal Myoblast Proliferation through the Paracrine Release of VEGF

    Science.gov (United States)

    Chellini, Flaminia; Mazzanti, Benedetta; Nistri, Silvia; Nosi, Daniele; Saccardi, Riccardo; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

    2012-01-01

    Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies. PMID:22815682

  12. Laminin-Coated Poly(Methyl Methacrylate (PMMA Nanofiber Scaffold Facilitates the Enrichment of Skeletal Muscle Myoblast Population

    Directory of Open Access Journals (Sweden)

    Nor Kamalia Zahari

    2017-10-01

    Full Text Available Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate (PMMA nanofiber (PM scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h−1 and migration (0.26 ± 0.04 μm/min, while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h−1 and migration (0.23 ± 0.03 μm/min. Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.

  13. Mesenchymal Stromal Cell Secreted Sphingosine 1-Phosphate (S1P) Exerts a Stimulatory Effect on Skeletal Myoblast Proliferation

    Science.gov (United States)

    Tani, Alessia; Anderloni, Giulia; Pierucci, Federica; Matteini, Francesca; Chellini, Flaminia; Zecchi Orlandini, Sandra; Meacci, Elisabetta

    2014-01-01

    Bone-marrow-derived mesenchymal stromal cells (MSCs) have the potential to significantly contribute to skeletal muscle healing through the secretion of paracrine factors that support proliferation and enhance participation of the endogenous muscle stem cells in the process of repair/regeneration. However, MSC-derived trophic molecules have been poorly characterized. The aim of this study was to investigate paracrine signaling effects of MSCs on skeletal myoblasts. It was found, using a biochemical and morphological approach that sphingosine 1-phosphate (S1P), a natural bioactive lipid exerting a broad range of muscle cell responses, is secreted by MSCs and represents an important factor by which these cells exert their stimulatory effects on C2C12 myoblast and satellite cell proliferation. Indeed, exposure to conditioned medium obtained from MSCs cultured in the presence of the selective sphingosine kinase inhibitor (iSK), blocked increased cell proliferation caused by the conditioned medium from untreated MSCs, and the addition of exogenous S1P in the conditioned medium from MSCs pre-treated with iSK further increased myoblast proliferation. Finally, we also demonstrated that the myoblast response to MSC-secreted vascular endothelial growth factor (VEGF) involves the release of S1P from C2C12 cells. Our data may have important implications in the optimization of cell-based strategies to promote skeletal muscle regeneration. PMID:25264785

  14. Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

    International Nuclear Information System (INIS)

    Roura-Ferrer, Meritxell; Sole, Laura; Martinez-Marmol, Ramon; Villalonga, Nuria; Felipe, Antonio

    2008-01-01

    Voltage-dependent K + channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G 1 -phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletal muscle cell proliferation

  15. Substrate stiffness affects skeletal myoblast differentiation in vitro

    Directory of Open Access Journals (Sweden)

    Sara Romanazzo, Giancarlo Forte, Mitsuhiro Ebara, Koichiro Uto, Stefania Pagliari, Takao Aoyagi, Enrico Traversa and Akiyoshi Taniguchi

    2012-01-01

    Full Text Available To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.

  16. Improvement of maternal vitamin D status with 25-hydroxycholecalciferol positively impacts porcine fetal skeletal muscle development and myoblast activity.

    Science.gov (United States)

    Hines, E A; Coffey, J D; Starkey, C W; Chung, T K; Starkey, J D

    2013-09-01

    There is little information available regarding the influence of maternal vitamin D status on fetal skeletal muscle development. Therefore, we investigated the effect of improved vitamin D status resulting from 25-hydroxycholecalciferol (25OHD3) supplementation of dams on fetal skeletal muscle developmental characteristics and myoblast activity using Camborough 22 gilts (n = 40) randomly assigned to 1 of 2 corn-soybean meal-based diets. The control diet (CTL) contained 2,500 IU cholecalciferol (D3)/kg diet, whereas the experimental diet contained 500 IU D3/kg diet plus 50 µg 25OHD3/kg diet. Gilts were fed 2.7 kg of their assigned diet once daily beginning 43 d before breeding through d 90 of gestation. On gestational d 90 (± 1), fetal LM and semitendinosus muscle samples were collected for analysis of developmental characteristics and myoblast activity, respectively. No treatment difference was observed in fetal LM cross-sectional area (P = 0.25). Fetuses from 25OHD3-supplemented gilts had more LM fibers (P = 0.04) that tended to be smaller in cross-sectional area compared with CTL fetuses (P = 0.11). A numerical increase in the total number of Pax7+ myoblasts was also observed in fetuses from 25OHD3-supplemented gilts (P = 0.12). Myoblasts derived from the muscles of fetuses from 25OHD3-fed dams displayed an extended proliferative phase in culture compared with those from fetuses of dams fed only D3 (P importance of maternal vitamin D status on the development of fetal skeletal muscle.

  17. Syncytin-1 in differentiating human myoblasts

    DEFF Research Database (Denmark)

    Bjerregard, Bolette; Ziomkiewicz, Iwona; Schulz, Alexander

    2014-01-01

    Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts...... fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma....

  18. Marked heterogeneity in growth characteristics of myoblast clonal cultures and myoblast mixed cultures obtained from the same individual.

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    Maier, Andrea B; Cohen, Ron; Blom, Joke; van Heemst, Diana; Westendorp, Rudi G J

    2012-01-01

    Sarcopenia is defined as an age-related decrease in skeletal muscle mass and function while adjacent satellite cells are unable to compensate for this loss. However, myoblast cultures can be established even in the presence of sarcopenia. It is yet unknown whether satellite cells from failing muscle in older age are equally affected, as human satellite cells have been assessed using myoblast mixed cultures and not by using myoblast clonal cultures. We questioned to what extent myoblast mixed cultures reflect the in vivo characteristics of single satellite cells from adult skeletal muscle. We established a myoblast mixed culture and three myoblast clonal cultures out of the same muscle biopsy and cultured these cells for 100 days. Replicative capacity and oxidative stress resistance were compared. We found marked heterogeneity between the myoblast clonal cultures that all had a significantly lower replicative capacity when compared to the mixed culture. Replicative capacity of the clonal cultures was inversely related to the β-galactosidase activity after exposure to oxidative stress. Addition of L-carnosine enhanced the remaining replicative capacity in all cultures with a concomitant marginal decrease in β-galactosidase activity. It is concluded that myoblast mixed cultures in vitro do not reflect the marked heterogeneity between single isolated satellite cells. The consequences of the heterogeneity on muscle performance remain to be established. Copyright © 2011 S. Karger AG, Basel.

  19. Isolation and Characterization of Human Myoblast Culture In Vitro for Technologies of Cell and Gene Therapy of Skeletal Muscle Pathologies.

    Science.gov (United States)

    Tabakov, V Yu; Zinov'eva, O E; Voskresenskaya, O N; Skoblov, M Yu

    2018-03-01

    We analyzed cultures of 5 independent myoblast lines from human skeletal muscles. It was shown that the content of desmin-positive cells in cultures at early passages exceeds 90%. Typical morphofunctional signs of myogenic differentiation disturbances were identified and their dynamics was studied. Signs of alternative adipogenic and chondrogenic differentiation of cells were revealed. Based on these data, limitations for the use of myoblast cultures of certain passages for biomedical research and cell therapy were evaluated.

  20. The use of cell-sheet technique eliminates arrhythmogenicity of skeletal myoblast-based therapy to the heart with enhanced therapeutic effects.

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    Narita, Takuya; Shintani, Yasunori; Ikebe, Chiho; Kaneko, Masahiro; Harada, Narumi; Tshuma, Nomathamsanqa; Takahashi, Kunihiko; Campbell, Niall G; Coppen, Steven R; Yashiro, Kenta; Sawa, Yoshiki; Suzuki, Ken

    2013-09-20

    Clinical application of skeletal myoblast transplantation has been curtailed due to arrhythmogenicity and inconsistent therapeutic benefits observed in previous studies. However, these issues may be solved by the use of a new cell-delivery mode. It is now possible to generate "cell-sheets" using temperature-responsive dishes without artificial scaffolds. This study aimed to validate the safety and efficacy of epicardial placement of myoblast-sheets (myoblast-sheet therapy) in treating heart failure. After coronary artery ligation in rats, the same numbers of syngeneic myoblasts were transplanted by intramyocardial injection or cell-sheet placement. Continuous radio-telemetry monitoring detected increased ventricular arrhythmias, including ventricular tachycardia, after intramyocardial injection compared to the sham-control, while these were abolished in myoblast-sheet therapy. This effect was conjunct with avoidance of islet-like cell-cluster formation that disrupts electrical conduction, and with prevention of increased arrhythmogenic substrates due to exaggerated inflammation. Persistent ectopic donor cells were found in the lung only after intramyocardial injection, strengthening the improved safety of myoblast-sheet therapy. In addition, myoblast-sheet therapy enhanced cardiac function, corresponding to a 9.2-fold increase in donor cell survival, compared to intramyocardial injection. Both methods achieved reduced infarct size, decreased fibrosis, attenuated cardiomyocyte hypertrophy, and increased neovascular formation, in association with myocardial upregulation of a group of relevant molecules. The pattern of these beneficial changes was similar between two methods, but the degree was more substantial after myoblast-sheet therapy. The cell-sheet technique enhanced safety and therapeutic efficacy of myoblast-based therapy, compared to the current method, thereby paving the way for clinical application. Copyright © 2012 Elsevier Ireland Ltd. All rights

  1. Enhanced contractile force generation by artificial skeletal muscle tissues using IGF-I gene-engineered myoblast cells.

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    Sato, Masanori; Ito, Akira; Kawabe, Yoshinori; Nagamori, Eiji; Kamihira, Masamichi

    2011-09-01

    The aim of this study was to investigate whether insulin-like growth factor (IGF)-I gene delivery to myoblast cells promotes the contractile force generated by hydrogel-based tissue-engineered skeletal muscles in vitro. Two retroviral vectors allowing doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into mouse myoblast C2C12 cells to evaluate the effects of IGF-I gene expression on these cells. IGF-I gene expression stimulated the proliferation of C2C12 cells, and a significant increase in the growth rate was observed for IGF-I-transduced C2C12 cells with Dox addition, designated C2C12/IGF (Dox+) cells. Quantitative morphometric analyses showed that the myotubes induced from C2C12/IGF (Dox+) cells had a larger area and a greater width than control myotubes induced from normal C2C12 cells. Artificial skeletal muscle tissues were prepared from the respective cells using hydrogels composed of type I collagen and Matrigel. Western blot analyses revealed that the C2C12/IGF (Dox+) tissue constructs showed activation of a skeletal muscle hypertrophy marker (Akt) and enhanced expression of muscle-specific markers (myogenin, myosin heavy chain and tropomyosin). Moreover, the creatine kinase activity was increased in the C2C12/IGF (Dox+) tissue constructs. The C2C12/IGF (Dox+) tissue constructs contracted in response to electrical pulses, and generated a significantly higher physical force than the control C2C12 tissue constructs. These findings indicate that IGF-I gene transfer has the potential to yield functional skeletal muscle substitutes that are capable of in vivo restoration of the load-bearing function of injured muscle or acting as in vitro electrically-controlled bio-actuators. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    International Nuclear Information System (INIS)

    Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

    2013-01-01

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP 3 /calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation

  3. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    Energy Technology Data Exchange (ETDEWEB)

    Zuloaga, R. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Fuentes, E.N.; Molina, A. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: jvaldes@unab.cl [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile)

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  4. MicroRNA-27a promotes myoblast proliferation by targeting myostatin

    International Nuclear Information System (INIS)

    Huang, Zhiqing; Chen, Xiaoling; Yu, Bing; He, Jun; Chen, Daiwen

    2012-01-01

    Highlights: ► We identified a myogenic role for miR-27a and a new target, myostatin. ► The miR-27a was confirmed to target myostatin 3′UTR. ► miR-27a is upregulated and myostatin is downregulated during myoblast proliferation. ► miR-27a promotes myoblast proliferation by reducing the expression of myostatin. -- Abstract: MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3′UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.

  5. MicroRNA-27a promotes myoblast proliferation by targeting myostatin

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Zhiqing; Chen, Xiaoling; Yu, Bing; He, Jun [Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014 (China); Chen, Daiwen, E-mail: dwchen@sicau.edu.cn [Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Institute of Animal Nutrition, Sichuan Agricultural University, Yaan, Sichuan 625014 (China)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer We identified a myogenic role for miR-27a and a new target, myostatin. Black-Right-Pointing-Pointer The miR-27a was confirmed to target myostatin 3 Prime UTR. Black-Right-Pointing-Pointer miR-27a is upregulated and myostatin is downregulated during myoblast proliferation. Black-Right-Pointing-Pointer miR-27a promotes myoblast proliferation by reducing the expression of myostatin. -- Abstract: MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs that play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. However, the role of miRNAs in myoblast proliferation remains poorly understood. Here we found that the expression of miR-27a was increased during proliferation of C2C12 myoblasts. Moreover, overexpression of miR-27a in C2C12 cells promoted myoblast proliferation by reducing the expression of myostatin, a critical inhibitor of skeletal myogenesis. In addition, the miR-27a was confirmed to target myostatin 3 Prime UTR by a luciferase reporter analysis. Together, these results suggest that miR-27a promotes myoblast proliferation through targeting myostatin.

  6. NEU3 sialidase strictly modulates GM3 levels in skeletal myoblasts C2C12 thus favoring their differentiation and protecting them from apoptosis.

    Science.gov (United States)

    Anastasia, Luigi; Papini, Nadia; Colazzo, Francesca; Palazzolo, Giacomo; Tringali, Cristina; Dileo, Loredana; Piccoli, Marco; Conforti, Erika; Sitzia, Clementina; Monti, Eugenio; Sampaolesi, Maurilio; Tettamanti, Guido; Venerando, Bruno

    2008-12-26

    Membrane-bound sialidase NEU3, often referred to as the "ganglioside sialidase," has a critical regulatory function on the sialoglycosphingolipid pattern of the cell membrane, with an anti-apoptotic function, especially in cancer cells. Although other sialidases have been shown to be involved in skeletal muscle differentiation, the role of NEU3 had yet to be disclosed. Herein we report that NEU3 plays a key role in skeletal muscle differentiation by strictly modulating the ganglioside content of adjacent cells, with special regard to GM3. Induced down-regulation of NEU3 in murine C2C12 myoblasts, even when partial, totally inhibits their capability to differentiate by increasing the GM3 level above a critical point, which causes epidermal growth factor receptor inhibition (and ultimately its down-regulation) and an higher responsiveness of myoblasts to the apoptotic stimuli.

  7. Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

    Science.gov (United States)

    Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

    1996-08-01

    To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

  8. TIPS to manipulate myogenesis: retention of myoblast differentiation capacity using microsphere culture

    Directory of Open Access Journals (Sweden)

    N Parmar

    2015-07-01

    Full Text Available Cell therapy is an emerging option for regenerating skeletal muscle. Improved delivery methods for anchorage-dependent myoblasts are likely to improve integration and function of transplanted muscle cells. Highly porous microspheres, produced using thermally induced phase separation (TIPS, have features ideally suited for minimally invasive cell delivery. The purpose of this study was to investigate, for the first time, the use of TIPS microspheres as highly porous microcarriers for manipulation of human skeletal muscle myoblasts (HSMM under defined culture conditions. HSMM cells readily attached to the surface of poly (DL-lactide-co-glycolide (PLGA TIPS microcarriers, where they were induced to continue proliferating or to be driven towards differentiation whilst under static-dynamic culture conditions for 7 days. Switching from proliferation medium to differentiation medium for 7 days, resulted in increased protein expression of skeletal muscle cell contractile apparatus components, MyoD and skeletal muscle myosin heavy chain, compared with cells cultured on conventional culture plasticware for the same duration (p < 0.001. Growth of myoblasts on the surface of the microcarriers and their migration following simulated delivery, caused no change to the proliferative capacity of cells over 7 days. Results from this study demonstrate that TIPS microspheres provide an ideal vehicle for the expansion and delivery of myoblasts for therapeutic applications. Transplantation of myoblasts anchored to a substrate, rather than in suspension, will reduce the amount of ex vivo manipulation required during preparation of the product and allows cells to be delivered in a more natural state. This will improve the ability to control cell dosage and increase the likelihood of efficacy.

  9. Marked heterogeneity in growth characteristics of myoblast clonal cultures and myoblast mixed cultures obtained from the same individual

    NARCIS (Netherlands)

    Maier, Andrea B.; Cohen, Ron; Blom, Joke; Van Heemst, Diana; Westendorp, Rudi G.J.

    Background: Sarcopenia is defined as an age-related decrease in skeletal muscle mass and function while adjacent satellite cells are unable to compensate for this loss. However, myoblast cultures can be established even in the presence of sarcopenia. Objective: It is yet unknown whether satellite

  10. Group I Paks Promote Skeletal Myoblast Differentiation In Vivo and In Vitro

    DEFF Research Database (Denmark)

    Joseph, Giselle A; Lu, Min; Radu, Maria

    2017-01-01

    fusion in Drosophila We report that both Pak1 and Pak2 are activated during mammalian myoblast differentiation. One pathway of activation is initiated by N-cadherin ligation and involves the cadherin coreceptor Cdo with its downstream effector, Cdc42. Individual genetic deletion of Pak1 and Pak2 in mice....... Furthermore, primary myoblasts lacking Pak1 and Pak2 display delayed expression of myogenic differentiation markers and myotube formation. These results identify Pak1 and Pak2 as redundant regulators of myoblast differentiation in vitro and in vivo and as components of the promyogenic Ncad/Cdo/Cdc42 signaling...

  11. Skeletal muscle tissue engineering: methods to form skeletal myotubes and their applications.

    Science.gov (United States)

    Ostrovidov, Serge; Hosseini, Vahid; Ahadian, Samad; Fujie, Toshinori; Parthiban, Selvakumar Prakash; Ramalingam, Murugan; Bae, Hojae; Kaji, Hirokazu; Khademhosseini, Ali

    2014-10-01

    Skeletal muscle tissue engineering (SMTE) aims to repair or regenerate defective skeletal muscle tissue lost by traumatic injury, tumor ablation, or muscular disease. However, two decades after the introduction of SMTE, the engineering of functional skeletal muscle in the laboratory still remains a great challenge, and numerous techniques for growing functional muscle tissues are constantly being developed. This article reviews the recent findings regarding the methodology and various technical aspects of SMTE, including cell alignment and differentiation. We describe the structure and organization of muscle and discuss the methods for myoblast alignment cultured in vitro. To better understand muscle formation and to enhance the engineering of skeletal muscle, we also address the molecular basics of myogenesis and discuss different methods to induce myoblast differentiation into myotubes. We then provide an overview of different coculture systems involving skeletal muscle cells, and highlight major applications of engineered skeletal muscle tissues. Finally, potential challenges and future research directions for SMTE are outlined.

  12. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

    International Nuclear Information System (INIS)

    Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T.; Pierre, Philippe; Chadee, Deborah N.; Pizza, Francis X.

    2015-01-01

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

  13. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T. [Department of Kinesiology, The University of Toledo, Toledo, OH (United States); Pierre, Philippe [Centre d’Immunologie de Marseille-Luminy U2M, Aix-Marseille Université, Marseille (France); INSERM U631, Institut National de la Santé et Recherche Médicale, Marseille (France); CNRS UMR6102, Centre National de la Recherche Scientifique, Marseille (France); Chadee, Deborah N. [Department of Biological Sciences, The University of Toledo, Toledo, OH (United States); Pizza, Francis X., E-mail: Francis.Pizza@utoledo.edu [Department of Kinesiology, The University of Toledo, Toledo, OH (United States)

    2015-02-15

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

  14. FSHD myoblasts fail to downregulate intermediate filament protein vimentin during myogenic differentiation.

    Directory of Open Access Journals (Sweden)

    Lipinski M.

    2011-10-01

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal dominant hereditary neuromuscular disorder. The clinical features of FSHD include weakness of the facial and shoulder girdle muscles followed by wasting of skeletal muscles of the pelvic girdle and lower extremities. Although FSHD myoblasts grown in vitro can be induced to differentiate into myotubes by serum starvation, the resulting FSHD myotubes have been shown previously to be morphologically abnormal. Aim. In order to find the cause of morphological anomalies of FSHD myotubes we compared in vitro myogenic differentiation of normal and FSHD myoblasts at the protein level. Methods. We induced myogenic differentiation of normal and FSHD myoblasts by serum starvation. We then compared protein extracts from proliferating myoblasts and differentiated myotubes using SDS-PAGE followed by mass spectrometry identification of differentially expressed proteins. Results. We demonstrated that the expression of vimentin was elevated at the protein and mRNA levels in FSHD myotubes as compared to normal myotubes. Conclusions. We demonstrate for the first time that in contrast to normal myoblasts, FSHD myoblasts fail to downregulate vimentin after induction of in vitro myogenic differentiation. We suggest that vimentin could be an easily detectable marker of FSHD myotubes

  15. Function of skeletal muscle tissue formed after myoblast transplantation into irradiated mouse muscles.

    Science.gov (United States)

    Wernig, A; Zweyer, M; Irintchev, A

    2000-01-15

    1. Pretreatment of muscles with ionising radiation enhances tissue formation by transplanted myoblasts but little is known about the effects on muscle function. We implanted myoblasts from an expanded, male-donor-derived, culture (i28) into X-ray irradiated (16 Gy) or irradiated and damaged soleus muscles of female syngeneic mice (Balb/c). Three to 6 months later the isometric contractile properties of the muscles were studied in vitro, and donor nuclei were visualised in muscle sections with a Y chromosome-specific DNA probe. 2. Irradiated sham-injected muscles had smaller masses than untreated solei and produced less twitch and tetanic force (all by about 18 %). Injection of 106 myoblasts abolished these deficiencies and innervation appeared normal. 3. Cryodamage of irradiated solei produced muscle remnants with few (1-50) or no fibres. Additional myoblast implantation led to formation of large muscles (25 % above normal) containing numerous small-diameter fibres. Upon direct electrical stimulation, these muscles produced considerable twitch (53 % of normal) and tetanic forces (35 % of normal) but innervation was insufficient as indicated by weak nerve-evoked contractions and elevated ACh sensitivity. 4. In control experiments on irradiated muscles, reinnervation was found to be less complete after botulinum toxin paralysis than after nerve crush indicating that proliferative arrest of irradiated Schwann cells may account for the observed innervation deficits. 5. Irradiation appears to be an effective pretreatment for improving myoblast transplantation. The injected cells can even produce organised contractile tissue replacing whole muscle. However, impaired nerve regeneration limits the functional performance of the new muscle.

  16. Myomaker, Regulated by MYOD, MYOG and miR-140-3p, Promotes Chicken Myoblast Fusion

    Directory of Open Access Journals (Sweden)

    Wen Luo

    2015-11-01

    Full Text Available The fusion of myoblasts is an important step during skeletal muscle differentiation. A recent study in mice found that a transmembrane protein called Myomaker, which is specifically expressed in muscle, is critical for myoblast fusion. However, the cellular mechanism of its roles and the regulatory mechanism of its expression remain unclear. Chicken not only plays an important role in meat production but is also an ideal model organism for muscle development research. Here, we report that Myomaker is also essential for chicken myoblast fusion. Forced expression of Myomaker in chicken primary myoblasts promotes myoblast fusion, whereas knockdown of Myomaker by siRNA inhibits myoblast fusion. MYOD and MYOG, which belong to the family of myogenic regulatory factors, can bind to a conserved E-box located proximal to the Myomaker transcription start site and induce Myomaker transcription. Additionally, miR-140-3p can inhibit Myomaker expression and myoblast fusion, at least in part, by binding to the 3ʹ UTR of Myomaker in vitro. These findings confirm the essential roles of Myomaker in avian myoblast fusion and show that MYOD, MYOG and miR-140-3p can regulate Myomaker expression.

  17. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  18. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    International Nuclear Information System (INIS)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven

    2014-01-01

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro

  19. Selenium regulates gene expression of selenoprotein W in chicken skeletal muscle system.

    Science.gov (United States)

    Ruan, Hongfeng; Zhang, Ziwei; Wu, Qiong; Yao, Haidong; Li, Jinlong; Li, Shu; Xu, Shiwen

    2012-01-01

    Selenoprotein W (SelW) is abundantly expressed in skeletal muscles of mammals and necessary for the metabolism of skeletal muscles. However, its expression pattern in skeletal muscle system of birds is still uncovered. Herein, to investigate the distribution of SelW mRNA in chicken skeletal muscle system and its response to different selenium (Se) status, 1-day-old chickens were exposed to various concentrations of Se as sodium selenite in the feed for 35 days. In addition, myoblasts were treated with different concentrations of Se in the medium for 72 h. Then the levels of SelW mRNA in skeletal muscles (wing muscle, pectoral muscle, thigh muscle) and myoblasts were determined on days 1, 15, 25, and 35 and at 0, 24, 48, and 72 h, respectively. The results showed that SelW was detected in all these muscle components and it increased both along with the growth of organism and the differentiation process of myoblasts. The thigh muscle is more responsive to Se intake than the other two skeletal muscle tissues while the optimal Se supplementation for SelW mRNA expression in chicken myoblasts was 10(-7) M. In summary, Se plays important roles in the development of chicken skeletal muscles. To effect optimal SelW gene expression, Se must be provided in the diet and the media in adequate amounts and neither at excessive nor deficient levels.

  20. Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology

    International Nuclear Information System (INIS)

    Shafey, Dina; Cote, Patrice D.; Kothary, Rashmi

    2005-01-01

    Dosage of the survival motor neuron (SMN) protein has been directly correlated with the severity of disease in patients diagnosed with spinal muscular atrophy (SMA). It is also clear that SMA is a neurodegenerative disorder characterized by the degeneration of the α-motor neurons in the anterior horn of the spinal cord and atrophy of the associated skeletal muscle. What is more controversial is whether it is neuronal and/or muscle-cell-autonomous defects that are responsible for the disease per se. Although motor neuron degeneration is generally accepted as the primary event in SMA, intrinsic muscle defects in this disease have not been ruled out. To gain a better understanding of the influence of SMN protein dosage in muscle, we have generated a hypomorphic series of myoblast (C2C12) stable cell lines with variable Smn knockdown. We show that depletion of Smn in these cells resulted in a decrease in the number of nuclear 'gems' (gemini of coiled bodies), reduced proliferation with no increase in cell death, defects in myoblast fusion, and malformed myotubes. Importantly, the severity of these abnormalities is directly correlated with the decrease in Smn dosage. Taken together, our work supports the view that there is an intrinsic defect in skeletal muscle cells of SMA patients and that this defect contributes to the overall pathogenesis in this devastating disease

  1. Combined use of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and platelet rich plasma (PRP) stimulates proliferation and differentiation of myoblasts in vitro: new therapeutic perspectives for skeletal muscle repair/regeneration.

    Science.gov (United States)

    Sassoli, Chiara; Vallone, Larissa; Tani, Alessia; Chellini, Flaminia; Nosi, Daniele; Zecchi-Orlandini, Sandra

    2018-02-05

    Satellite cell-mediated skeletal muscle repair/regeneration is compromised in cases of extended damage. Bone marrow mesenchymal stromal cells (BM-MSCs) hold promise for muscle healing but some criticisms hamper their clinical application, including the need to avoid animal serum contamination for expansion and the scarce survival after transplant. In this context, platelet-rich plasma (PRP) could offer advantages. Here, we compare the effects of PRP or standard culture media on C2C12 myoblast, satellite cell and BM-MSC viability, survival, proliferation and myogenic differentiation and evaluate PRP/BM-MSC combination effects in promoting myogenic differentiation. PRP induced an increase of mitochondrial activity and Ki67 expression comparable or even greater than that elicited by standard media and promoted AKT signaling activation in myoblasts and BM-MSCs and Notch-1 pathway activation in BM-MSCs. It stimulated MyoD, myogenin, α-sarcomeric actin and MMP-2 expression in myoblasts and satellite cell activation. Notably, PRP/BM-MSC combination was more effective than PRP alone. We found that BM-MSCs influenced myoblast responses through a paracrine activation of AKT signaling, contributing to shed light on BM-MSC action mechanisms. Our results suggest that PRP represents a good serum substitute for BM-MSC manipulation in vitro and could be beneficial towards transplanted cells in vivo. Moreover, it might influence muscle resident progenitors' fate, thus favoring the endogenous repair/regeneration mechanisms. Finally, within the limitations of an in vitro experimentation, this study provides an experimental background for considering the PRP/BM-MSC combination as a potential therapeutic tool for skeletal muscle damage, combining the beneficial effects of BM-MSCs and PRP on muscle tissue, while potentiating BM-MSC functionality.

  2. Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities.

    Science.gov (United States)

    Pavlath, G K; Thaloor, D; Rando, T A; Cheong, M; English, A W; Zheng, B

    1998-08-01

    Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.

  3. Differentiation-Associated Downregulation of Poly(ADP-Ribose Polymerase-1 Expression in Myoblasts Serves to Increase Their Resistance to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Gábor Oláh

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP-1, the major isoform of the poly (ADP-ribose polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose (PAR groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12 and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6. Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant

  4. Effect of ionizing radiation on human skeletal muscle precursor cells

    International Nuclear Information System (INIS)

    Jurdana, Mihaela; Cemazar, Maja; Pegan, Katarina; Mars, Tomaz

    2013-01-01

    Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures. Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin – 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death. Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70). Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions

  5. Sphingosine kinase/sphingosine 1-phosphate axis: a new player for insulin-like growth factor-1-induced myoblast differentiation

    Directory of Open Access Journals (Sweden)

    Bernacchioni Caterina

    2012-07-01

    Full Text Available Abstract Background Insulin-like growth factor-1 (IGF-1 is the most important physiological regulator of skeletal muscle progenitor cells, which are responsible for adult skeletal muscle regeneration. The ability of IGF-1 to affect multiple aspects of skeletal muscle cell biology such as proliferation, differentiation, survival and motility is well recognized, although the molecular mechanisms implicated in its complex biological action are not fully defined. Since sphingosine 1-phosphate (S1P has recently emerged as a key player in skeletal muscle regeneration, we investigated the possible involvement of the sphingosine kinase (SK/S1P receptor axis on the biological effects of IGF-1 in murine myoblasts. Methods RNA interference, chemical inhibition and immunofluorescence approaches were used to assess the role of the SK/S1P axis on the myogenic and mitogenic effects of IGF-1 in C2C12 myoblasts. Results We show that IGF-1 increases SK activity in mouse myoblasts. The effect of the growth factor does not involve transcriptional regulation of SK1 or SK2, since the protein content of both isoforms is not affected; rather, IGF-1 enhances the fraction of the active form of SK. Moreover, transactivation of the S1P2 receptor induced by IGF-1 via SK activation appears to be involved in the myogenic effect of the growth factor. Indeed, the pro-differentiating effect of IGF-1 in myoblasts is impaired when SK activity is pharmacologically inhibited, or SK1 or SK2 are specifically silenced, or the S1P2 receptor is downregulated. Furthermore, in this study we show that IGF-1 transactivates S1P1/S1P3 receptors via SK activation and that this molecular event negatively regulates the mitogenic effect elicited by the growth factor, since the specific silencing of S1P1 or S1P3 receptors increases cell proliferation induced by IGF-1. Conclusions We demonstrate a dual role of the SK/S1P axis in response to myoblast challenge with IGF-1, that likely is important to

  6. Spatial and functional restriction of regulatory molecules during mammalian myoblast fusion

    International Nuclear Information System (INIS)

    Pavlath, Grace K.

    2010-01-01

    Myoblast fusion is a highly regulated process that is key for forming skeletal muscle during development and regeneration in mammals. Much remains to be understood about the molecular regulation of myoblast fusion. Some molecules that influence mammalian muscle fusion display specific cellular localization during myogenesis. Such molecules can be localized to the contact region between two fusing cells either in both cells or only in one of the cells. How distinct localization of molecules contributes to fusion is not clear. Further complexity exists as other molecules are functionally restricted to myoblasts at later stages of myogenesis to regulate their fusion with multinucleated myotubes. This review examines these three categories of molecules and discusses how spatial and functional restriction may contribute to the formation of a multinucleated cell. Understanding how and why molecules become restricted in location or function is likely to provide further insights into the mechanisms regulating mammalian muscle fusion.

  7. Short-hairpin Mediated Myostatin Knockdown Resulted in Altered Expression of Myogenic Regulatory Factors with Enhanced Myoblast Proliferation in Fetal Myoblast Cells of Goats.

    Science.gov (United States)

    Kumar, Rohit; Singh, Satyendra Pal; Mitra, Abhijit

    2018-01-02

    Myostatin (MSTN) is a well-known negative regulator of skeletal muscle development. Reduced expression due to natural mutations in the coding region and knockout as well as knockdown of MSTN results in an increase in the muscle mass. In the present study, we demonstrated as high as 60 and 52% downregulation (p < 0.01) of MSTN mRNA and protein in the primary fetal myoblast cells of goats using synthetic shRNAs (n = 3), without any interferon response. We, for the first time, evaluated the effect of MSTN knockdown on the expression of MRFs (namely, MyoD, Myf5), follistatin (FST), and IGFs (IGF-1 & IGF-2) in goat myoblast cells. MSTN knockdown caused an upregulation (p < 0.05) of MyoD and downregulation (p < 0.01) of MYf5 and FST expression. Moreover, we report up to ∼four fold (p < 0.001) enhanced proliferation in myoblasts after four days of culture. The anti-MSTN shRNA demonstrated in the present study could be used for the production of transgenic goats to increase the muscle mass.

  8. Perturbations of NAD+ salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle

    DEFF Research Database (Denmark)

    Andersen, Marianne Agerholm; Dall, Morten; Jensen, Benjamin Anderschou Holbech

    2018-01-01

    Nicotinamide adenine dinucleotide (NAD+) can be synthesized by nicotinamide phosphoribosyltransferase (NAMPT). We aimed to determine the role of NAMPT for maintaining NAD+ levels, mitochondrial function, and metabolic homeostasis in skeletal muscle cells. We generated stable Nampt knockdown (sh......Nampt KD) C2C12 cells using a shRNA lentiviral approach. Moreover, we applied gene electrotransfer to express cre recombinase in tibialis anterior muscle of floxed Nampt mice. In shNampt KD C2C12 myoblasts, Nampt and NAD+ levels were reduced by 70% and 50%, respectively, and maximal respiratory capacity...... was reduced by 25%. Moreover, anaerobic glycolytic flux increased by 55% and 2-deoxyglucose uptake increased by 25% in shNampt KD cells. Treatment with the NAD+ precursor nicotinamide riboside restored NAD+ levels in shNampt cells and increased maximal respiratory capacity by 18% and 32% in control and sh...

  9. Let-7b Regulates Myoblast Proliferation by Inhibiting IGF2BP3 Expression in Dwarf and Normal Chicken

    Science.gov (United States)

    Lin, Shumao; Luo, Wen; Ye, Yaqiong; Bekele, Endashaw J.; Nie, Qinghua; Li, Yugu; Zhang, Xiquan

    2017-01-01

    The sex-linked dwarf chicken is caused by the mutation of growth hormone receptor (GHR) gene and characterized by shorter shanks, lower body weight, smaller muscle fiber diameter and fewer muscle fiber number. However, the precise regulatory pathways that lead to the inhibition of skeletal muscle growth in dwarf chickens still remain unclear. Here we found a let-7b mediated pathway might play important role in the regulation of dwarf chicken skeletal muscle growth. Let-7b has higher expression in the skeletal muscle of dwarf chicken than in normal chicken, and the expression of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which is a translational activator of IGF2, showed opposite expression trend to let-7b. In vitro cellular assays validated that let-7b directly inhibits IGF2BP3 expression through binding to its 3′UTR region, and the protein level but not mRNA level of IGF2 would be reduced in let-7b overexpressed chicken myoblast. Let-7b can inhibit cell proliferation and induce cell cycle arrest in chicken myoblast through let-7b-IGF2BP3-IGF2 signaling pathway. Additionally, let-7b can also regulate skeletal muscle growth through let-7b-GHR-GHR downstream genes pathway, but this pathway is non-existent in dwarf chicken because of the deletion mutation of GHR 3′UTR. Notably, as the loss binding site of GHR for let-7b, let-7b has enhanced its binding and inhibition on IGF2BP3 in dwarf myoblast, suggesting that the miRNA can balance its inhibiting effect through dynamic regulate its binding to target genes. Collectively, these results not only indicate that let-7b can inhibit skeletal muscle growth through let-7b-IGF2BP3-IGF2 signaling pathway, but also show that let-7b regulates myoblast proliferation by inhibiting IGF2BP3 expression in dwarf and normal chickens. PMID:28736533

  10. The critical role of myostatin in differentiation of sheep myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chenxi [College of Life Science and Technology, Xinjiang University, Urumqi (China); Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Li, Wenrong; Zhang, Xuemei; Zhang, Ning; He, Sangang; Huang, Juncheng [Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Laboratory of Grass-fed Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Urumqi (China); Animal Biotechnological Research Center, Xinjiang Academy of Animal Science, Urumqi (China); Ge, Yubin [The State Engineering Laboratory of AIDS Vaccine, College of Life Science, Jilin University, Changchun (China); Liu, Mingjun, E-mail: xjlmj2004@yahoo.com.cn [Xinjiang Laboratory of Animal Biotechnology, Urumqi (China); Laboratory of Grass-fed Animal Genetics, Breeding and Reproduction of Ministry of Agriculture, Urumqi (China); Animal Biotechnological Research Center, Xinjiang Academy of Animal Science, Urumqi (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Identification of the effective and specific shRNA to knockdown MSTN. Black-Right-Pointing-Pointer Overexpression of MSTN reversibly suppressed myogenic differentiation. Black-Right-Pointing-Pointer shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. Black-Right-Pointing-Pointer MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. Black-Right-Pointing-Pointer Provides a promise for the generation of transgenic sheep to improve meat productivity. -- Abstract: Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

  11. The critical role of myostatin in differentiation of sheep myoblasts

    International Nuclear Information System (INIS)

    Liu, Chenxi; Li, Wenrong; Zhang, Xuemei; Zhang, Ning; He, Sangang; Huang, Juncheng; Ge, Yubin; Liu, Mingjun

    2012-01-01

    Highlights: ► Identification of the effective and specific shRNA to knockdown MSTN. ► Overexpression of MSTN reversibly suppressed myogenic differentiation. ► shRNA knockdown of endogenous MSTN promoted ovine myoblast differentiation. ► MSTN inhibits myogenic differentiation through down-regulation of MyoD and Myogenin and up-regulation of Smad3. ► Provides a promise for the generation of transgenic sheep to improve meat productivity. -- Abstract: Myostatin [MSTN, also known as growth differentiation factor 8 (GDF8)], is an inhibitor of skeletal muscle growth. Blockade of MSTN function has been reported to result in increased muscle mass in mice. However, its role in myoblast differentiation in farm animals has not been determined. In the present study, we sought to determine the role of MSTN in the differentiation of primary sheep myoblasts. We found that ectopic overexpression of MSTN resulted in lower fusion index in sheep myoblasts, which indicated the repression of myoblast differentiation. This phenotypic change was reversed by shRNA knockdown of the ectopically expressed MSTN in the cells. In contrast, shRNA knockdown of the endogenous MSTN resulted in induction of myogenic differentiation. Additional studies revealed that the induction of differentiation by knocking down the ectopically or endogenously expressed MSTN was accompanied by up-regulation of MyoD and myogenin, and down-regulation of Smad3. Our results demonstrate that MSTN plays critical role in myoblast differentiation in sheep, analogous to that in mice. This study also suggests that shRNA knockdown of MSTN could be a potentially promising approach to improve sheep muscle growth, so as to increase meat productivity.

  12. MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis.

    Science.gov (United States)

    Tagawa, Masashi; Ueyama, Tomomi; Ogata, Takehiro; Takehara, Naofumi; Nakajima, Norio; Isodono, Koji; Asada, Satoshi; Takahashi, Tomosaburo; Matsubara, Hiroaki; Oh, Hidemasa

    2008-08-01

    Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.

  13. Skeletal Myoblast Cell Sheet Implantation Ameliorates Both Systolic and Diastolic Cardiac Performance in Canine Dilated Cardiomyopathy Model.

    Science.gov (United States)

    Shirasaka, Tomonori; Miyagawa, Shigeru; Fukushima, Satsuki; Kawaguchi, Naomasa; Nakatani, Satoshi; Daimon, Takashi; Okita, Yutaka; Sawa, Yoshiki

    2016-02-01

    Improving both systolic and diastolic function may be the most important factor in treating heart failure. In this study, we hypothesized that cell-sheet transplantation could improve these function in the damaged heart. We generated a dilated cardiomyopathy model in beagles by continuous ventricle pacing at 240 beats per minute. After 4 weeks, the beagles underwent skeletal myoblast cell sheet transplantation (SMCST) or a sham operation, and rapid ventricle pacing continued for an additional 4 weeks. Six of the e8 beagles treated by SMCST were still alive 4 weeks after the procedure. We evaluated SMCST's cardiotherapeutic effects by comparing beagles treated by SMCST with beagles that underwent a sham operation (control, n = 5). Diastolic function, as well as systolic function improved significantly in the SMCST group as compared with the sham group (control vs SMCST group, median [interquartile range]: E/E', 16 [0.9] vs 11 [1.0]; P dilated cardiomyopathy heart.

  14. β‐Taxilin participates in differentiation of C2C12 myoblasts into myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Sakane, Hiroshi; Makiyama, Tomohiko; Nogami, Satoru; Horii, Yukimi [Department of Molecular and Cell Biology, Graduate school of Medicine, Dokkyo Medical University, 880 Kitakobayashi, Mibu-town, Tochigi 321-0293 (Japan); Akasaki, Kenji [Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292 (Japan); Shirataki, Hiromichi, E-mail: hiro-sh@dokkyomed.ac.jp [Department of Molecular and Cell Biology, Graduate school of Medicine, Dokkyo Medical University, 880 Kitakobayashi, Mibu-town, Tochigi 321-0293 (Japan)

    2016-07-15

    Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that β-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of β-taxilin in myogenesis. In C2C12 cells, knockdown of β-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also found that β-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of β-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that β-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes. - Highlights: • β‐Taxilin is progressively expressed during differentiation of C2C12 cell. • Knockdown of β-taxilin impaired C2C12 myotube formation. • β‐Taxilin interacted with dysbindin. • Knockdown of dysbindin promoted C2C12 myotube formation. • The function of β-taxilin in C2C12 myotube formation depends on dysbindin.

  15. Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

    Directory of Open Access Journals (Sweden)

    Côté Claude H

    2011-10-01

    Full Text Available Abstract Background Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2. The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade. Methods Proliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF2α production. Proliferation assays were also performed in presence of different prostaglandins (PGs. Results Tryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor in vivo in skeletal muscle cells and in satellite cells and in vitro in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ-12,14-prostaglandin J2 (15Δ-PGJ2, a product of COX-2-derived prostaglandin D2, stimulated myoblast proliferation, but not PGE2 and PGF2α. Conclusions Taken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream

  16. Macrophages improve survival, proliferation and migration of engrafted myogenic precursor cells into MDX skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Pierre-François Lesault

    Full Text Available Transplantation of muscle precursor cells is of therapeutic interest for focal skeletal muscular diseases. However, major limitations of cell transplantation are the poor survival, expansion and migration of the injected cells. The massive and early death of transplanted myoblasts is not fully understood although several mechanisms have been suggested. Various attempts have been made to improve their survival or migration. Taking into account that muscle regeneration is associated with the presence of macrophages, which are helpful in repairing the muscle by both cleansing the debris and deliver trophic cues to myoblasts in a sequential way, we attempted in the present work to improve myoblast transplantation by coinjecting macrophages. The present data showed that in the 5 days following the transplantation, macrophages efficiently improved: i myoblast survival by limiting their massive death, ii myoblast expansion within the tissue and iii myoblast migration in the dystrophic muscle. This was confirmed by in vitro analyses showing that macrophages stimulated myoblast adhesion and migration. As a result, myoblast contribution to regenerating host myofibres was increased by macrophages one month after transplantation. Altogether, these data demonstrate that macrophages are beneficial during the early steps of myoblast transplantation into skeletal muscle, showing that coinjecting these stromal cells may be used as a helper to improve the efficiency of parenchymal cell engraftment.

  17. Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts.

    Science.gov (United States)

    Sin, Jon; Andres, Allen M; Taylor, David J R; Weston, Thomas; Hiraumi, Yoshimi; Stotland, Aleksandr; Kim, Brandon J; Huang, Chengqun; Doran, Kelly S; Gottlieb, Roberta A

    2016-01-01

    Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

  18. Myoblast replication is reduced in the IUGR fetus despite maintained proliferative capacity in vitro.

    Science.gov (United States)

    Soto, Susan M; Blake, Amy C; Wesolowski, Stephanie R; Rozance, Paul J; Barthel, Kristen B; Gao, Bifeng; Hetrick, Byron; McCurdy, Carrie E; Garza, Natalia G; Hay, William W; Leinwand, Leslie A; Friedman, Jacob E; Brown, Laura D

    2017-03-01

    Adults who were affected by intrauterine growth restriction (IUGR) suffer from reductions in muscle mass and insulin resistance, suggesting muscle growth may be restricted by molecular events that occur during fetal development. To explore the basis of restricted fetal muscle growth, we used a sheep model of progressive placental insufficiency-induced IUGR to assess myoblast proliferation within intact skeletal muscle in vivo and isolated myoblasts stimulated with insulin in vitro Gastrocnemius and soleus muscle weights were reduced by 25% in IUGR fetuses compared to those in controls (CON). The ratio of PAX7+ nuclei (a marker of myoblasts) to total nuclei was maintained in IUGR muscle compared to CON, but the fraction of PAX7+ myoblasts that also expressed Ki-67 (a marker of cellular proliferation) was reduced by 23%. Despite reduced proliferation in vivo, fetal myoblasts isolated from IUGR biceps femoris and cultured in enriched media in vitro responded robustly to insulin in a dose- and time-dependent manner to increase proliferation. Similarly, insulin stimulation of IUGR myoblasts upregulated key cell cycle genes and DNA replication. There were no differences in the expression of myogenic regulatory transcription factors that drive commitment to muscle differentiation between CON and IUGR groups. These results demonstrate that the molecular machinery necessary for transcriptional control of proliferation remains intact in IUGR fetal myoblasts, indicating that in vivo factors such as reduced insulin and IGF1, hypoxia and/or elevated counter-regulatory hormones may be inhibiting muscle growth in IUGR fetuses. © 2017 Society for Endocrinology.

  19. A Simplified Method for Tissue Engineering Skeletal Muscle Organoids in Vitro

    Science.gov (United States)

    Shansky, Janet; DelTatto, Michael; Chromiak, Joseph; Vandenburgh, Herman

    1996-01-01

    Tissue-engineered three dimensional skeletal muscle organ-like structures have been formed in vitro from primary myoblasts by several different techniques. This report describes a simplified method for generating large numbers of muscle organoids from either primary embryonic avian or neonatal rodent myoblasts, which avoids the requirements for stretching and other mechanical stimulation.

  20. Dynamics of the Skeletal Muscle Secretome during Myoblast Differentiation

    DEFF Research Database (Denmark)

    Henningsen, Jeanette; Rigbolt, Kristoffer T G; Blagoev, Blagoy

    2010-01-01

    During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative...... proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells. Using triple encoding stable isotope labeling by amino acids in cell culture, we compared the secretomes at three different time points of muscle differentiation and followed the dynamics...... of the skeletal muscle as a prominent secretory organ. In addition to previously reported molecules, we identified many secreted proteins that have not previously been shown to be released from skeletal muscle cells nor shown to be differentially released during the process of myogenesis. We found 188...

  1. Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

    Science.gov (United States)

    Powell, C.; Shansky, J.; Del Tatto, M.; Forman, D. E.; Hennessey, J.; Sullivan, K.; Zielinski, B. A.; Vandenburgh, H. H.

    1999-01-01

    Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.

  2. Enhancement of contractile force generation of artificial skeletal muscle tissues by mild and transient heat treatment.

    Science.gov (United States)

    Sato, Masanori; Ikeda, Kazushi; Kanno, Shota; Ito, Akira; Kawabe, Yoshinori; Kamihira, Masamichi

    2014-01-01

    Artificial skeletal muscle tissues composed of cells are expected to be used for applications of regenerative medicine and drug screening. Generally, however, the physical forces generated by tissue-engineered skeletal muscle are lower than those of skeletal muscle tissues found in the body. Local hyperthermia is used for many diseases including muscle injuries. It was recently reported that mild heat treatment improved skeletal muscle functions. In this study, we investigated the effects of mild heat treatment on the tissue-engineered skeletal muscle tissues in vitro. We used magnetite cationic liposomes to label C2C12 myoblast cells magnetically, and constructed densely packed artificial skeletal muscle tissues by using magnetic force. Cell culture at 39°C promoted the differentiation of myoblast cells into myotubes. Moreover, the mild and transient heat treatment improved the contractile properties of artificial skeletal muscle tissue constructs. These findings indicate that the culture method using heat treatment is a useful approach to enhance functions of artificial skeletal muscle tissue.

  3. Growth of Limb Muscle is Dependent on Skeletal-Derived Indian Hedgehog

    Science.gov (United States)

    Bren-Mattison, Yvette; Hausburg, Melissa; Olwin, Bradley B.

    2011-01-01

    During embryogenesis, muscle and bone develop in close temporal and spatial proximity. We show that Indian Hedgehog, a bone-derived signaling molecule, participates in growth of skeletal muscle. In Ihh−/− embryos, skeletal muscle development appears abnormal at embryonic day 14.5 and at later ages through embryonic day 20.5, dramatic losses of hindlimb muscle occur. To further examine the role of Ihh in myogenesis, we manipulated Ihh expression in the developing chick hindlimb. Reduction of Ihh in chicken embryo hindlimbs reduced skeletal muscle mass similar to that seen in Ihh−/− mouse embryos. The reduction in muscle mass appears to be a direct effect of Ihh since ectopic expression of Ihh by RCAS retroviral infection of chicken embryo hindlimbs restores muscle mass. These effects are independent of bone length, and occur when Shh is not expressed, suggesting Ihh acts directly on fetal myoblasts to regulate secondary myogenesis. Loss of muscle mass in Ihh null mouse embryos is accompanied by a dramatic increase in myoblast apoptosis accompanied by a loss of p21 protein. Our data suggest that Ihh promotes fetal myoblast survival during their differentiation into secondary myofibers by maintaining p21 protein levels. PMID:21683695

  4. Possible role of TIEG1 as a feedback regulator of myostatin and TGF-{beta} in myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan); Yamaguchi, Takahiro, E-mail: ty1010@bios.tohoku.ac.jp [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan)

    2010-03-19

    Myostatin and TGF-{beta} negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-{beta} signaling remains unclear. TGF-{beta} inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-{beta} signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-{beta} signaling using C2C12 myoblasts. Myostatin and TGF-{beta} induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-{beta} enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-{beta} in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-{beta}. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-{beta} that prevents excess action in myoblasts.

  5. Possible role of TIEG1 as a feedback regulator of myostatin and TGF-β in myoblasts

    International Nuclear Information System (INIS)

    Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi; Yamaguchi, Takahiro

    2010-01-01

    Myostatin and TGF-β negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-β signaling remains unclear. TGF-β inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-β signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-β signaling using C2C12 myoblasts. Myostatin and TGF-β induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-β enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-β in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-β. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-β that prevents excess action in myoblasts.

  6. Injectable scaffold materials differ in their cell instructive effects on primary human myoblasts

    DEFF Research Database (Denmark)

    Hejbøl, Eva Kildall; Sellathurai, Jeeva; Nair, Prabha Damodaran

    2017-01-01

    Scaffolds are materials used for delivery of cells for regeneration of tissues. They support three-dimensional organization and improve cell survival. For the repair of small skeletal muscles, injections of small volumes of cells are attractive, and injectable scaffolds for delivery of cells offer...... a minimally invasive technique. In this study, we examined in vitro the cell instructive effects of three types of injectable scaffolds, fibrin, alginate, and poly(lactic-co-glycolic acid)-based microparticles on primary human myoblasts. The myoblast morphology and progression in the myogenic program differed......, depending on the type of scaffold material. In alginate gel, the cells obtained a round morphology, they ceased to proliferate, and entered quiescence. In the fibrin gels, differentiation was promoted, and myotubes were observed within a few days in culture, while poly(lactic-co-glycolic acid...

  7. mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Pengpeng [Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Liang, Xinrong; Shan, Tizhong [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); Jiang, Qinyang [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States); College of Animal Science and Technology, Guangxi University, Nanning 530004 (China); Deng, Changyan [Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Zheng, Rong, E-mail: zhengrong@mail.hzau.edu.cn [Key Laboratory of Swine Genetics and Breeding of Agricultural Ministry & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Kuang, Shihuan, E-mail: skuang@purdue.edu [Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 (United States)

    2015-07-17

    The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7{sup CreER} and Mtor{sup flox/flox} mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes. - Highlights: • Pax7{sup CreER} was used to delete Mtor gene in satellite cells. • Satellite cell specific deletion of Mtor impairs muscle regeneration. • mTOR is necessary for satellite cell proliferation and differentiation. • Deletion of Mtor leads to reduced expression of key myogenic genes.

  8. mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration

    International Nuclear Information System (INIS)

    Zhang, Pengpeng; Liang, Xinrong; Shan, Tizhong; Jiang, Qinyang; Deng, Changyan; Zheng, Rong; Kuang, Shihuan

    2015-01-01

    The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7 CreER and Mtor flox/flox mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes. - Highlights: • Pax7 CreER was used to delete Mtor gene in satellite cells. • Satellite cell specific deletion of Mtor impairs muscle regeneration. • mTOR is necessary for satellite cell proliferation and differentiation. • Deletion of Mtor leads to reduced expression of key myogenic genes

  9. Chiral Orientation of Skeletal Muscle Cells Requires Rigid Substrate

    Directory of Open Access Journals (Sweden)

    Ninghao Zhu

    2017-06-01

    Full Text Available Reconstitution of tissue morphology with inherent left–right (LR asymmetry is essential for tissue/organ functions. For skeletal muscle, the largest tissue in mammalian organisms, successful myogenesis requires the regulation of the LR asymmetry to form the appropriate muscle alignment. However, the key factor for reproducing the LR asymmetry of skeletal tissues in a controllable, engineering context remains largely unknown. Recent reports indicate that cell chirality may underlie the LR development in tissue morphogenesis. Here, we report that a rigid substrate is required for the chirality of skeletal muscle cells. By using alternating micropatterned cell-adherent and cell-repellent stripes on a rigid substrate, we found that C2C12 skeletal muscle myoblasts exhibited a unidirectional tilted orientation with respect to the stripe boundary. Importantly, such chiral orientation was reduced when soft substrates were used instead. In addition, we demonstrated the key role of actin stress fibers in the formation of the chiral orientation. This study reveals that a rigid substrate is required for the chiral pattern of myoblasts, paving the way for reconstructing damaged muscle tissue with inherent LR asymmetry in the future.

  10. Expression of androgen receptor target genes in skeletal muscle

    Directory of Open Access Journals (Sweden)

    Kesha Rana

    2014-10-01

    Full Text Available We aimed to determine the mechanisms of the anabolic actions of androgens in skeletal muscle by investigating potential androgen receptor (AR-regulated genes in in vitro and in vivo models. The expression of the myogenic regulatory factor myogenin was significantly decreased in skeletal muscle from testosterone-treated orchidectomized male mice compared to control orchidectomized males, and was increased in muscle from male AR knockout mice that lacked DNA binding activity (ARΔZF2 versus wildtype mice, demonstrating that myogenin is repressed by the androgen/AR pathway. The ubiquitin ligase Fbxo32 was repressed by 12 h dihydrotestosterone treatment in human skeletal muscle cell myoblasts, and c-Myc expression was decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle, and increased in AR∆ZF2 muscle. The expression of a group of genes that regulate the transition from myoblast proliferation to differentiation, Tceal7 , p57 Kip2, Igf2 and calcineurin Aa, was increased in AR∆ZF2 muscle, and the expression of all but p57 Kip2 was also decreased in testosterone-treated orchidectomized male muscle compared to control orchidectomized male muscle. We conclude that in males, androgens act via the AR in part to promote peak muscle mass by maintaining myoblasts in the proliferative state and delaying the transition to differentiation during muscle growth and development, and by suppressing ubiquitin ligase-mediated atrophy pathways to preserve muscle mass in adult muscle.

  11. Impact of Hepatocyte Growth Factor on Skeletal Myoblast Transplantation Late after Myocardial Infarction

    Directory of Open Access Journals (Sweden)

    Stacy B. O'blenes

    2013-01-01

    Full Text Available In clinical studies, skeletal myoblast (SKMB transplantation late after myocardial infarction (MI has minimal impact on left ventricular (LV function. This may be related to our previous observation that the extent of SKMB engraftment is minimal in chronic MI when compared to acute MI, which correlates with decreased hepatocyte growth factor (HGF expression, an important regulator of SKMB function. Here, we investigated delivery of exogenous HGF as a strategy for augmenting SKMB engraftment late after MI. Rats underwent SKMB transplantation 4 weeks after coronary ligation. HGF or vehicle control was delivered intravenously during the subsequent 2 weeks. LV function was assessed by MRI before and 2 weeks after SKMB transplantation. We evaluated HGF delivery, SKMB engraftment, and expression of genes associated with post-MI remodeling. Serum HGF was 6.2 ± 2.4 ng/mL after 2 weeks of HGF infusion (n = 7, but undetectable in controls (n = 7. LV end-diastolic volume and ejection fraction did not improve with HGF treatment (321 ± 27 mm 3 , 42% ± 2% vs. 285 ± 33 mm 3 , 43% ± 2%, HGF vs. control. MIs were larger in HGF-treated animals (50 ± 7 vs. 30 ± 6 mm 3 , P = 0.046, but the volume of engrafted SKMBs or percentage of MIs occupied by SKMBs did not increase with HGF (1.7 ± 0.3 mm 3 , 4.7% ± 1.9% vs. 1.4 ± 0.4 mm 3 , 5.3% ± 1.6%, HGF vs. control. Expression of genes associated with post-infarction remodeling was not altered by HGF. Delivery of exogenous HGF failed to augment SKMB engraftment and functional recovery in chronic MI. Expression of genes associated with LV remodeling was not altered by HGF. Alternative strategies to enhance engraftment of SKMB must be explored to optimize the clinical efficacy of SKMB transplantation.

  12. Identification of gene expression modifications in myostatin-stimulated myoblasts

    International Nuclear Information System (INIS)

    Yang Wei; Zhang Yong; Ma Guoda; Zhao Xinyi; Chen Yan; Zhu Dahai

    2005-01-01

    Myostatin belongs to the transforming growth factor beta superfamily and has been shown to function as an inhibitor of skeletal muscle proliferation and differentiation. To gain insight into the molecular mechanisms of myostatin function during myogenesis, differential display reverse transcription PCR was employed to identify altered gene expressions associated with myostatin inhibitory function in chicken fetal myoblasts (CFMs). In this work, we have identified seven up-regulated and 12 down-regulated genes in myostatin stimulated CFMs. Those genes are involved in myogenic differentiation, cell architecture, energy metabolism, signal transduction, and apoptosis. The down-regulation of muscle creatine kinase B, troponin C, and myosin regulatory light chain is in agreement with the myostatin negative role in myocyte differentiation. In addition, the expression alteration of skeletal muscle-specific cardiac ankyrin repeat protein and the bcl-2 related anti-apoptotic protein Nr-13 suggests possible unique roles for myostatin in regulating myogenesis by controlling cofactors participated transcriptional regulation and apoptosis

  13. Cardiac cell therapy: overexpression of connexin43 in skeletal myoblasts and prevention of ventricular arrhythmias

    NARCIS (Netherlands)

    Fernandes, Sarah; van Rijen, Harold V. M.; Forest, Virginie; Evain, Stéphane; Leblond, Anne-Laure; Mérot, Jean; Charpentier, Flavien; de Bakker, Jacques M. T.; Lemarchand, Patricia

    2009-01-01

    Cell-based therapies have great potential for the treatment of cardiovascular diseases. Recently, using a transgenic mouse model Roell et al. reported that cardiac engraftment of connexin43 (Cx43)-overexpressing myoblasts in vivo prevents post-infarct arrhythmia, a common cause of death in patients

  14. Liposome-based vascular endothelial growth factor-165 transfection with skeletal myoblast for treatment of ischaemic limb disease.

    Science.gov (United States)

    Ye, Lei; Haider, Husnain Kh; Esa, Wahidah Bte; Su, Liping; Law, Peter K; Zhang, Wei; Lim, Yeanteng; Poh, Kian Keong; Sim, Eugene K W

    2010-01-01

    The study aims to use cholesterol (Chol) + DOTAP liposome (CD liposome) based human vascular endothelial growth factor-165 (VEGF(165)) gene transfer into skeletal myoblasts (SkMs) for treatment of acute hind limb ischaemia in a rabbit model. The feasibility and efficacy of CD liposome mediated gene transfer with rabbit SkMs were characterized using plasmid carrying enhanced green fluorescent protein (pEGFP) and assessed by flow cytometry. After optimization, SkMs were transfected with CD lipoplexes carrying plasmid-VEGF(165) (CD-pVEGF(165)) and transplanted into rabbit ischaemic limb. Animals were randomized to receive intramuscular injection of Medium199 (M199; group 1), non-transfected SkM (group 2) or CD-pVEGF(165) transfected SkM (group 3). Flow cytometry revealed that up to 16% rabbit SkMs were successfully transfected with pEGFP. Based on the optimized transfection condition, transfected rabbit SkM expressed VEGF(165) up to day 18 with peak at day 2. SkMs were observed in all cell-transplanted groups, as visualized with 6-diamidino-2-phenylindole and bromodeoxyuridine. Angiographic blood vessel score revealed increased collateral vessel development in group 3 (39.7 +/- 2.0) compared with group 2 (21.6 +/- 1.1%, P limb and may serve as a safe and new therapeutic modality for the repair of acute ischaemic limb disease.

  15. α-linolenic acid reduces TNF-induced apoptosis in C2C12 myoblasts by regulating expression of apoptotic proteins

    Directory of Open Access Journals (Sweden)

    Felicia Carotenuto

    2016-11-01

    Full Text Available Impaired regeneration and consequent muscle wasting is a major feature of muscle degenerative diseases. Nutritional interventions as adjuvant strategy for preventing such conditions are recently gaining increasing attention. Ingestion of n3-polyunsaturated fatty acids has been suggested to have a positive impact on muscle diseases. We recently demonstrated that the dietary n3-fatty acid, alpha-linolenic acid (ALA, exerts potent beneficial effects in preserving skeletal muscle regeneration in models of muscle dystrophy. To better elucidate the underlying mechanism we investigate here on the expression level of the anti- and pro-apototic proteins, as well as caspase-3 activity, in C2C12 myoblasts challenged with pathological levels of TNF. The results demonstrated that ALA protective effect on C2C12 myoblasts was associated to an increased Bcl-2/Bax ratio. Indeed, the effect of ALA was directed to rescue Bcl-2 expression and decrease Bax expression both affected in an opposite way by TNF treatment. This effect was associated with a decrease in caspase-3 activity by ALA. TNF is a major pro-inflammatory cytokine that is expressed in damaged skeletal muscle, therefore, counteract inflammatory signals in the muscle microenvironment represents a critical strategy to ameliorate skeletal muscle pathologies

  16. Comparison of arrhythmogenicity and proinflammatory activity induced by intramyocardial or epicardial myoblast sheet delivery in a rat model of ischemic heart failure.

    Directory of Open Access Journals (Sweden)

    Tommi Pätilä

    route may serve as the preferred method of skeletal myoblast transplantation to treat heart failure.

  17. Electrotransfection and lipofection show comparable efficiency for in vitro gene delivery of primary human myoblasts.

    Science.gov (United States)

    Mars, Tomaz; Strazisar, Marusa; Mis, Katarina; Kotnik, Nejc; Pegan, Katarina; Lojk, Jasna; Grubic, Zoran; Pavlin, Mojca

    2015-04-01

    Transfection of primary human myoblasts offers the possibility to study mechanisms that are important for muscle regeneration and gene therapy of muscle disease. Cultured human myoblasts were selected here because muscle cells still proliferate at this developmental stage, which might have several advantages in gene therapy. Gene therapy is one of the most sought-after tools in modern medicine. Its progress is, however, limited due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of the presently used techniques, two non-viral transfection methods--lipofection and electroporation--were compared. The parameters that can influence transfection efficiency and cell viability were systematically approached and compared. Cultured myoblasts were transfected with the pEGFP-N1 plasmid either using Lipofectamine 2000 or with electroporation. Various combinations for the preparation of the lipoplexes and the electroporation media, and for the pulsing protocols, were tested and compared. Transfection efficiency and cell viability were inversely proportional for both approaches. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection, while for electroporation, RPMI medium and a pulsing protocol using eight pulses of 2 ms at E = 0.8 kV/cm proved to be the optimal combination. The transfection efficiencies for the optimal lipofection and optimal electrotransfection protocols were similar (32 vs. 32.5%, respectively). Both of these methods are effective for transfection of primary human myoblasts; however, electroporation might be advantageous for in vivo application to skeletal muscle.

  18. Identification of Histone Deacetylase 2 as a Functional Gene for Skeletal Muscle Development in Chickens

    Directory of Open Access Journals (Sweden)

    Md. Shahjahan

    2016-04-01

    Full Text Available A previous genome-wide association study (GWAS exposed histone deacetylase 2 (HDAC2 as a possible candidate gene for breast muscle weight in chickens. The present research has examined the possible role of HDAC2 in skeletal muscle development in chickens. Gene expression was measured by quantitative polymerase chain reaction in breast and thigh muscles during both embryonic (four ages and post-hatch (five ages development and in cultures of primary myoblasts during both proliferation and differentiation. The expression of HDAC2 increased significantly across embryonic days (ED in breast (ED 14, 16, 18, and 21 and thigh (ED 14 and 18, and ED 14 and 21 muscles suggesting that it possibly plays a role in myoblast hyperplasia in both breast and thigh muscles. Transcript abundance of HDAC2 identified significantly higher in fast growing muscle than slow growing in chickens at d 90 of age. Expression of HDAC2 during myoblast proliferation in vitro declined between 24 h and 48 h when expression of the marker gene paired box 7 (PAX7 increased and cell numbers increased throughout 72 h of culture. During induced differentiation of myoblasts to myotubes, the abundance of HDAC2 and the marker gene myogenic differentiation 1 (MYOD1, both increased significantly. Taken together, it is suggested that HDAC2 is most likely involved in a suppressive fashion in myoblast proliferation and may play a positive role in myoblast differentiation. The present results confirm the suggestion that HDAC2 is a functional gene for pre-hatch and post-hatch (fast growing muscle development of chicken skeletal muscle.

  19. Effects of heat stimulation and l-ascorbic acid 2-phosphate supplementation on myogenic differentiation of artificial skeletal muscle tissue constructs.

    Science.gov (United States)

    Ikeda, Kazushi; Ito, Akira; Sato, Masanori; Kanno, Shota; Kawabe, Yoshinori; Kamihira, Masamichi

    2017-05-01

    Although skeletal muscle tissue engineering has been extensively studied, the physical forces produced by tissue-engineered skeletal muscles remain to be improved for potential clinical utility. In this study, we examined the effects of mild heat stimulation and supplementation of a l-ascorbic acid derivative, l-ascorbic acid 2-phosphate (AscP), on myoblast differentiation and physical force generation of tissue-engineered skeletal muscles. Compared with control cultures at 37°C, mouse C2C12 myoblast cells cultured at 39°C enhanced myotube diameter (skeletal muscle hypertrophy), whereas mild heat stimulation did not promote myotube formation (differentiation rate). Conversely, AscP supplementation resulted in an increased differentiation rate but did not induce skeletal muscle hypertrophy. Following combined treatment with mild heat stimulation and AscP supplementation, both skeletal muscle hypertrophy and differentiation rate were enhanced. Moreover, the active tension produced by the tissue-engineered skeletal muscles was improved following combined treatment. These findings indicate that tissue culture using mild heat stimulation and AscP supplementation is a promising approach to enhance the function of tissue-engineered skeletal muscles. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  20. MiRNA-199a-3p Regulates C2C12 Myoblast Differentiation through IGF-1/AKT/mTOR Signal Pathway

    Directory of Open Access Journals (Sweden)

    Long Jia

    2013-12-01

    Full Text Available MicroRNAs constitute a class of ~22-nucleotide non-coding RNAs. They modulate gene expression by associating with the 3' untranslated regions (3' UTRs of messenger RNAs (mRNAs. Although multiple miRNAs are known to be regulated during myoblast differentiation, their individual roles in muscle development are still not fully understood. In this study, we showed that miR-199a-3p was highly expressed in skeletal muscle and was induced during C2C12 myoblasts differentiation. We also identified and confirmed several genes of the IGF-1/AKT/mTOR signal pathway, including IGF-1, mTOR, and RPS6KA6, as important cellular targets of miR-199a-3p in myoblasts. Overexpression of miR-199a-3p partially blocked C2C12 myoblast differentiation and the activation of AKT/mTOR signal pathway, while interference of miR-199a-3p by antisense oligonucleotides promoted C2C12 differentiation and myotube hypertrophy. Thus, our studies have established miR-199a-3p as a potential regulator of myogenesis through the suppression of IGF-1/AKT/mTOR signal pathway.

  1. Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

    Directory of Open Access Journals (Sweden)

    Sindhu Subramaniam

    Full Text Available Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2. Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation. Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for

  2. Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

    Science.gov (United States)

    Subramaniam, Sindhu; Sreenivas, Prethish; Cheedipudi, Sirisha; Reddy, Vatrapu Rami; Shashidhara, Lingadahalli Subrahmanya; Chilukoti, Ravi Kumar; Mylavarapu, Madhavi; Dhawan, Jyotsna

    2014-01-01

    Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological

  3. Lipopolysaccharide inhibits myogenic differentiation of C2C12 myoblasts through the Toll-like receptor 4-nuclear factor-κB signaling pathway and myoblast-derived tumor necrosis factor-α.

    Directory of Open Access Journals (Sweden)

    Yuko Ono

    Full Text Available Circulating lipopolysaccharide (LPS concentrations are often elevated in patients with sepsis or with various endogenous diseases that are associated with metabolic endotoxemia. Involuntary loss of skeletal muscle, termed muscle wasting, is commonly observed in these conditions, suggesting that circulating LPS might play an essential role in its development. Although impairment of muscle regeneration is an important determinant of skeletal muscle wasting, it is unclear whether LPS affects this process and, if so, by what mechanism. Here, we used the C2C12 myoblast cell line to investigate the effects of LPS on myogenesis.C2C12 myoblasts were grown to 80% confluence and induced to differentiate in the absence or presence of LPS (0.1 or 1 μg/mL; TAK-242 (1 μM, a specific inhibitor of Toll-like receptor 4 (TLR4 signaling; and a tumor necrosis factor (TNF-α neutralizing antibody (5 μg/mL. Expression of a skeletal muscle differentiation marker (myosin heavy chain II, two essential myogenic regulatory factors (myogenin and MyoD, and a muscle negative regulatory factor (myostatin was analyzed by western blotting. Nuclear factor-κB (NF-κB DNA-binding activity was measured using an enzyme-linked immunosorbent assay.LPS dose-dependently and significantly decreased the formation of multinucleated myotubes and the expression of myosin heavy chain II, myogenin, and MyoD, and increased NF-κB DNA-binding activity and myostatin expression. The inhibitory effect of LPS on myogenic differentiation was reversible, suggesting that it was not caused by nonspecific toxicity. Both TAK-242 and anti-TNF-α reduced the LPS-induced increase in NF-κB DNA-binding activity, downregulation of myogenic regulatory factors, and upregulation of myostatin, thereby partially rescuing the impairment of myogenesis.Our data suggest that LPS inhibits myogenic differentiation via a TLR4-NF-κB-dependent pathway and an autocrine/paracrine TNF-α-induced pathway. These pathways

  4. Human skeletal muscle drug transporters determine local exposure and toxicity of statins.

    Science.gov (United States)

    Knauer, Michael J; Urquhart, Bradley L; Meyer zu Schwabedissen, Henriette E; Schwarz, Ute I; Lemke, Christopher J; Leake, Brenda F; Kim, Richard B; Tirona, Rommel G

    2010-02-05

    The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro. We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance-associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1. These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.

  5. Uniaxial cyclic strain enhances adipose-derived stem cell fusion with skeletal myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Jens Isak; Juhl, Morten; Nielsen, Thøger; Emmersen, Jeppe; Fink, Trine; Zachar, Vladimir; Pennisi, Cristian Pablo, E-mail: cpennisi@hst.aau.dk

    2014-07-25

    Highlights: • Uniaxial cyclic tensile strain (CTS) applied to ASCs alone or in coculture with myogenic precursors. • CTS promoted the formation of a highly ordered array of parallel ASCs. • Without biochemical supplements, CTS did not support advanced myogenic differentiation of ASCs. • Mechanical stimulation of cocultures boosted fusion of ASCs with skeletal myoblasts. - Abstract: Although adult muscle tissue possesses an exceptional capacity for regeneration, in the case of large defects, the restoration to original state is not possible. A well-known source for the de novo regeneration is the adipose-derived stem cells (ASCs), which can be readily isolated and have been shown to have a broad differentiation and regenerative potential. In this work, we employed uniaxial cyclic tensile strain (CTS), to mechanically stimulate human ASCs to participate in the formation skeletal myotubes in an in vitro model of myogenesis. The application of CTS for 48 h resulted in the formation of a highly ordered array of parallel ASCs, but failed to support skeletal muscle terminal differentiation. When the same stimulation paradigm was applied to cocultures with mouse skeletal muscle myoblasts, the percentage of ASCs contributing to the formation of myotubes significantly exceeded the levels reported in the literature hitherto. In perspective, the mechanical strain may be used to increase the efficiency of incorporation of ASCs in the skeletal muscles, which could be found useful in diverse traumatic or pathologic scenarios.

  6. Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression

    DEFF Research Database (Denmark)

    Grossi, Alberto Blak; Lametsch, Rene; Karlsson, Anders H

    2011-01-01

    Mechanical forces are crucial in the regulation of cell morphology and function. At the cellular level, these forces influence myoblast differentiation and fusion. In this study we applied mechanical stimuli to embryonic muscle cells using magnetic microbeads, a method shown to apply stress...... by mechanical stimulation including Galectin-1, Annexin III, and RhoGDI. In this study we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms...... during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of Galectin-1, in mechanically stimulated myoblasts. A potential involvement of Galectin-1 in myoblast differentiation is presented....

  7. Skeletal muscle atrophy in bioengineered skeletal muscle: a new model system.

    Science.gov (United States)

    Lee, Peter H U; Vandenburgh, Herman H

    2013-10-01

    Skeletal muscle atrophy has been well characterized in various animal models, and while certain pathways that lead to disuse atrophy and its associated functional deficits have been well studied, available drugs to counteract these deficiencies are limited. An ex vivo tissue-engineered skeletal muscle offers a unique opportunity to study skeletal muscle physiology in a controlled in vitro setting. Primary mouse myoblasts isolated from adult muscle were tissue engineered into bioartificial muscles (BAMs) containing hundreds of aligned postmitotic muscle fibers expressing sarcomeric proteins. When electrically stimulated, BAMs generated measureable active forces within 2-3 days of formation. The maximum isometric tetanic force (Po) increased for ∼3 weeks to 2587±502 μN/BAM and was maintained at this level for greater than 80 days. When BAMs were reduced in length by 25% to 50%, muscle atrophy occurred in as little as 6 days. Length reduction resulted in significant decreases in Po (50.4%), mean myofiber cross-sectional area (21.7%), total protein synthesis rate (22.0%), and noncollagenous protein content (6.9%). No significant changes occurred in either the total metabolic activity or protein degradation rates. This study is the first in vitro demonstration that length reduction alone can induce skeletal muscle atrophy, and establishes a novel in vitro model for the study of skeletal muscle atrophy.

  8. Peripheral endocannabinoids regulate skeletal muscle development and maintenance

    Directory of Open Access Journals (Sweden)

    Dongjiao Zhao

    2010-12-01

    Full Text Available As a principal tissue responsible for insulin-mediated glucose uptake, skeletal muscle is important for whole-body health. The role of peripheral endocannabinoids as regulators of skeletal muscle metabolism has recently gained a lot of interest, as endocannabinoid system disorders could cause peripheral insulin resistance. We investigated the role of the peripheral endocannabinoid system in skeletal muscle development and maintenance. Cultures of C2C12 cells, primary satellite cells and mouse skeletal muscle single fibers were used as model systems for our studies. We found an increase in cannabinoid receptor type 1 (CB1 mRNA and endocannabinoid synthetic enzyme mRNA skeletal muscle cells during differentiation. We also found that activation of CB1 inhibited myoblast differentiation, expanded the number of satellite cells, and stimulated the fast-muscle oxidative phenotype. Our findings contribute to understanding of the role of the endocannabinoid system in skeletal muscle metabolism and muscle oxygen consumption, and also help to explain the effects of the peripheral endocannabinoid system on whole-body energy balance.

  9. The role of Na+/K+-ATPase during chick skeletal myogenesis.

    Directory of Open Access Journals (Sweden)

    Taissa Neustadt Oliveira

    Full Text Available The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na+/K+-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na+/K+-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na+/K+-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na+/K+-ATPase.

  10. Nuclear translocation of the cytoskeleton-associated protein, smALP, upon induction of skeletal muscle differentiation

    International Nuclear Information System (INIS)

    Cambier, Linda; Pomies, Pascal

    2011-01-01

    Highlights: → The cytoskeleton-associated protein, smALP, is expressed in differentiated skeletal muscle. → smALP is translocated from the cytoplasm to the nucleus of C2C12 myoblasts upon induction of myogenesis. → The differentiation-dependent nuclear translocation of smALP occurs in parallel with the nuclear accumulation of myogenin. → The LIM domain of smALP is essential for the nuclear accumulation of the protein. → smALP might act in the nucleus to control some critical aspect of the muscle differentiation process. -- Abstract: The skALP isoform has been shown to play a critical role in actin organization and anchorage within the Z-discs of skeletal muscles, but no data is available on the function of the smALP isoform in skeletal muscle cells. Here, we show that upon induction of differentiation a nuclear translocation of smALP from the cytoplasm to the nucleus of C2C12 myoblasts, concomitant to an up-regulation of the protein expression, occurs in parallel with the nuclear accumulation of myogenin. Moreover, we demonstrate that the LIM domain of smALP is essential for the nuclear translocation of the protein.

  11. SPARC is up-regulated during skeletal muscle regeneration and inhibits myoblast differentiation

    DEFF Research Database (Denmark)

    Petersson, Stine Juhl; Jørgensen, Louise Helskov; Andersen, Ditte C.

    2013-01-01

    Skeletal muscle repair is mediated primarily by the muscle stem cell, the satellite cell. Several factors, including extracellular matrix, are known to regulate satellite cell function and regeneration. One factor, the matricellular Secreted Protein Acidic and Rich in Cysteine (SPARC) is highly up......-regulated during skeletal muscle disease, but its function remains elusive. In the present study, we demonstrate a prominent yet transient increase in SPARC mRNA and protein content during skeletal muscle regeneration that correlates with the expression profile of specific muscle factors like MyoD, Myf5, Myf6......, Myogenin, NCAM, CD34, and M-Cadherin, all known to be implicated in satellite cell activation/proliferation following muscle damage. This up regulation was detected in more cell types. Ectopic expression of SPARC in the muscle progenitor cell line C2C12 was performed to mimic the high levels of SPARC seen...

  12. Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy

    Directory of Open Access Journals (Sweden)

    Isart Roca

    2015-01-01

    Full Text Available The use of adult myogenic stem cells as a cell therapy for skeletal muscle regeneration has been attempted for decades, with only moderate success. Myogenic progenitors (MP made from induced pluripotent stem cells (iPSCs are promising candidates for stem cell therapy to regenerate skeletal muscle since they allow allogenic transplantation, can be produced in large quantities, and, as compared to adult myoblasts, present more embryonic-like features and more proliferative capacity in vitro, which indicates a potential for more self-renewal and regenerative capacity in vivo. Different approaches have been described to make myogenic progenitors either by gene overexpression or by directed differentiation through culture conditions, and several myopathies have already been modeled using iPSC-MP. However, even though results in animal models have shown improvement from previous work with isolated adult myoblasts, major challenges regarding host response have to be addressed and clinically relevant transplantation protocols are lacking. Despite these challenges we are closer than we think to bringing iPSC-MP towards clinical use for treating human muscle disease and sporting injuries.

  13. Isolation, culture and biological characteristics of multipotent porcine skeletal muscle satellite cells.

    Science.gov (United States)

    Yang, Jinjuan; Liu, Hao; Wang, Kunfu; Li, Lu; Yuan, Hongyi; Liu, Xueting; Liu, Yingjie; Guan, Weijun

    2017-12-01

    Skeletal muscle has a huge regenerative potential for postnatal muscle growth and repair, which mainly depends on a kind of muscle progenitor cell population, called satellite cell. Nowadays, the majority of satellite cells were obtained from human, mouse, rat and other animals but rarely from pig. In this article, the porcine skeletal muscle satellite cells were isolated and cultured in vitro. The expression of surface markers of satellite cells was detected by immunofluorescence and RT-PCR assays. The differentiation capacity was assessed by inducing satellite cells into adipocytes, myoblasts and osteoblasts. The results showed that satellite cells isolated from porcine tibialis anterior were subcultured up to 12 passages and were positive for Pax7, Myod, c-Met, desmin, PCNA and NANOG but were negative for Myogenin. Satellite cells were also induced to differentiate into adipocytes, osteoblasts and myoblasts, respectively. These findings indicated that porcine satellite cells possess similar biological characteristics of stem cells, which may provide theoretical basis and experimental evidence for potential therapeutic application in the treatment of dystrophic muscle and other muscle injuries.

  14. Activation of estrogen response elements is mediated both via estrogen and muscle contractions in rat skeletal muscle myotubes

    DEFF Research Database (Denmark)

    Wiik, A.; Hellsten, Ylva; Berthelson, P.

    2009-01-01

    is ER independent. The muscle contraction-induced transactivation of ERE and increase in ERbeta mRNA were instead found to be MAP kinase (MAPK) dependent. This study demonstrates for the first time that muscle contractions have a similar functional effect as estrogen in skeletal muscle myotubes, causing......The aim of the present study was to investigate the activation of estrogen response elements (EREs) by estrogen and muscle contractions in rat myotubes in culture and to assess whether the activation is dependent on the estrogen receptors (ERs). In addition, the effect of estrogen and contraction...... on the mRNA levels of ERalpha and ERbeta was studied to determine the functional consequence of the transactivation. Myoblasts were isolated from rat skeletal muscle and transfected with a vector consisting of sequences of EREs coupled to the gene for luciferase. The transfected myoblasts were...

  15. Extra-anatomic transplantations in autologous adult cell therapies aiding anatomical regeneration and physiological recovery – An insight and categorization

    Directory of Open Access Journals (Sweden)

    Editorial

    2015-12-01

    Full Text Available Autologous mature adult cells as well as stem cells, which are not considered pluripotent, have been reported to be safe and efficacious in clinical applications for regenerating cartilage [1] and corneal epithelium [2]. Use of primary autologous cells and stem cells expanded in number from cartilage and corneal epithelial tissues have shown abilities to reconstruct and regenerate tissues, de novo. It is to be noted that in both these cases, the source of the cells that have been used for transplantation into the cornea and cartilage have been from the same organ and tissue. The replacement cells for regeneration have also been sourced from the same germ layer, as that of the cells of the target tissue; corneal epithelial tissue embryologically originating from the ectoderm has been replaced with corneal limbal stem cells that are also of ectodermal origin from the unaffected healthy eye of the same individual. Similarly, the cartilage which developmentally is from the mesoderm has been replaced with mature chondrocytes from the non-weight bearing area of the cartilage, again of the same individual. Figure 1: Autologous, in vitro cultured, adult cell based therapies; An overview and categorization. (Click here for High Resol. Image The proceedings of the IIDIAS session published in this issue have described two novel cell therapies, where cells taken from a tissue or organ, after normal in vitro expansion, have been clinically applied to aid the regeneration of a different tissue or organ, i.e skeletal myoblasts having been used for myocardial regeneration and buccal mucosal epithelium having been used for corneal epithelial regeneration heralding the birth of a new paradigm called ‘extra-anatomic cell therapy’. The myocardium is a specialized muscle in that it works as an electrical synctitium with an intrinsic capacity to generate and propagate action potentials (involuntary as opposed to the skeletal muscles that are dependent on neuronal

  16. Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

    International Nuclear Information System (INIS)

    Sassoli, Chiara; Nosi, Daniele; Tani, Alessia; Chellini, Flaminia; Mazzanti, Benedetta; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

    2014-01-01

    Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7 + satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration

  17. Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Sassoli, Chiara; Nosi, Daniele; Tani, Alessia; Chellini, Flaminia [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Mazzanti, Benedetta [Dept. of Experimental and Clinical Medicine—Section of Haematology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Quercioli, Franco [CNR-National Institute of Optics (INO), Largo Enrico Fermi 6, 50125 Arcetri-Florence (Italy); Zecchi-Orlandini, Sandra [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy); Formigli, Lucia, E-mail: formigli@unifi.it [Dept. of Experimental and Clinical Medicine—Section of Anatomy and Histology, University of Florence, Largo Brambilla, 3, 50134, Florence (Italy)

    2014-05-01

    Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the single muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7{sup +} satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration.

  18. Intercellular adhesion molecule-1 augments myoblast adhesion and fusion through homophilic trans-interactions.

    Science.gov (United States)

    Pizza, Francis X; Martin, Ryan A; Springer, Evan M; Leffler, Maxwell S; Woelmer, Bryce R; Recker, Isaac J; Leaman, Douglas W

    2017-07-11

    The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.

  19. Myoblast fusion in Drosophila

    Energy Technology Data Exchange (ETDEWEB)

    Haralalka, Shruti [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Abmayr, Susan M., E-mail: sma@stowers.org [Stowers Institute for Medical Research, Kansas City, MO 64110 (United States); Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, MO 66160 (United States)

    2010-11-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  20. Myoblast fusion in Drosophila

    International Nuclear Information System (INIS)

    Haralalka, Shruti; Abmayr, Susan M.

    2010-01-01

    The body wall musculature of a Drosophila larva is composed of an intricate pattern of 30 segmentally repeated muscle fibers in each abdominal hemisegment. Each muscle fiber has unique spatial and behavioral characteristics that include its location, orientation, epidermal attachment, size and pattern of innervation. Many, if not all, of these properties are dictated by founder cells, which determine the muscle pattern and seed the fusion process. Myofibers are then derived from fusion between a specific founder cell and several fusion competent myoblasts (FCMs) fusing with as few as 3-5 FCMs in the small muscles on the most ventral side of the embryo and as many as 30 FCMs in the larger muscles on the dorsal side of the embryo. The focus of the present review is the formation of the larval muscles in the developing embryo, summarizing the major issues and players in this process. We have attempted to emphasize experimentally-validated details of the mechanism of myoblast fusion and distinguish these from the theoretically possible details that have not yet been confirmed experimentally. We also direct the interested reader to other recent reviews that discuss myoblast fusion in Drosophila, each with their own perspective on the process . With apologies, we use gene nomenclature as specified by Flybase (http://flybase.org) but provide Table 1 with alternative names and references.

  1. Enhanced glucose metabolism in cultured human skeletal muscle after Roux-en-Y gastric bypass surgery.

    Science.gov (United States)

    Nascimento, Emmani B M; Riedl, Isabelle; Jiang, Lake Qunfeng; Kulkarni, Sameer S; Näslund, Erik; Krook, Anna

    2015-01-01

    Roux-en-Y gastric bypass (RYGB) surgery rapidly increases whole body insulin sensitivity, with changes in several organs including skeletal muscle. Objectives were to determine whether improvements in insulin action in skeletal muscle may occur directly at the level of the myocyte or secondarily from changes in systemic factors associated with weight loss. Myotubes were derived before and after RYGB surgery. The setting was Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden. Eight patients (body mass index (BMI) 41.8 kg/m(2); age 41 yr) underwent RYGB surgery. Before and 6 months after RYGB surgery, skeletal muscle biopsies were collected from vastus lateralis muscle. Satellite cells derived from skeletal muscle biopsies were propagated in vitro as myoblasts and differentiated into myotubes. Expression of myogenic markers is increased in myoblasts derived from biopsies taken 6 months after bypass surgery, compared with their respective presurgery condition. Furthermore, glycogen synthesis, tyrosine phosphorylation of insulin receptor (IRS)-1-Tyr612 and Interleukin (IL)-8 secretion were increased, while fatty acid oxidation and circulating IL8 levels remain unaltered. Myotubes derived from muscle biopsies obtained after RYGB surgery displayed increased insulin-stimulated phosphorylation of protein kinase B (PKB)-Thr308 and proline-rich Akt substrate of 40 kDa (PRAS40)-Thr246. RYGB surgery is accompanied by enhanced glucose metabolism and insulin signaling, altered IL8 secretion and changes in mRNA levels and myogenic markers in cultured skeletal muscle cells. Thus, RYGB surgery involves intrinsic reprogramming of skeletal muscle to increase peripheral insulin sensitivity and glucose metabolism. Copyright © 2015 American Society for Bariatric Surgery. Published by Elsevier Inc. All rights reserved.

  2. Isolation of human foetal myoblasts and its application for microencapsulation

    Science.gov (United States)

    Li, Anna Aihua; Bourgeois, Jacqueline; Potter, Murray; Chang, Patricia L

    2008-01-01

    Abstract Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non-collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi-nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18–24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell-based therapy in human clinical applications with microencapsulation technology. PMID:18366454

  3. Use of Flow Focusing Technique for Microencapsulation of Myoblasts.

    Science.gov (United States)

    Ciriza, J; Saenz del Burgo, L; Hernández, R M; Orive, G; Pedraz, J L

    2017-01-01

    Alginate cell microencapsulation implies the immobilization of cells within a polymeric membrane that allows the bidirectional diffusion of nutrients and oxygen inside the microcapsules and the release of waste and therapeutic molecules outside them. This technology has been applied to several cell types and it has been extensively described with pancreatic islets. However, other cells such as myoblasts are being currently studied and showing high interest. Moreover, different systems and approaches have been developed for cell encapsulation such as electrostatic extrusion and Flow focusing technology. When Flow focusing technology is applied for myoblast encapsulation, several factors should be considered, such as the pressure, the flow of the system, or the diameter size of the nebulizer, which will determine the final diameter size and shape of the microcapsules containing the myoblasts. Finally, viability of encapsulated myoblasts needs to be assessed before further studies are performed.

  4. YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Song, Young Joon [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of); Lee, Hansol, E-mail: hlee@inha.ac.kr [Department of Biological Sciences, College of Natural Science, Inha University, 253 Yonghyun-dong, Nam-Gu, Incheon, Korea, 402-751 (Korea, Republic of)

    2010-02-15

    Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

  5. YB1/p32, a nuclear Y-box binding protein 1, is a novel regulator of myoblast differentiation that interacts with Msx1 homeoprotein

    International Nuclear Information System (INIS)

    Song, Young Joon; Lee, Hansol

    2010-01-01

    Precisely controlled cellular differentiation is essential for the proper development of vertebrate embryo and deregulated differentiation is a major cause of many human congenital diseases as well as cancer. Msx1 is a member of the homeoprotein family implicated in these processes, which inhibits the differentiation of skeletal muscle and other cell types, presumably by regulating transcription of target genes through interaction with other cellular factors. We presently show that YB1/p32, a nuclear Y-box binding protein 1, interacts with Msx1 homeoprotein and functions as a regulator of C2C12 myoblast differentiation. We demonstrate that YB1/p32 functionally interacts with Msx1 through its N-terminal region and colocalizes with Msx1 at the nuclear periphery. Moreover, we find that YB1/p32 is competent for inhibition of C2C12 myoblast differentiation, which is correlated with its activity as a negative regulator of MyoD gene expression and binding to the MyoD core enhancer region (CER). Furthermore, YB1/p32 cooperates with Msx1 in transcriptional repression and knocking down the expression of endogenous YB1 attenuates the effects of Msx1. Taken together, our study has uncovered a new function of YB1/p32, a regulator of skeletal muscle differentiation.

  6. Improved Cell Culture Method for Growing Contracting Skeletal Muscle Models

    Science.gov (United States)

    Marquette, Michele L.; Sognier, Marguerite A.

    2013-01-01

    An improved method for culturing immature muscle cells (myoblasts) into a mature skeletal muscle overcomes some of the notable limitations of prior culture methods. The development of the method is a major advance in tissue engineering in that, for the first time, a cell-based model spontaneously fuses and differentiates into masses of highly aligned, contracting myotubes. This method enables (1) the construction of improved two-dimensional (monolayer) skeletal muscle test beds; (2) development of contracting three-dimensional tissue models; and (3) improved transplantable tissues for biomedical and regenerative medicine applications. With adaptation, this method also offers potential application for production of other tissue types (i.e., bone and cardiac) from corresponding precursor cells.

  7. Secreted Protein Acidic and Rich in Cysteine (SPARC) in Human Skeletal Muscle

    Science.gov (United States)

    Jørgensen, Louise H.; Petersson, Stine J.; Sellathurai, Jeeva; Andersen, Ditte C.; Thayssen, Susanne; Sant, Dorte J.; Jensen, Charlotte H.; Schrøder, Henrik D.

    2009-01-01

    Secreted protein acidic and rich in cysteine (SPARC)/osteonectin is expressed in different tissues during remodeling and repair, suggesting a function in regeneration. Several gene expression studies indicated that SPARC was expressed in response to muscle damage. Studies on myoblasts further indicated a function of SPARC in skeletal muscle. We therefore found it of interest to study SPARC expression in human skeletal muscle during development and in biopsies from Duchenne and Becker muscular dystrophy and congenital muscular dystrophy, congenital myopathy, inclusion body myositis, and polymyositis patients to analyze SPARC expression in a selected range of inherited and idiopathic muscle wasting diseases. SPARC-positive cells were observed both in fetal and neonatal muscle, and in addition, fetal myofibers were observed to express SPARC at the age of 15–16 weeks. SPARC protein was detected in the majority of analyzed muscle biopsies (23 of 24), mainly in mononuclear cells of which few were pax7 positive. Myotubes and regenerating myofibers also expressed SPARC. The expression-degree seemed to reflect the severity of the lesion. In accordance with these in vivo findings, primary human-derived satellite cells were found to express SPARC both during proliferation and differentiation in vitro. In conclusion, this study shows SPARC expression both during muscle development and in regenerating muscle. The expression is detected both in satellite cells/myoblasts and in myotubes and muscle fibers, indicating a role for SPARC in the skeletal muscle compartment. (J Histochem Cytochem 57:29–39, 2009) PMID:18796407

  8. Defective myoblasts identified in Duchenne muscular dystrophy.

    OpenAIRE

    Blau, H M; Webster, C; Pavlath, G K

    1983-01-01

    A defect in the proliferative capacity of satellite cells, mononucleated precursors of mature muscle fibers, was found in clonal analyses of cells cultured from Duchenne muscular dystrophy (DMD) patients. The total yield of myoblasts per gram of muscle biopsy was decreased to 5% of normal. Of the DMD myoblast clones obtained, a large proportion contained a morphological class of flat distended cells that had an increased generation time and ceased to proliferate beyond 100-1,000 cells but cou...

  9. The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle

    DEFF Research Database (Denmark)

    Hansen, Lise K; Schrøder, Henrik D; Lund, Lars

    2017-01-01

    , proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after...... incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro....

  10. Obscurin Depletion Impairs Organization of Skeletal Muscle in Developing Zebrafish Embryos

    Directory of Open Access Journals (Sweden)

    Maide Ö. Raeker

    2011-01-01

    Full Text Available During development, skeletal myoblasts differentiate into myocytes and skeletal myotubes with mature contractile structures that are precisely oriented with respect to surrounding cells and tissues. Establishment of this highly ordered structure requires reciprocal interactions between the differentiating myocytes and the surrounding extracellular matrix to form correctly positioned and well-organized attachments from the skeletal muscle to the bony skeleton. Using the developing zebrafish embryo as a model, we examined the relationship between new myofibril assembly and the organization of the membrane domains involved in cell-extracellular matrix interactions. We determined that depletion of obscurin, a giant muscle protein, resulted in irregular cell morphology and disturbed extracellular matrix organization during skeletal muscle development. The resulting impairment of myocyte organization was associated with disturbance of the internal architecture of the myocyte suggesting that obscurin participates in organizing the internal structure of the myocyte and translating those structural cues to surrounding cells and tissues.

  11. Obscurin Depletion Impairs Organization of Skeletal Muscle in Developing Zebrafish Embryos

    Science.gov (United States)

    Raeker, Maide Ö.; Russell, Mark W.

    2011-01-01

    During development, skeletal myoblasts differentiate into myocytes and skeletal myotubes with mature contractile structures that are precisely oriented with respect to surrounding cells and tissues. Establishment of this highly ordered structure requires reciprocal interactions between the differentiating myocytes and the surrounding extracellular matrix to form correctly positioned and well-organized attachments from the skeletal muscle to the bony skeleton. Using the developing zebrafish embryo as a model, we examined the relationship between new myofibril assembly and the organization of the membrane domains involved in cell-extracellular matrix interactions. We determined that depletion of obscurin, a giant muscle protein, resulted in irregular cell morphology and disturbed extracellular matrix organization during skeletal muscle development. The resulting impairment of myocyte organization was associated with disturbance of the internal architecture of the myocyte suggesting that obscurin participates in organizing the internal structure of the myocyte and translating those structural cues to surrounding cells and tissues. PMID:22190853

  12. Tissue-Engineered Skeletal Muscle Organoids for Reversible Gene Therapy

    Science.gov (United States)

    Vandenburgh, Herman; DelTatto, Michael; Shansky, Janet; Lemaire, Julie; Chang, Albert; Payumo, Francis; Lee, Peter; Goodyear, Amy; Raven, Latasha

    1996-01-01

    Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myofibers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid Implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postimtotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.

  13. Phosphorylation of Lbx1 controls lateral myoblast migration into the limb.

    Science.gov (United States)

    Masselink, Wouter; Masaki, Megumi; Sieiro, Daniel; Marcelle, Christophe; Currie, Peter D

    2017-10-15

    The migration of limb myogenic precursors from limb level somites to their ultimate site of differentiation in the limb is a paradigmatic example of a set of dynamic and orchestrated migratory cell behaviours. The homeobox containing transcription factor ladybird homeobox 1 (Lbx1) is a central regulator of limb myoblast migration, null mutations of Lbx1 result in severe disruptions to limb muscle formation, particularly in the distal region of the limb in mice (Gross et al., 2000). As such Lbx1 has been hypothesized to control lateral migration of myoblasts into the distal limb anlage. It acts as a core regulator of the limb myoblast migration machinery, controlled by Pax3. A secondary role for Lbx1 in the differentiation and commitment of limb musculature has also been proposed (Brohmann et al., 2000; Uchiyama et al., 2000). Here we show that lateral migration, but not differentiation or commitment of limb myoblasts, is controlled by the phosphorylation of three adjacent serine residues of LBX1. Electroporation of limb level somites in the chick embryo with a dephosphomimetic form of Lbx1 results in a specific defect in the lateral migration of limb myoblasts. Although the initial delamination and migration of myoblasts is unaffected, migration into the distal limb bud is severely disrupted. Interestingly, myoblasts undergo normal differentiation independent of their migratory status, suggesting that the differentiation potential of hypaxial muscle is not regulated by the phosphorylation state of LBX1. Furthermore, we show that FGF8 and ERK mediated signal transduction, both critical regulators of the developing limb bud, have the capacity to induce the phosphorylation of LBX1 at these residues. Overall, this suggests a mechanism whereby the phosphorylation of LBX1, potentially through FGF8 and ERK signalling, controls the lateral migration of myoblasts into the distal limb bud. Copyright © 2017. Published by Elsevier Inc.

  14. Neonatal epicardial-derived progenitors aquire myogenic traits in skeletal muscle, but not cardiac muscle

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Jensen, Charlotte H; Skovrind, Ida

    2016-01-01

    heart missing regenerative signals essential for directed differentiation of EPDCs. Herein, we aimed to evaluate the myogenic potential of neonatal EPDCs in adult and neonatal mouse myocardium, as well as in skeletal muscle. The two latter tissues have an intrinsic capability to develop and regenerate......, in contrast to the adult heart. METHODS: Highly purified mouse EPDCs were transplanted into damaged neonatal and adult myocardium as well as regenerating skeletal muscle. Co-cultures with skeletal myoblasts were used to distinguish fusion independent myogenic conversion. RESULTS: No donor EPDC...... that EPDCs may be more myogenic than previously anticipated. But, the heart may lack factors for induction of myogenesis of EPDCs, a scenario that should be taken into consideration when aiming for repair of damaged myocardium by stem cell transplantation....

  15. IGF-II-mediated downregulation of peroxisome proliferator-activated receptor-γ coactivator-1α in myoblast cells involves PI3K/Akt/FoxO1 signaling pathway.

    Science.gov (United States)

    Mu, Xiaoyu; Qi, Weihong; Liu, Yunzhang; Zhou, Jianfeng; Li, Yun; Rong, Xiaozhi; Lu, Ling

    2017-08-01

    Insulin-like growth factor II (IGF-II) can stimulate myogenesis and is critically involved in skeletal muscle differentiation. The presence of negative regulators of this process, however, is not well explored. Here, we showed that in myoblast cells, IGF-II negatively regulated peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA expression, while constitutive expression of PGC-1α induced myoblast differentiation. These results suggest that the negative regulation of PGC-1α by IGF-II may act as a negative feedback mechanism in IGF-II-induced myogenic differentiation. Reporter assays demonstrated that IGF-II suppresses the basal PGC-1α promoter activity. Blocking the IGF-II signaling pathway increased the endogenous PGC-1α levels. In addition, pharmacological inhibition of PI3 kinase activity prevented the downregulation of PGC-1α but the activation of mTOR was not required for this process. Importantly, further analysis showed that forkhead transcription factor FoxO1 contributes to mediating the effects of IGF-II on PGC-1 promoter activity. These findings indicate that IGF-II reduces PGC-1α expression in skeletal muscle cells through a mechanism involving PI3K-Akt-FoxO1 but not p38 MAPK or Erk1/2 MAPK pathways.

  16. Abelson tyrosine-protein kinase 2 Regulates Myoblast Proliferation and Controls Muscle Fiber Length

    OpenAIRE

    Burden, Steven; Lee, Jennifer

    2017-01-01

    Muscle fiber length is nearly uniform within a muscle but widely different among muscles. Here, we show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm and other muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of available myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but expansion of the diaphragm ...

  17. Novel Endogenous, Insulin-Stimulated Akt2 Protein Interaction Partners in L6 Myoblasts.

    Directory of Open Access Journals (Sweden)

    Michael Caruso

    Full Text Available Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2's role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 49 were detected with a significantly increased (47 or decreased (2 association with Akt2 following insulin administration (n = 4; p<0.05. Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network. Data are available via ProteomeXchange with identifier PXD002557.

  18. A preclinical evaluation of an autologous living hyaline-like cartilaginous graft for articular cartilage repair: a pilot study

    OpenAIRE

    Yvonne Peck; Pengfei He; Geetha Soujanya V. N. Chilla; Chueh Loo Poh; Dong-An Wang

    2015-01-01

    In this pilot study, an autologous synthetic scaffold-free construct with hyaline quality, termed living hyaline cartilaginous graft (LhCG), was applied for treating cartilage lesions. Implantation of autologous LhCG was done at load-bearing regions of the knees in skeletally mature mini-pigs for 6 months. Over the course of this study, significant radiographical improvement in LhCG treated sites was observed via magnetic resonance imaging. Furthermore, macroscopic repair was effected by LhCG...

  19. Establishment of bipotent progenitor cell clone from rat skeletal muscle.

    Science.gov (United States)

    Murakami, Yousuke; Yada, Erica; Nakano, Shin-ichi; Miyagoe-Suzuki, Yuko; Hosoyama, Tohru; Matsuwaki, Takashi; Yamanouchi, Keitaro; Nishihara, Masugi

    2011-12-01

    The present study describes the isolation, cloning and characterization of adipogenic progenitor cells from rat skeletal muscle. Among the obtained 10 clones, the most highly adipogenic progenitor, 2G11 cells, were further characterized. In addition to their adipogenicity, 2G11 cells retain myogenic potential as revealed by formation of multinucleated myotubes when co-cultured with myoblasts. 2G11 cells were resistant to an inhibitory effect of basic fibroblast growth factor on adipogenesis, while adipogenesis of widely used preadipogenic cell line, 3T3-L1 cells, was suppressed almost completely by the same treatment. In vivo transplantation experiments revealed that 2G11 cells are able to possess both adipogenicity and myogenicity in vivo. These results indicate the presence of bipotent progenitor cells in rat skeletal muscle, and suggest that such cells may contribute to ectopic fat formation in skeletal muscle. © 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

  20. Analysis of differential lipofection efficiency in primary and established myoblasts.

    Science.gov (United States)

    Pampinella, Francesca; Lechardeur, Delphine; Zanetti, Elena; MacLachlan, Ian; Benharouga, Mohammed; Lukacs, Gergely L; Vitiello, Libero

    2002-02-01

    In this study we have compared the process of lipid-mediated transfection in primary and established myoblasts, in an attempt to elucidate the mechanisms responsible for the scarce transfectability of the former. We determined the metabolic stability of cytoplasmically injected and lipofected DNA in primary and established myoblasts and carried out a comparative time course analysis of luciferase reporter-gene expression and DNA stability. The efficiency of the transcription-translation machinery of the two cell types was compared by intranuclear injection of naked plasmid DNA encoding luciferase. Subcellular colocalization of fluorescein-labeled lipopolyplexes with specific endosomal and lysosomal markers was performed by confocal microscopy to monitor the intracellular trafficking of plasmid DNA during transfection. The metabolic stability of plasmid DNA was similar in primary and established myoblasts after both lipofection and cytoplasmic injection. In both cell types, lipofection had no detectable effect on the rate of cell proliferation. Confocal analysis showed that nuclear translocation of transfected DNA coincided with localization in a compartment devoid of endosome- or lysosome-specific marker proteins. The residency time of plasmid DNA in this compartment differed for primary and established myoblasts. Our findings suggest that the lower transfectability of primary myoblasts is mostly due to a difference in the intracellular delivery pathway that correlates with more rapid delivery of internalized complex to the lysosomal compartment.

  1. Mesenchymal stem cells (MSCs) as skeletal therapeutics-an update

    DEFF Research Database (Denmark)

    Saeed, H.; Ahsan, M.; Saleem, Z.

    2016-01-01

    Mesenchymal stem cells hold the promise to treat not only several congenital and acquired bone degenerative diseases but also to repair and regenerate morbid bone tissues. Utilizing MSCs, several lines of evidences advocate promising clinical outcomes in skeletal diseases and skeletal tissue repair....../regeneration. In this context, both, autologous and allogeneic cell transfer options have been utilized. Studies suggest that MSCs are transplanted either alone by mixing with autogenous plasma/serum or by loading onto repair/induction supportive resorb-able scaffolds. Thus, this review is aimed at highlighting a wide range...

  2. [Human myoblast culture as muscle stem cells in medical and biological studies].

    Science.gov (United States)

    Terekhov, S M; Krokhina, T B; Shishkin, S S; Krakhmaleva, I N; Zakharov, S F; Ershova, E S

    2001-01-01

    The method for obtaining human myoblast culture has been modified to consider the specific histological localization of the satellite cells as well as their growth properties; the cultivation conditions have been selected to grow up to 150000 cells/cm2. At high densities, the cells remain mononuclear and preserve their typical myoblast morphology as well as the capacity for fusion and the formation of myotubes. By contrast to fibroblasts, up to 80% of the cells in the myoblast culture were positive in the acid phosphatase test, which indicates their stem nature. The obtained myoblast cultures were used in the clinical tests of cell-mediated gene therapy of Duchenne's muscular dystrophy as well as in the bioassay for the effects of biologically active compounds.

  3. The Recent Understanding of the Neurotrophin's Role in Skeletal Muscle Adaptation

    Directory of Open Access Journals (Sweden)

    Kunihiro Sakuma

    2011-01-01

    Full Text Available This paper summarizes the various effects of neurotrophins in skeletal muscle and how these proteins act as potential regulators of the maintenance, function, and regeneration of skeletal muscle fibers. Increasing evidence suggests that this family of neurotrophic factors influence not only the survival and function of innervating motoneurons but also the development and differentiation of myoblasts and muscle fibers. Muscle contractions (e.g., exercise produce BDNF mRNA and protein in skeletal muscle, and the BDNF seems to play a role in enhancing glucose metabolism and may act for myokine to improve various brain disorders (e.g., Alzheimer's disease and major depression. In adults with neuromuscular disorders, variations in neurotrophin expression are found, and the role of neurotrophins under such conditions is beginning to be elucidated. This paper provides a basis for a better understanding of the role of these factors under such pathological conditions and for treatment of human neuromuscular disease.

  4. Disruption of myoblast alignment by highly motile rhabdomyosarcoma cell in tissue structure.

    Science.gov (United States)

    Li, Menglu; Nagamori, Eiji; Kino-Oka, Masahiro

    2017-02-01

    Rhabdomyosarcoma (RMS) is a highly malignant tumor type of skeletal muscle origin, hallmarked by local invasion. Interaction between invasive tumor cells and normal cells plays a major role in tumor invasion and metastasis. Culturing tumor cells in a three-dimensional (3D) model can translate tumor malignancy relevant cell-cell interaction. To mimic tumor heterogeneity in vitro, a co-culture system consisting of a malignant embryonal rhabdomyosarcoma (ERMS) cell line RD and a normal human skeletal muscle myoblast (HSMM) cell line was established by cell sheet technology. Various ratios of RDs to HSMMs were employed to understand the quantitative effect on intercellular interactions. Disruption of sheet structure was observed in heterogeneous cell sheets having a low ratio of RDs to HSMMs, whereas homogeneous HSMM or RD sheets maintained intact structure. Deeper exploration of dynamic tumor cell behavior inside HSMM sheets revealed that HSMM cell alignment was disrupted by highly motile RDs. This study demonstrated that RMS cells are capable of compromising their surrounding environment through induced decay of HSMMs alignment in a cell-based 3D system. This suggests that muscle disruption might be a major consequence of RMS cell invasion into muscles, which could be a promising target to preventing tumor invasion. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

    Science.gov (United States)

    Torgan, C. E.; Burge, S. S.; Collinsworth, A. M.; Truskey, G. A.; Kraus, W. E.

    2000-01-01

    The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

  6. Elastic hydrogel substrate supports robust expansion of murine myoblasts and enhances their engraftment

    International Nuclear Information System (INIS)

    Ding, Ke; Yang, Zhong; Xu, Jian-zhong; Liu, Wen-ying; Zeng, Qiang; Hou, Fang; Lin, Sen

    2015-01-01

    The application of satellite cell-derived myoblasts in regenerative medicine has been restricted by the rapid loss of stemness during in vitro cell expansion using traditional culture systems. However, studies published in the past decade have highlighted the influence of substrate elasticity on stem cell fate and revealed that culture on a soft hydrogel substrate can promote self-renewal and prolong the regenerative potential of muscle stem cells. Whether hydrogel substrates have similar effects after long-term robust expansion remains to be determined. Herein we prepared an elastic chitosan/beta-glycerophosphate/collagen hydrogel mimicking the soft microenvironment of muscle tissues for use as the substrate for satellite cell culture and investigated its influence on long-term cell expansion. After 20 passages in culture, satellite cell-derived myoblasts cultured on our hydrogel substrate exhibited significant improvements in proliferation capability, cell viability, colony forming frequency, and potential for myogenic differentiation compared to those cultured on a routine rigid culture surface. Immunochemical staining and western blot analysis both confirmed that myoblasts cultured on the hydrogel substrate expressed higher levels of several differentiation-related markers, including Pax7, Pax3, and SSEA-1, and a lower level of MyoD compared to myoblasts cultured on rigid culture plates (all p<0.05). After transplantation into the tibialis anterior of nude mice, myoblasts that had been cultured on the hydrogel substrate demonstrated a significantly greater engraftment efficacy than those cultured on the traditional surface. Collectively, these results indicate that the elastic hydrogel substrate supported robust expansion of murine myoblasts and enhanced their engraftment in vivo. - Highlights: • An elastic hydrogel was designed to mimic the pliable muscle tissue microenvironment. • Myoblasts retained their stemness in long-term culture on the elastic

  7. Elastic hydrogel substrate supports robust expansion of murine myoblasts and enhances their engraftment

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Ke, E-mail: dk1118@yeah.net [Department of Pediatric Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072 (China); Yang, Zhong [Department of Clinical Hematology, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Xu, Jian-zhong, E-mail: xjzspine@163.com [Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Liu, Wen-ying; Zeng, Qiang; Hou, Fang [Department of Pediatric Surgery, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072 (China); Lin, Sen [Department of Anatomy and Histology & Embryology, Chengdu Medical College, Chengdu 610500 (China)

    2015-09-10

    The application of satellite cell-derived myoblasts in regenerative medicine has been restricted by the rapid loss of stemness during in vitro cell expansion using traditional culture systems. However, studies published in the past decade have highlighted the influence of substrate elasticity on stem cell fate and revealed that culture on a soft hydrogel substrate can promote self-renewal and prolong the regenerative potential of muscle stem cells. Whether hydrogel substrates have similar effects after long-term robust expansion remains to be determined. Herein we prepared an elastic chitosan/beta-glycerophosphate/collagen hydrogel mimicking the soft microenvironment of muscle tissues for use as the substrate for satellite cell culture and investigated its influence on long-term cell expansion. After 20 passages in culture, satellite cell-derived myoblasts cultured on our hydrogel substrate exhibited significant improvements in proliferation capability, cell viability, colony forming frequency, and potential for myogenic differentiation compared to those cultured on a routine rigid culture surface. Immunochemical staining and western blot analysis both confirmed that myoblasts cultured on the hydrogel substrate expressed higher levels of several differentiation-related markers, including Pax7, Pax3, and SSEA-1, and a lower level of MyoD compared to myoblasts cultured on rigid culture plates (all p<0.05). After transplantation into the tibialis anterior of nude mice, myoblasts that had been cultured on the hydrogel substrate demonstrated a significantly greater engraftment efficacy than those cultured on the traditional surface. Collectively, these results indicate that the elastic hydrogel substrate supported robust expansion of murine myoblasts and enhanced their engraftment in vivo. - Highlights: • An elastic hydrogel was designed to mimic the pliable muscle tissue microenvironment. • Myoblasts retained their stemness in long-term culture on the elastic

  8. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activates promyogenic signaling pathways, thereby promoting myoblast differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Seo, Dong-Wan [College of Pharmacy, Dankook University, Cheonan 330-714 (Korea, Republic of); Kang, Jong-Sun [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 440-746 (Korea, Republic of); Bae, Gyu-Un, E-mail: gbae@sookmyung.ac.kr [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2016-01-29

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) regulates postnatal myogenesis by alleviating myostatin activity, but the molecular mechanisms by which it regulates myogenesis are not fully understood. In this study, we investigate molecular mechanisms of PPARβ/δ in myoblast differentiation. C2C12 myoblasts treated with a PPARβ/δ agonist, GW0742 exhibit enhanced myotube formation and muscle-specific gene expression. GW0742 treatment dramatically activates promyogenic kinases, p38MAPK and Akt, in a dose-dependent manner. GW0742-stimulated myoblast differentiation is mediated by p38MAPK and Akt, since it failed to restore myoblast differentiation repressed by inhibition of p38MAPK and Akt. In addition, GW0742 treatment enhances MyoD-reporter activities. Consistently, overexpression of PPARβ/δ enhances myoblast differentiation accompanied by elevated activation of p38MAPK and Akt. Collectively, these results suggest that PPARβ/δ enhances myoblast differentiation through activation of promyogenic signaling pathways. - Highlights: • A PPARβ/δ agonist, GW0742 promotes myoblast differentiation. • GW0742 activates both p38MAPK and Akt activation in myogenic differentiation. • GW0742 enhances MyoD activity for myogenic differentiation. • Overexpression of PPARβ/δ enhances myoblast differentiation via activating promyogenic signaling pathways. • This is the first finding for agonistic mechanism of PPARβ/δ in myogenesis.

  9. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activates promyogenic signaling pathways, thereby promoting myoblast differentiation

    International Nuclear Information System (INIS)

    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee; Seo, Dong-Wan; Kang, Jong-Sun; Bae, Gyu-Un

    2016-01-01

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) regulates postnatal myogenesis by alleviating myostatin activity, but the molecular mechanisms by which it regulates myogenesis are not fully understood. In this study, we investigate molecular mechanisms of PPARβ/δ in myoblast differentiation. C2C12 myoblasts treated with a PPARβ/δ agonist, GW0742 exhibit enhanced myotube formation and muscle-specific gene expression. GW0742 treatment dramatically activates promyogenic kinases, p38MAPK and Akt, in a dose-dependent manner. GW0742-stimulated myoblast differentiation is mediated by p38MAPK and Akt, since it failed to restore myoblast differentiation repressed by inhibition of p38MAPK and Akt. In addition, GW0742 treatment enhances MyoD-reporter activities. Consistently, overexpression of PPARβ/δ enhances myoblast differentiation accompanied by elevated activation of p38MAPK and Akt. Collectively, these results suggest that PPARβ/δ enhances myoblast differentiation through activation of promyogenic signaling pathways. - Highlights: • A PPARβ/δ agonist, GW0742 promotes myoblast differentiation. • GW0742 activates both p38MAPK and Akt activation in myogenic differentiation. • GW0742 enhances MyoD activity for myogenic differentiation. • Overexpression of PPARβ/δ enhances myoblast differentiation via activating promyogenic signaling pathways. • This is the first finding for agonistic mechanism of PPARβ/δ in myogenesis.

  10. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan); Gojo, Satoshi [Department of Cardiac Support, Kyoto Prefectural University of Medicine, Kyoto (Japan); Mazda, Osam, E-mail: mazda@koto.kpu-m.ac.jp [Department of Immunology, Kyoto Prefectural University of Medicine, Kyoto (Japan)

    2013-02-08

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.

  11. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-01-01

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases

  12. Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length

    OpenAIRE

    Lee, Jennifer K; Hallock, Peter T; Burden, Steven J

    2017-01-01

    Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myobla...

  13. Live cell imaging reveals marked variability in myoblast proliferation and fate

    Science.gov (United States)

    2013-01-01

    Background During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear. Methods We have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line. Results Analyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls. Conclusion Our results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated. PMID:23638706

  14. Role of an inward rectifier K+ current and of hyperpolarization in human myoblast fusion

    Science.gov (United States)

    Liu, J-H; Bijlenga, P; Fischer-Lougheed, J; Occhiodoro, T; Kaelin, A; Bader, C R; Bernheim, L

    1998-01-01

    The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion-competent myoblasts (FCMBs), and freshly formed myotubes. Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non-inactivating delayed rectifier, and IK(IR), an inward rectifier. IK(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28 % of the FCMBs and in all myotubes. IK(IR) is reversibly blocked by Ba2+ or Cs+. Cells expressing IK(IR) have resting membrane potentials of −65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (−32 mV). Cs+ and Ba2+ ions reduce myoblast fusion. It is hypothesized that the IK(IR)-mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T-type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion. PMID:9705997

  15. PEDF-derived peptide promotes skeletal muscle regeneration through its mitogenic effect on muscle progenitor cells.

    Science.gov (United States)

    Ho, Tsung-Chuan; Chiang, Yi-Pin; Chuang, Chih-Kuang; Chen, Show-Li; Hsieh, Jui-Wen; Lan, Yu-Wen; Tsao, Yeou-Ping

    2015-08-01

    In response injury, intrinsic repair mechanisms are activated in skeletal muscle to replace the damaged muscle fibers with new muscle fibers. The regeneration process starts with the proliferation of satellite cells to give rise to myoblasts, which subsequently differentiate terminally into myofibers. Here, we investigated the promotion effect of pigment epithelial-derived factor (PEDF) on muscle regeneration. We report that PEDF and a synthetic PEDF-derived short peptide (PSP; residues Ser(93)-Leu(112)) induce satellite cell proliferation in vitro and promote muscle regeneration in vivo. Extensively, soleus muscle necrosis was induced in rats by bupivacaine, and an injectable alginate gel was used to release the PSP in the injured muscle. PSP delivery was found to stimulate satellite cell proliferation in damaged muscle and enhance the growth of regenerating myofibers, with complete regeneration of normal muscle mass by 2 wk. In cell culture, PEDF/PSP stimulated C2C12 myoblast proliferation, together with a rise in cyclin D1 expression. PEDF induced the phosphorylation of ERK1/2, Akt, and STAT3 in C2C12 myoblasts. Blocking the activity of ERK, Akt, or STAT3 with pharmacological inhibitors attenuated the effects of PEDF/PSP on the induction of C2C12 cell proliferation and cyclin D1 expression. Moreover, 5-bromo-2'-deoxyuridine pulse-labeling demonstrated that PEDF/PSP stimulated primary rat satellite cell proliferation in myofibers in vitro. In summary, we report for the first time that PSP is capable of promoting the regeneration of skeletal muscle. The signaling mechanism involves the ERK, AKT, and STAT3 pathways. These results show the potential utility of this PEDF peptide for muscle regeneration. Copyright © 2015 the American Physiological Society.

  16. Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle.

    Directory of Open Access Journals (Sweden)

    Nobuyuki Takenaka

    Full Text Available Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

  17. HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

    Science.gov (United States)

    González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

    2017-10-10

    The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving

  18. The Satellite Cell Niche Regulates the Balance between Myoblast Differentiation and Self-Renewal via p53.

    Science.gov (United States)

    Flamini, Valentina; Ghadiali, Rachel S; Antczak, Philipp; Rothwell, Amy; Turnbull, Jeremy E; Pisconti, Addolorata

    2018-03-13

    Satellite cells are adult muscle stem cells residing in a specialized niche that regulates their homeostasis. How niche-generated signals integrate to regulate gene expression in satellite cell-derived myoblasts is poorly understood. We undertook an unbiased approach to study the effect of the satellite cell niche on satellite cell-derived myoblast transcriptional regulation and identified the tumor suppressor p53 as a key player in the regulation of myoblast quiescence. After activation and proliferation, a subpopulation of myoblasts cultured in the presence of the niche upregulates p53 and fails to differentiate. When satellite cell self-renewal is modeled ex vivo in a reserve cell assay, myoblasts treated with Nutlin-3, which increases p53 levels in the cell, fail to differentiate and instead become quiescent. Since both these Nutlin-3 effects are rescued by small interfering RNA-mediated p53 knockdown, we conclude that a tight control of p53 levels in myoblasts regulates the balance between differentiation and return to quiescence. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Absence of muscle regeneration after implantation of a collagen matrix seeded with myoblasts

    NARCIS (Netherlands)

    van Wachem, PB; Brouwer, LA; van Luyn, MJA

    Collagens are widely used as biomaterials for e.g. soft tissue reconstruction. The present study was aimed at reconstruction of abdominal wall muscle using processed dermal sheep collagen (DSC) and myoblast seeding. Myoblasts were harvested from foetal quadriceps muscle of an inbred rat strain,

  20. Insulin-like growth factor-1 suppresses the Myostatin signaling pathway during myogenic differentiation

    International Nuclear Information System (INIS)

    Retamales, A.; Zuloaga, R.; Valenzuela, C.A.; Gallardo-Escarate, C.; Molina, A.; Valdés, J.A.

    2015-01-01

    Myogenic differentiation is a complex and well-coordinated process for generating mature skeletal muscle fibers. This event is autocrine/paracrine regulated by growth factors, principally Myostatin (MSTN) and Insulin-like Growth Factor-1 (IGF-1). Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of skeletal muscle growth in vertebrates that exerts its inhibitory function by activating Smad transcription factors. In contrast, IGF-1 promotes the differentiation of skeletal myoblasts by activating the PI3K/Akt signaling pathway. This study reports on a novel functional crosstalk between the IGF-1 and MSTN signaling pathways, as mediated through interaction between PI3K/Akt and Smad3. Stimulation of skeletal myoblasts with MSTN resulted in a transient increase in the pSmad3:Smad3 ratio and Smad-dependent transcription. Moreover, MSTN inhibited myod gene expression and myoblast fusion in an Activin receptor-like kinase/Smad3-dependent manner. Preincubation of skeletal myoblasts with IGF-1 blocked MSTN-induced Smad3 activation, promoting myod expression and myoblast differentiation. This inhibitory effect of IGF-1 on the MSTN signaling pathway was dependent on IGF-1 receptor, PI3K, and Akt activities. Finally, immunoprecipitation assay analysis determined that IGF-1 pretreatment increased Akt and Smad3 interaction. These results demonstrate that the IGF-1/PI3K/Akt pathway may inhibit MSTN signaling during myoblast differentiation, providing new insight to existing knowledge on the complex crosstalk between both growth factors. - Highlights: • IGF-1 inhibits Myostatin canonical signaling pathway through IGF-1R/PI3K/Akt pathway. • IGF-1 promotes myoblast differentiation through a direct blocking of Myostatin signaling pathway. • IGF-1 induces the interaction of Akt with Smad3 in skeletal myoblast

  1. Insulin-like growth factor-1 suppresses the Myostatin signaling pathway during myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Retamales, A.; Zuloaga, R.; Valenzuela, C.A. [Laboratorio de Biotecnología Molecular, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago (Chile); Gallardo-Escarate, C. [Laboratory of Biotechnology and Aquatic Genomics, Universidad de Concepción, Concepción (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), P.O. Box 160-C, Concepción (Chile); Molina, A. [Laboratorio de Biotecnología Molecular, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), P.O. Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: jvaldes@unab.cl [Laboratorio de Biotecnología Molecular, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), P.O. Box 160-C, Concepción (Chile)

    2015-08-21

    Myogenic differentiation is a complex and well-coordinated process for generating mature skeletal muscle fibers. This event is autocrine/paracrine regulated by growth factors, principally Myostatin (MSTN) and Insulin-like Growth Factor-1 (IGF-1). Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of skeletal muscle growth in vertebrates that exerts its inhibitory function by activating Smad transcription factors. In contrast, IGF-1 promotes the differentiation of skeletal myoblasts by activating the PI3K/Akt signaling pathway. This study reports on a novel functional crosstalk between the IGF-1 and MSTN signaling pathways, as mediated through interaction between PI3K/Akt and Smad3. Stimulation of skeletal myoblasts with MSTN resulted in a transient increase in the pSmad3:Smad3 ratio and Smad-dependent transcription. Moreover, MSTN inhibited myod gene expression and myoblast fusion in an Activin receptor-like kinase/Smad3-dependent manner. Preincubation of skeletal myoblasts with IGF-1 blocked MSTN-induced Smad3 activation, promoting myod expression and myoblast differentiation. This inhibitory effect of IGF-1 on the MSTN signaling pathway was dependent on IGF-1 receptor, PI3K, and Akt activities. Finally, immunoprecipitation assay analysis determined that IGF-1 pretreatment increased Akt and Smad3 interaction. These results demonstrate that the IGF-1/PI3K/Akt pathway may inhibit MSTN signaling during myoblast differentiation, providing new insight to existing knowledge on the complex crosstalk between both growth factors. - Highlights: • IGF-1 inhibits Myostatin canonical signaling pathway through IGF-1R/PI3K/Akt pathway. • IGF-1 promotes myoblast differentiation through a direct blocking of Myostatin signaling pathway. • IGF-1 induces the interaction of Akt with Smad3 in skeletal myoblast.

  2. Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length

    Science.gov (United States)

    Lee, Jennifer K; Hallock, Peter T

    2017-01-01

    Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but as a consequence of expansion of the diaphragm muscle, the diaphragm central tendon is reduced in size, likely contributing to reduced stamina of Abl2 mutant mice. Ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon of Abl2+/− mice. Specialized tendon cells, resembling tendon cells at myotendinous junctions, form at the ends of these muscle islands, suggesting that myofibers induce differentiation of tendon cells, which reciprocally regulate myofiber length and orientation. PMID:29231808

  3. Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length.

    Science.gov (United States)

    Lee, Jennifer K; Hallock, Peter T; Burden, Steven J

    2017-12-12

    Muscle fiber length is nearly uniform within a muscle but widely different among different muscles. We show that Abelson tyrosine-protein kinase 2 (Abl2) has a key role in regulating myofiber length, as a loss of Abl2 leads to excessively long myofibers in the diaphragm, intercostal and levator auris muscles but not limb muscles. Increased myofiber length is caused by enhanced myoblast proliferation, expanding the pool of myoblasts and leading to increased myoblast fusion. Abl2 acts in myoblasts, but as a consequence of expansion of the diaphragm muscle, the diaphragm central tendon is reduced in size, likely contributing to reduced stamina of Abl2 mutant mice. Ectopic muscle islands, each composed of myofibers of uniform length and orientation, form within the central tendon of Abl2 +/- mice. Specialized tendon cells, resembling tendon cells at myotendinous junctions, form at the ends of these muscle islands, suggesting that myofibers induce differentiation of tendon cells, which reciprocally regulate myofiber length and orientation.

  4. Generation of a vascularized organoid using skeletal muscle as the inductive source.

    LENUS (Irish Health Repository)

    Messina, Aurora

    2005-09-01

    The technology required for creating an in vivo microenvironment and a neovasculature that can grow with and service new tissue is lacking, precluding the possibility of engineering complex three-dimensional organs. We have shown that when an arterio-venous (AV) loop is constructed in vivo in the rat groin, and placed inside a semisealed chamber, an extensive functional vasculature is generated. To test whether this unusually angiogenic environment supports the survival and growth of implanted tissue or cells, we inserted various preparations of rat and human skeletal muscle. We show that after 6 weeks incubation of muscle tissue, the chamber filled with predominantly well-vascularized recipient-derived adipose tissue, but some new donor-derived skeletal muscle and connective tissue were also evident. When primary cultured myoblasts were inserted into the chamber with the AV loop, they converted to mature striated muscle fibers. Furthermore, we identify novel adipogenesis-inducing properties of skeletal muscle. This represents the first report of a specific three-dimensional tissue grown on its own vascular supply.

  5. The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse

    DEFF Research Database (Denmark)

    Vasyutina, Elena; Martarelli, Benedetta; Brakebusch, Cord

    2009-01-01

    Rac1 and Cdc42 are small G-proteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. We used conditional mutagenesis in mice to demonstrate that Rac1 and Cdc42 are essential for myoblast fusion in vivo and in vitro. The deficit in fusion of Rac1 or Cdc42 mut...... genetic analysis demonstrates thus that the function of Rac in myoblast fusion is evolutionarily conserved from insects to mammals and that Cdc42, a molecule hitherto not implicated in myoblast fusion, is essential for the fusion of murine myoblasts....

  6. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells.

    Science.gov (United States)

    Gao, Fei; Kishida, Tsunao; Ejima, Akika; Gojo, Satoshi; Mazda, Osam

    2013-02-08

    Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Sirtuin-3 (Sirt3) regulates skeletal muscle metabolism and insulin signaling via altered mitochondrial oxidation and reactive oxygen species production

    DEFF Research Database (Denmark)

    Jing, Enxuan; Emanuelli, Brice; Hirschey, Matthew D

    2011-01-01

    Sirt3 is a member of the sirtuin family of protein deacetylases that is localized in mitochondria and regulates mitochondrial function. Sirt3 expression in skeletal muscle is decreased in models of type 1 and type 2 diabetes and regulated by feeding, fasting, and caloric restriction. Sirt3 knockout...... mice exhibit decreased oxygen consumption and develop oxidative stress in skeletal muscle, leading to JNK activation and impaired insulin signaling. This effect is mimicked by knockdown of Sirt3 in cultured myoblasts, which exhibit reduced mitochondrial oxidation, increased reactive oxygen species......, activation of JNK, increased serine and decreased tyrosine phosphorylation of IRS-1, and decreased insulin signaling. Thus, Sirt3 plays an important role in diabetes through regulation of mitochondrial oxidation, reactive oxygen species production, and insulin resistance in skeletal muscle....

  8. Expression of Pannexin 1 and Pannexin 3 during skeletal muscle development, regeneration, and Duchenne muscular dystrophy.

    Science.gov (United States)

    Pham, Tammy L; St-Pierre, Marie-Eve; Ravel-Chapuis, Aymeric; Parks, Tara E C; Langlois, Stéphanie; Penuela, Silvia; Jasmin, Bernard J; Cowan, Kyle N

    2018-05-10

    Pannexin 1 (Panx1) and Pannexin 3 (Panx3) are single membrane channels recently implicated in myogenic commitment, as well as myoblast proliferation and differentiation in vitro. However, their expression patterns during skeletal muscle development and regeneration had yet to be investigated. Here, we show that Panx1 levels increase during skeletal muscle development becoming highly expressed together with Panx3 in adult skeletal muscle. In adult mice, Panx1 and Panx3 were differentially expressed in fast- and slow-twitch muscles. We also report that Panx1/PANX1 and Panx3/PANX3 are co-expressed in mouse and human satellite cells, which play crucial roles in skeletal muscle regeneration. Interestingly, Panx1 and Panx3 levels were modulated in muscle degeneration/regeneration, similar to the pattern seen during skeletal muscle development. As Duchenne muscular dystrophy is characterized by skeletal muscle degeneration and impaired regeneration, we next used mild and severe mouse models of this disease and found a significant dysregulation of Panx1 and Panx3 levels in dystrophic skeletal muscles. Together, our results are the first demonstration that Panx1 and Panx3 are differentially expressed amongst skeletal muscle types with their levels being highly modulated during skeletal muscle development, regeneration, and dystrophy. These findings suggest that Panx1 and Panx3 channels may play important and distinct roles in healthy and diseased skeletal muscles. © 2018 Wiley Periodicals, Inc.

  9. The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion.

    Science.gov (United States)

    Estrada, Beatriz; Maeland, Anne D; Gisselbrecht, Stephen S; Bloor, James W; Brown, Nicholas H; Michelson, Alan M

    2007-07-15

    Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where--as in myoblast fusion--membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells are unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane.

  10. Membrane glycoproteins of differentiating skeletal muscle cells

    International Nuclear Information System (INIS)

    Miller, K.R.; Remy, C.N.; Smith, P.B.

    1987-01-01

    The composition of N-linked glycoprotein oligosaccharides was studied in myoblasts and myotubes of the C2 muscle cell line. Oligosaccharides were radioactively labelled for 15 hr with [ 3 H] mannose and plasma membranes isolated. Ten glycopeptides were detected by SDS-PAGE and fluorography. The extent of labelling was 4-6 fold greater in myoblasts vs myotubes. A glycopeptide of Mr > 100,000 was found exclusively in myoblast membranes. Lectin chromatography revealed that the proportion of tri-, tetranntenary, biantennary and high mannose chains was similar throughout differentiation. The high mannose chain fraction was devoid of hybrid chains. The major high mannose chain contained nine mannose residues. The higher level of glycopeptide labelling in myoblasts vs myotubes corresponded to a 5-fold greater rate of protein synthesis. Pulse-chase experiments were used to follow the synthesis of the Dol-oligosaccharides. Myoblasts and myotubes labelled equivalently the glucosylated tetradecasaccharide but myoblasts labelled the smaller intermediates 3-4 greater than myotubes. Myoblasts also exhibited a 2-3 fold higher Dol-P dependent glycosyl transferase activity for chain elongation and Dol-sugar synthesis. Together these results show that the degree of protein synthesis and level of Dol-P are contributing factors in the higher capacity of myoblasts to produce N-glycoproteins compared to myotubes

  11. Novel lncRNAs in myogenesis: a miR-31 overlapping transcript controls myoblast differentiation.

    KAUST Repository

    Ballarino, Monica; Cazzella, Valentina; D'Andrea, Daniel; Grassi, Luigi; Bisceglie, Lavinia; Cipriano, Andrea; Santini, Tiziana; Pinnarò , Chiara; Morlando, Mariangela; Tramontano, Anna; Bozzoni, Irene

    2014-01-01

    Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.

  12. Novel lncRNAs in myogenesis: a miR-31 overlapping transcript controls myoblast differentiation.

    KAUST Repository

    Ballarino, Monica

    2014-12-15

    Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.

  13. Degree of Suppression of Mouse Myoblast Cell Line C₂C12 Differentiation Varies According to Chondroitin Sulfate Subtype.

    Science.gov (United States)

    Warita, Katsuhiko; Oshima, Nana; Takeda-Okuda, Naoko; Tamura, Jun-Ichi; Hosaka, Yoshinao Z

    2016-10-21

    Chondroitin sulfate (CS), a type of glycosaminoglycan (GAG), is a factor involved in the suppression of myogenic differentiation. CS comprises two repeating sugars and has different subtypes depending on the position and number of bonded sulfate groups. However, the effect of each subtype on myogenic differentiation remains unclear. In this study, we spiked cultures of C₂C 12 myoblasts, cells which are capable of undergoing skeletal muscle differentiation, with one of five types of CS (CS-A, -B, -C, -D, or -E) and induced differentiation over a fixed time. After immunostaining of the formed myotubes with an anti-MHC antibody, we counted the number of nuclei in the myotubes and then calculated the fusion index (FI) as a measure of myotube differentiation. The FI values of all the CS-treated groups were lower than the FI value of the control group, especially the group treated with CS-E, which displayed notable suppression of myotube formation. To confirm that the sugar chain in CS-E is important in the suppression of differentiation, chondroitinase ABC (ChABC), which catabolizes CS, was added to the media. The addition of ChABC led to the degradation of CS-E, and neutralized the suppression of myotube formation by CS-E. Collectively, it can be concluded that the degree of suppression of differentiation depends on the subtype of CS and that CS-E strongly suppresses myogenic differentiation. We conclude that the CS sugar chain has inhibitory action against myoblast cell fusion.

  14. Functional evaluation of artificial skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique.

    Science.gov (United States)

    Yamamoto, Yasunori; Ito, Akira; Fujita, Hideaki; Nagamori, Eiji; Kawabe, Yoshinori; Kamihira, Masamichi

    2011-01-01

    Skeletal muscle tissue engineering is currently applied in a variety of research fields, including regenerative medicine, drug screening, and bioactuator development, all of which require the fabrication of biomimic and functional skeletal muscle tissues. In the present study, magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs. Histological studies revealed that elongated and multinucleated myotubes were observed within the tissue. Expression of muscle-specific markers, such as myogenin, myosin heavy chain and tropomyosin, were detected in the tissue constructs by western blot analysis. Further, creatine kinase activity increased during differentiation. In response to electric pulses, the artificial tissue constructs contracted to generate a physical force (the maximum twitch force, 33.2 μN [1.06 mN/mm2]). Rheobase and chronaxie of the tissue were determined as 4.45 V and 0.72 ms, respectively. These results indicate that the artificial skeletal muscle tissue constructs fabricated in this study were physiologically functional and the data obtained for the evaluation of their functional properties may provide useful information for future skeletal muscle tissue engineering studies.

  15. Loss of MyoD and Myf5 in Skeletal Muscle Stem Cells Results in Altered Myogenic Programming and Failed Regeneration

    Directory of Open Access Journals (Sweden)

    Masakazu Yamamoto

    2018-03-01

    Full Text Available Summary: MyoD and Myf5 are fundamental regulators of skeletal muscle lineage determination in the embryo, and their expression is induced in satellite cells following muscle injury. MyoD and Myf5 are also expressed by satellite cell precursors developmentally, although the relative contribution of historical and injury-induced expression to satellite cell function is unknown. We show that satellite cells lacking both MyoD and Myf5 (double knockout [dKO] are maintained with aging in uninjured muscle. However, injured muscle fails to regenerate and dKO satellite cell progeny accumulate in damaged muscle but do not undergo muscle differentiation. dKO satellite cell progeny continue to express markers of myoblast identity, although their myogenic programming is labile, as demonstrated by dramatic morphological changes and increased propensity for non-myogenic differentiation. These data demonstrate an absolute requirement for either MyoD or Myf5 in muscle regeneration and indicate that their expression after injury stabilizes myogenic identity and confers the capacity for muscle differentiation. : In this article, Goldhamer and colleagues show that loss of both MyoD and Myf5 in skeletal muscle satellite cells results in regenerative failure following injury. Satellite cell progeny accumulate in injured muscle and continue to express markers of myoblast identity, but do not undergo muscle differentiation, and exhibit a propensity for non-myogenic differentiation. Keywords: skeletal muscle regeneration, muscle stem cell programming, muscle differentiation, satellite cell, MyoD, Myf5, adipogenesis, fibrosis, conditional knockout, Cre/loxP

  16. Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids

    Science.gov (United States)

    Chromiak, J. A.; Shansky, J.; Perrone, C.; Vandenburgh, H. H.

    1998-01-01

    Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.

  17. Detection of Pancreatic Cancer-Induced Cachexia Using a Fluorescent Myoblast Reporter System and Analysis of Metabolite Abundance.

    Science.gov (United States)

    Winnard, Paul T; Bharti, Santosh K; Penet, Marie-France; Marik, Radharani; Mironchik, Yelena; Wildes, Flonne; Maitra, Anirban; Bhujwalla, Zaver M

    2016-03-15

    The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body "holistic" approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice-bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real-time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight losses of only 1.2% to 2.7% and tumor burdens of only approximately 79 to 170 mm(3). Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma into a reporter-carrying mouse may be able to provide a risk assessment of cachexia, with possible implications for therapeutic development. ©2015 American Association for Cancer Research.

  18. Transdifferentiation of myoblasts into osteoblasts - possible use for bone therapy.

    Science.gov (United States)

    Lin, Daphne P L; Carnagarin, Revathy; Dharmarajan, Arun; Dass, Crispin R

    2017-12-01

    Transdifferentiation is defined as the conversion of one cell type to another and is an ever-expanding field with a growing number of cells found to be capable of such a process. To date, the fact remains that there are limited treatment options for fracture healing, osteoporosis and bone repair post-destruction by bone tumours. Hence, this review focuses on the transdifferentiation of myoblast to osteoblast as a means to further understand the transdifferentiation process and to investigate a potential therapeutic option if successful. The potent osteoinductive effects of the bone morphogenetic protein-2 are largely implicated in the transdifferentiation of myoblast to osteoblast. Bone morphogenetic protein-2-induced activation of the Smad1 protein ultimately results in JunB synthesis, the first transcriptional step in myoblast dedifferentiation. The upregulation of the activating protein-1 binding activity triggers the transcription of the runt-related transcription factor 2 gene, a transcription factor that plays a major role in osteoblast differentiation. This potential transdifferentiation treatment may be utilised for dental implants, fracture healing, osteoporosis and bone repair post-destruction by bone tumours. © 2017 Royal Pharmaceutical Society.

  19. CD36 is required for myoblast fusion during myogenic differentiation

    International Nuclear Information System (INIS)

    Park, Seung-Yoon; Yun, Youngeun; Kim, In-San

    2012-01-01

    Highlights: ► CD36 expression was induced during myogenic differentiation. ► CD36 expression was localized in multinucleated myotubes. ► The expression of myogenic markers is attenuated in CD36 knockdown C2C12 cells. ► Knockdown of CD36 significantly inhibited myotube formation during differentiation. -- Abstract: Recently, CD36 has been found to be involved in the cytokine-induced fusion of macrophage. Myoblast fusion to form multinucleated myotubes is required for myogenesis and muscle regeneration. Because a search of gene expression database revealed the attenuation of CD36 expression in the muscles of muscular dystrophy patients, the possibility that CD36 could be required for myoblast fusion was investigated. CD36 expression was markedly up-regulated during myoblast differentiation and localized in multinucleated myotubes. Knockdown of endogenous CD36 significantly decreased the expression of myogenic markers as well as myotube formation. These results support the notion that CD36 plays an important role in cell fusion during myogenic differentiation. Our finding will aid the elucidation of the common mechanism governing cell-to-cell fusion in various fusion models.

  20. CD36 is required for myoblast fusion during myogenic differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Seung-Yoon [Department of Biochemistry, College of Medicine, Dongguk University and Medical Institute of Dongguk University, Gyeongju 780-714 (Korea, Republic of); Yun, Youngeun [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, In-San, E-mail: iskim@knu.ac.kr [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Biomedical Research Institute, Korea Institute Science and Technology, Seoul (Korea, Republic of)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer CD36 expression was induced during myogenic differentiation. Black-Right-Pointing-Pointer CD36 expression was localized in multinucleated myotubes. Black-Right-Pointing-Pointer The expression of myogenic markers is attenuated in CD36 knockdown C2C12 cells. Black-Right-Pointing-Pointer Knockdown of CD36 significantly inhibited myotube formation during differentiation. -- Abstract: Recently, CD36 has been found to be involved in the cytokine-induced fusion of macrophage. Myoblast fusion to form multinucleated myotubes is required for myogenesis and muscle regeneration. Because a search of gene expression database revealed the attenuation of CD36 expression in the muscles of muscular dystrophy patients, the possibility that CD36 could be required for myoblast fusion was investigated. CD36 expression was markedly up-regulated during myoblast differentiation and localized in multinucleated myotubes. Knockdown of endogenous CD36 significantly decreased the expression of myogenic markers as well as myotube formation. These results support the notion that CD36 plays an important role in cell fusion during myogenic differentiation. Our finding will aid the elucidation of the common mechanism governing cell-to-cell fusion in various fusion models.

  1. hHGF overexpression in myoblast sheets enhances their angiogenic potential in rat chronic heart failure.

    Directory of Open Access Journals (Sweden)

    Antti Siltanen

    2011-04-01

    Full Text Available After severe myocardial infarction (MI, heart failure results from ischemia, fibrosis, and remodeling. A promising therapy to enhance cardiac function and induce therapeutic angiogenesis via a paracrine mechanism in MI is myoblast sheet transplantation. We hypothesized that in a rat model of MI-induced chronic heart failure, this therapy could be further improved by overexpression of the antiapoptotic, antifibrotic, and proangiogenic hepatocyte growth factor (HGF in the myoblast sheets. We studied the ability of wild type (L6-WT and human HGF-expressing (L6-HGF L6 myoblast sheet-derived paracrine factors to stimulate cardiomyocyte, endothelial cell, or smooth muscle cell migration in culture. Further, we studied the autocrine effect of hHGF-expression on myoblast gene expression profiles by use of microarray analysis. We induced MI in Wistar rats by left anterior descending coronary artery (LAD ligation and allowed heart failure to develop for 4 weeks. Thereafter, we administered L6-WT (n = 15 or L6-HGF (n = 16 myoblast sheet therapy. Control rats (n = 13 underwent LAD ligation and rethoracotomy without therapy, and five rats underwent a sham operation in both surgeries. We evaluated cardiac function with echocardiography at 2 and 4 weeks after therapy, and analyzed cardiac angiogenesis and left ventricular architecture from histological sections at 4 weeks. Paracrine mediators from L6-HGF myoblast sheets effectively induced migration of cardiac endothelial and smooth muscle cells but not cardiomyocytes. Microarray data revealed that hHGF-expression modulated myoblast gene expression. In vivo, L6-HGF sheet therapy effectively stimulated angiogenesis in the infarcted and non-infarcted areas. Both L6-WT and L6-HGF therapies enhanced cardiac function and inhibited remodeling in a similar fashion. In conclusion, L6-HGF therapy effectively induced angiogenesis in the chronically failing heart. Cardiac function, however, was not further

  2. Brain and muscle Arnt-like 1 promotes skeletal muscle regeneration through satellite cell expansion

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, Somik [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yin, Hongshan [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Cardiovascular Medicine, Third Affiliated Hospital, Hebei Medical University, Shijiazhuang 050051, Hebei (China); Nam, Deokhwa [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Li, Yong [Department of Pediatric Surgery, Center for Stem Cell Research and Regenerative Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030 (United States); Ma, Ke, E-mail: kma@houstonmethodist.org [Center for Diabetes Research, Department of Medicine, Houston Methodist Research Institute, Houston, TX 77030 (United States)

    2015-02-01

    Circadian clock is an evolutionarily conserved timing mechanism governing diverse biological processes and the skeletal muscle possesses intrinsic functional clocks. Interestingly, although the essential clock transcription activator, Brain and muscle Arnt-like 1 (Bmal1), participates in maintenance of muscle mass, little is known regarding its role in muscle growth and repair. In this report, we investigate the in vivo function of Bmal1 in skeletal muscle regeneration using two muscle injury models. Bmal1 is highly up-regulated by cardiotoxin injury, and its genetic ablation significantly impairs regeneration with markedly suppressed new myofiber formation and attenuated myogenic induction. A similarly defective regenerative response is observed in Bmal1-null mice as compared to wild-type controls upon freeze injury. Lack of satellite cell expansion accounts for the regeneration defect, as Bmal1{sup −/−} mice display significantly lower satellite cell number with nearly abolished induction of the satellite cell marker, Pax7. Furthermore, satellite cell-derived primary myoblasts devoid of Bmal1 display reduced growth and proliferation ex vivo. Collectively, our results demonstrate, for the first time, that Bmal1 is an integral component of the pro-myogenic response that is required for muscle repair. This mechanism may underlie its role in preserving adult muscle mass and could be targeted therapeutically to prevent muscle-wasting diseases. - Highlights: • Bmal1 is highly inducible by muscle injury and myogenic stimuli. • Genetic ablation of Bmal1 significantly impairs muscle regeneration. • Bmal1 promotes satellite cell expansion during muscle regeneration. • Bmal1-deficient primary myoblasts display attenuated growth and proliferation.

  3. Brain and muscle Arnt-like 1 promotes skeletal muscle regeneration through satellite cell expansion

    International Nuclear Information System (INIS)

    Chatterjee, Somik; Yin, Hongshan; Nam, Deokhwa; Li, Yong; Ma, Ke

    2015-01-01

    Circadian clock is an evolutionarily conserved timing mechanism governing diverse biological processes and the skeletal muscle possesses intrinsic functional clocks. Interestingly, although the essential clock transcription activator, Brain and muscle Arnt-like 1 (Bmal1), participates in maintenance of muscle mass, little is known regarding its role in muscle growth and repair. In this report, we investigate the in vivo function of Bmal1 in skeletal muscle regeneration using two muscle injury models. Bmal1 is highly up-regulated by cardiotoxin injury, and its genetic ablation significantly impairs regeneration with markedly suppressed new myofiber formation and attenuated myogenic induction. A similarly defective regenerative response is observed in Bmal1-null mice as compared to wild-type controls upon freeze injury. Lack of satellite cell expansion accounts for the regeneration defect, as Bmal1 −/− mice display significantly lower satellite cell number with nearly abolished induction of the satellite cell marker, Pax7. Furthermore, satellite cell-derived primary myoblasts devoid of Bmal1 display reduced growth and proliferation ex vivo. Collectively, our results demonstrate, for the first time, that Bmal1 is an integral component of the pro-myogenic response that is required for muscle repair. This mechanism may underlie its role in preserving adult muscle mass and could be targeted therapeutically to prevent muscle-wasting diseases. - Highlights: • Bmal1 is highly inducible by muscle injury and myogenic stimuli. • Genetic ablation of Bmal1 significantly impairs muscle regeneration. • Bmal1 promotes satellite cell expansion during muscle regeneration. • Bmal1-deficient primary myoblasts display attenuated growth and proliferation

  4. Human myotubes from myoblast cultures undergoing senescence exhibit defects in glucose and lipid metabolism

    DEFF Research Database (Denmark)

    Nehlin, Jan O; Just, Marlene; Rustan, Arild C

    2011-01-01

    Adult stem cells are known to have a finite replication potential. Muscle biopsy-derived human satellite cells (SCs) were grown at different passages and differentiated to human myotubes in culture to analyze the functional state of various carbohydrate and lipid metabolic pathways. As the prolif......Adult stem cells are known to have a finite replication potential. Muscle biopsy-derived human satellite cells (SCs) were grown at different passages and differentiated to human myotubes in culture to analyze the functional state of various carbohydrate and lipid metabolic pathways...... number and could be explained by reduced incorporation into diacyl- and triacylglycerols. The levels of long-chain acyl-CoA esters decreased with increased passage number. Late-passage, non-proliferating, myoblast cultures showed strong senescence-associated β-galactosidase activity indicating...... that the observed metabolic defects accompany the induction of a senescent state. The main function of SCs is regeneration and skeletal muscle-build up. Thus, the metabolic defects observed during aging of SC-derived myotubes could have a role in sarcopenia, the gradual age-related loss of muscle mass and strength....

  5. Ductile electroactive biodegradable hyperbranched polylactide copolymers enhancing myoblast differentiation.

    Science.gov (United States)

    Xie, Meihua; Wang, Ling; Guo, Baolin; Wang, Zhong; Chen, Y Eugene; Ma, Peter X

    2015-12-01

    Myotube formation is crucial to restoring muscular functions, and biomaterials that enhance the myoblast differentiation into myotubes are highly desirable for muscular repair. Here, we report the synthesis of electroactive, ductile, and degradable copolymers and their application in enhancing the differentiation of myoblasts to myotubes. A hyperbranched ductile polylactide (HPLA) was synthesized and then copolymerized with aniline tetramer (AT) to produce a series of electroactive, ductile and degradable copolymers (HPLAAT). The HPLA and HPLAAT showed excellent ductility with strain to failure from 158.9% to 42.7% and modulus from 265.2 to 758.2 MPa. The high electroactivity of the HPLAAT was confirmed by UV spectrometer and cyclic voltammogram measurements. These HPLAAT polymers also showed improved thermal stability and controlled biodegradation rate compared to HPLA. Importantly, when applying these polymers for myotube formation, the HPLAAT significantly improved the proliferation of C2C12 myoblasts in vitro compared to HPLA. Furthermore, these polymers greatly promoted myogenic differentiation of C2C12 cells as measured by quantitative analysis of myotube number, length, diameter, maturation index, and gene expression of MyoD and TNNT. Together, our study shows that these electroactive, ductile and degradable HPLAAT copolymers represent significantly improved biomaterials for muscle tissue engineering compared to HPLA. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Estrogens enhance myoblast differentiation in facioscapulohumeral muscular dystrophy by antagonizing DUX4 activity

    OpenAIRE

    Teveroni, Emanuela; Pellegrino, Marsha; Sacconi, Sabrina; Calandra, Patrizia; Cascino, Isabella; Farioli-Vecchioli, Stefano; Puma, Angela; Garibaldi, Matteo; Morosetti, Roberta; Tasca, Giorgio; Ricci, Enzo; Trevisan, Carlo Pietro; Galluzzi, Giuliana; Pontecorvi, Alfredo; Crescenzi, Marco

    2017-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that...

  7. Advancements in stem cells treatment of skeletal muscle wasting

    Directory of Open Access Journals (Sweden)

    mirella emeregalli

    2014-02-01

    Full Text Available Muscular dystrophies (MDs are a heterogeneous group of inherited disorders, in which progressive muscle wasting and weakness is often associated with exhaustion of muscle regeneration potential. Although physiological properties of skeletal muscle tissue are now well known, no treatments are effective for these diseases. Muscle regeneration was attempted by means transplantation of myogenic cells (from myoblast to embryonic stem cells and also by interfering with the malignant processes that originate in pathological tissues, such as uncontrolled fibrosis and inflammation. Taking into account the advances in the isolation of new subpopulation of stem cells and in the creation of artificial stem cell niches, we discuss how these emerging technologies offer great promises for therapeutic approaches to muscle diseases and muscle wasting associated with aging.

  8. Dynamic Support Culture of Murine Skeletal Muscle-Derived Stem Cells Improves Their Cardiogenic Potential In Vitro

    Directory of Open Access Journals (Sweden)

    Klaus Neef

    2015-01-01

    Full Text Available Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells.

  9. Myoblast transplantation for heart repair: A review of the state of the field

    Institute of Scientific and Technical Information of China (English)

    Howard J. Leonhardt; Michael Brown

    2006-01-01

    Over 200 humans have been treated with myoblast transplantation for heart muscle repair since June 2000. Bioheart sponsored percutaneous delivery studies began in May 2001 in Europe. Approximately one third of the patients have exhibited substantial improvement in left ventricular ejection fraction (LVEF) of over 30% and two heart failure class improvements. Over 80% of the patients have exhibited one heart failure class improvement with moderate improvement of LVEF. Clinical trials seem to demonstrate a marked reduction in emergency hospitalizations in myoblast treated patients. Many years of careful studies have lead to randomized controlled studies that are enrolling patients now at numerous centers worldwide. A firm conclusion on the safety and efficacy of myoblast transplantation cannot be determined until these randomized studies are completed. Final results from randomized controlled studies should be available soon. (J Geriatr Cardiol 2006;3:165-7.)

  10. Irisin is a pro-myogenic factor that induces skeletal muscle hypertrophy and rescues denervation-induced atrophy.

    Science.gov (United States)

    Reza, Musarrat Maisha; Subramaniyam, Nathiya; Sim, Chu Ming; Ge, Xiaojia; Sathiakumar, Durgalakshmi; McFarlane, Craig; Sharma, Mridula; Kambadur, Ravi

    2017-10-24

    Exercise induces expression of the myokine irisin, which is known to promote browning of white adipose tissue and has been shown to mediate beneficial effects following exercise. Here we show that irisin induces expression of a number of pro-myogenic and exercise response genes in myotubes. Irisin increases myogenic differentiation and myoblast fusion via activation of IL6 signaling. Injection of irisin in mice induces significant hypertrophy and enhances grip strength of uninjured muscle. Following skeletal muscle injury, irisin injection improves regeneration and induces hypertrophy. The effects of irisin on hypertrophy are due to activation of satellite cells and enhanced protein synthesis. In addition, irisin injection rescues loss of skeletal muscle mass following denervation by enhancing satellite cell activation and reducing protein degradation. These data suggest that irisin functions as a pro-myogenic factor in mice.

  11. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Young Woo [Cell Therapy and Tissue Engineering Center, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin [Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Park, Won Jin [Dr. Park' s Aesthetic Clinic, Seoul (Korea, Republic of); Kong, Jee Hyun; Shim, Kwang Yong [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Lee, Jong In, E-mail: oncochem@yonsei.ac.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Kim, Hyun Soo, E-mail: khsmd@unitel.co.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of)

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  12. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    International Nuclear Information System (INIS)

    Eom, Young Woo; Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin; Park, Won Jin; Kong, Jee Hyun; Shim, Kwang Yong; Lee, Jong In; Kim, Hyun Soo

    2011-01-01

    Highlights: → hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. → Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. → hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  13. Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system.

    Science.gov (United States)

    Mu, Xiaodong; Peng, Hairong; Pan, Haiying; Huard, Johnny; Li, Yong

    2011-02-03

    Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

  14. Phosphoinositide 3-kinase/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

    Science.gov (United States)

    Lim, Jeong A; Woo, Joo Hong; Kim, Hye Sun

    2008-09-01

    In this study, it was found that undifferentiated myoblasts were more vulnerable to menadione-induced oxidative stress than differentiated myotubes. Cell death occurred with a relatively low concentration of menadione in myoblasts compared to myotubes. With the same concentration of menadione, the Bcl-2/Bax ratio decreased and nuclei containing condensed chromatin were observed in myoblasts to a greater extent than in myotubes. However, myotubes became increasingly susceptible to menadione when phosphoinositide 3-kinase (PI3-K) was blocked by pre-incubation with LY294002, a PI3-K inhibitor. Actually, PI3-K activity was reduced by menadione in myoblasts but not in myotubes. In addition, the phosphorylation of Akt, a downstream effector of PI3-K, was inhibited in myoblasts by menadione but increased in myotubes. Both LY294002 and API-2, an Akt inhibitor, decreased the Bcl-2/Bax ratio in menadione-exposed myotubes. These results suggest that the differential activity of PI3-K/Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress.

  15. The effect of caffeine on skeletal muscle anabolic signaling and hypertrophy.

    Science.gov (United States)

    Moore, Timothy M; Mortensen, Xavier M; Ashby, Conrad K; Harris, Alexander M; Kump, Karson J; Laird, David W; Adams, Aaron J; Bray, Jeremy K; Chen, Ting; Thomson, David M

    2017-06-01

    Caffeine is a widely consumed stimulant with the potential to enhance physical performance through multiple mechanisms. However, recent in vitro findings have suggested that caffeine may block skeletal muscle anabolic signaling through AMP-activated protein kinase (AMPK)-mediated inhibition of mechanistic target of rapamycin (mTOR) signaling pathway. This could negatively affect protein synthesis and the capacity for muscle growth. The primary purpose of this study was to assess the effect of caffeine on in vivo AMPK and mTOR pathway signaling, protein synthesis, and muscle growth. In cultured C2C12 muscle cells, physiological levels of caffeine failed to impact mTOR activation or myoblast proliferation or differentiation. We found that caffeine administration to mice did not significantly enhance the phosphorylation of AMPK or inhibit signaling proteins downstream of mTOR (p70S6k, S6, or 4EBP1) or protein synthesis after a bout of electrically stimulated contractions. Skeletal muscle-specific knockout of LKB1, the primary AMPK activator in skeletal muscle, on the other hand, eliminated AMPK activation by contractions and enhanced S6k, S6, and 4EBP1 activation before and after contractions. In rats, the addition of caffeine did not affect plantaris hypertrophy induced by the tenotomy of the gastrocnemius and soleus muscles. In conclusion, caffeine administration does not impair skeletal muscle load-induced mTOR signaling, protein synthesis, or muscle hypertrophy.

  16. Productive infection of human skeletal muscle cells by pandemic and seasonal influenza A(H1N1 viruses.

    Directory of Open Access Journals (Sweden)

    Marion Desdouits

    Full Text Available Besides the classical respiratory and systemic symptoms, unusual complications of influenza A infection in humans involve the skeletal muscles. Numerous cases of acute myopathy and/or rhabdomyolysis have been reported, particularly following the outbreak of pandemic influenza A(H1N1 in 2009. The pathogenesis of these influenza-associated myopathies (IAM remains unkown, although the direct infection of muscle cells is suspected. Here, we studied the susceptibility of cultured human primary muscle cells to a 2009 pandemic and a 2008 seasonal influenza A(H1N1 isolate. Using cells from different donors, we found that differentiated muscle cells (i. e. myotubes were highly susceptible to infection by both influenza A(H1N1 isolates, whereas undifferentiated cells (i. e. myoblasts were partially resistant. The receptors for influenza viruses, α2-6 and α2-3 linked sialic acids, were detected on the surface of myotubes and myoblasts. Time line of viral nucleoprotein (NP expression and nuclear export showed that the first steps of the viral replication cycle could take place in muscle cells. Infected myotubes and myoblasts exhibited budding virions and nuclear inclusions as observed by transmission electron microscopy and correlative light and electron microscopy. Myotubes, but not myoblasts, yielded infectious virus progeny that could further infect naive muscle cells after proteolytic treatment. Infection led to a cytopathic effect with the lysis of muscle cells, as characterized by the release of lactate dehydrogenase. The secretion of proinflammatory cytokines by muscle cells was not affected following infection. Our results are compatible with the hypothesis of a direct muscle infection causing rhabdomyolysis in IAM patients.

  17. An integrated strategy for analyzing the unique developmental programs of different myoblast subtypes.

    Directory of Open Access Journals (Sweden)

    2006-02-01

    Full Text Available An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets--based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes--provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides

  18. Deficient leukemia inhibitory factor signaling in muscle precursor cells from patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Broholm, Christa; Brandt, Claus; Schultz, Ninna S

    2012-01-01

    The cytokine leukemia-inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of muscle precursor cells, an important feature of skeletal muscle maintenance and repair. We hypothesized that muscle precursor cells from patients with type 2 diabetes had a deficient response...... nor proliferation rate was affected. In conclusion, although LIF and LIFR proteins were increased in muscle tissue and myoblasts from diabetic patients, LIF signaling and LIF-stimulated cell proliferation were impaired in diabetic myoblasts, suggesting a novel mechanism by which muscle function......RNA knockdown of suppressor of cytokine signaling (SOCS)3 in myoblast cultures established from healthy individuals and patients with type 2 diabetes. Myoblast proliferation rate was assessed by bromodeoxyuridine incorporation. LIF and LIFR proteins were increased in both muscle tissue and cultured myoblasts...

  19. Efficient direct conversion of human fibroblasts into myogenic lineage induced by co-transduction with MYCL and MYOD1.

    Science.gov (United States)

    Wakao, Junko; Kishida, Tsunao; Fumino, Shigehisa; Kimura, Koseki; Yamamoto, Kenta; Kotani, Shin-Ichiro; Mizushima, Katsura; Naito, Yuji; Yoshikawa, Toshikazu; Tajiri, Tatsuro; Mazda, Osam

    2017-06-24

    The skeletal muscle consists of contractile myofibers and plays essential roles for maintenance of body posture, movement, and metabolic regulation. During the development and regeneration of the skeletal muscle tissue, the myoblasts fuse into multinucleated myotubes that subsequently form myofibers. Transplantation of myoblasts may make possible a novel regenerative therapy against defects or dysfunction of the skeletal muscle. It is reported that rodent fibroblasts are converted into myoblast-like cells and fuse to form syncytium after forced expression of exogenous myogenic differentiation 1 (MYOD1) that is a key transcription factor for myoblast differentiation. But human fibroblasts are less efficiently converted into myoblasts and rarely fused by MYOD1 alone. Here we found that transduction of v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog (MYCL) gene in combination with MYOD1 gene induced myoblast-like phenotypes in human fibroblasts more strongly than MYOD1 gene alone. The rate of conversion was approximately 90%. The directly converted myoblasts (dMBs) underwent fusion in an ERK5 pathway-dependent manner. The dMBs also formed myofiber-like structure in vivo after an inoculation into mice at the subcutaneous tissue. The present results strongly suggest that the combination of MYCL plus MYOD1 may promote direct conversion of human fibroblasts into functional myoblasts that could potentially be used for regenerative therapy for muscle diseases and congenital muscle defects. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. First intron of nestin gene regulates its expression during C2C12 myoblast ifferentiation

    Institute of Scientific and Technical Information of China (English)

    Hua Zhong; Zhigang Jin; Yongfeng Chen; Ting Zhang; Wei Bian; Xing Cui; Naihe Jing

    2008-01-01

    Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China Nestin is an intermediate filament protein expressed in neural progenitor cells and in developing skeletal muscle. Nestin has been widely used as a neural progenitor cell marker. It is well established that the specific expression of the nestin gene in neural progenitor cells is conferred by the neural-specific enhancer located in the second intron of the nestin gene. However, the transcriptional mechanism of nestin expression in developing muscle is still unclear. In this study, we identified a muscle cell-specific enhancer in the first intron of mouse nestin gene in mouse myoblast C2C12 cells.We localized the core enhancer activity to the 291-661 region of the first intron, and showed that the two E-boxes in the core enhancer region were important for enhancer activity in differentiating C2C12 cells. We also showed that MyoD protein was involved in the regulation of nestin expression in the myogenic differentiation of C2C12 cells.

  1. Biomarker-free dielectrophoretic sorting of differentiating myoblast multipotent progenitor cells and their membrane analysis by Raman spectroscopy.

    Science.gov (United States)

    Muratore, Massimo; Srsen, Vlastimil; Waterfall, Martin; Downes, Andrew; Pethig, Ronald

    2012-09-01

    Myoblasts are muscle derived mesenchymal stem cell progenitors that have great potential for use in regenerative medicine, especially for cardiomyogenesis grafts and intracardiac cell transplantation. To utilise such cells for pre-clinical and clinical applications, and especially for personalized medicine, it is essential to generate a synchronised, homogenous, population of cells that display phenotypic and genotypic homogeneity within a population of cells. We demonstrate that the biomarker-free technique of dielectrophoresis (DEP) can be used to discriminate cells between stages of differentiation in the C2C12 myoblast multipotent mouse model. Terminally differentiated myotubes were separated from C2C12 myoblasts to better than 96% purity, a result validated by flow cytometry and Western blotting. To determine the extent to which cell membrane capacitance, rather than cell size, determined the DEP response of a cell, C2C12 myoblasts were co-cultured with GFP-expressing MRC-5 fibroblasts of comparable size distributions (mean diameter ∼10 μm). A DEP sorting efficiency greater than 98% was achieved for these two cell types, a result concluded to arise from the fibroblasts possessing a larger membrane capacitance than the myoblasts. It is currently assumed that differences in membrane capacitance primarily reflect differences in the extent of folding or surface features of the membrane. However, our finding by Raman spectroscopy that the fibroblast membranes contained a smaller proportion of saturated lipids than those of the myoblasts suggests that the membrane chemistry should also be taken into account.

  2. Deletion of the Ste20-like kinase SLK in skeletal muscle results in a progressive myopathy and muscle weakness.

    Science.gov (United States)

    Pryce, Benjamin R; Al-Zahrani, Khalid N; Dufresne, Sébastien; Belkina, Natalya; Labrèche, Cédrik; Patino-Lopez, Genaro; Frenette, Jérôme; Shaw, Stephen; Sabourin, Luc A

    2017-02-02

    The Ste20-like kinase, SLK, plays an important role in cell proliferation and cytoskeletal remodeling. In fibroblasts, SLK has been shown to respond to FAK/Src signaling and regulate focal adhesion turnover through Paxillin phosphorylation. Full-length SLK has also been shown to be essential for embryonic development. In myoblasts, the overexpression of a dominant negative SLK is sufficient to block myoblast fusion. In this study, we crossed the Myf5-Cre mouse model with our conditional SLK knockout model to delete SLK in skeletal muscle. A thorough analysis of skeletal muscle tissue was undertaken in order to identify defects in muscle development caused by the lack of SLK. Isometric force analysis was performed on adult knockout mice and compared to age-matched wild-type mice. Furthermore, cardiotoxin injections were performed followed by immunohistochemistry for myogenic markers to assess the efficiency muscle regeneration following SLK deletion. We show here that early deletion of SLK from the myogenic lineage does not markedly impair skeletal muscle development but delays the regenerative process. Interestingly, adult mice (~6 months) display an increase in the proportion of central nuclei and increased p38 activation. Furthermore, mice as young as 3 months old present with decreased force generation, suggesting that the loss of SLK impairs myofiber stability and function. Assessment of structural components revealed aberrant localization of focal adhesion proteins, such as FAK and paxillin. Our data show that the loss of SLK results in unstable myofibers resulting in a progressive myopathy. Additionally, the loss of SLK resulted in a delay in muscle regeneration following cardiotoxin injections. Our results show that SLK is dispensable for muscle development and regeneration but is required for myofiber stability and optimal force generation.

  3. Delta-like 1 homolog (dlk1: a marker for rhabdomyosarcomas implicated in skeletal muscle regeneration.

    Directory of Open Access Journals (Sweden)

    Louise H Jørgensen

    Full Text Available Dlk1, a member of the Epidermal Growth Factor family, is expressed in multiple tissues during development, and has been detected in carcinomas and neuroendocrine tumors. Dlk1 is paternally expressed and belongs to a group of imprinted genes associated with rhabdomyosarcomas but not with other primitive childhood tumors to date. Here, we investigate the possible roles of Dlk1 in skeletal muscle tumor formation. We analyzed tumors of different mesenchymal origin for expression of Dlk1 and various myogenic markers and found that Dlk1 was present consistently in myogenic tumors. The coincident observation of Dlk1 with a highly proliferative state in myogenic tumors led us to subsequently investigate the involvement of Dlk1 in the control of the adult myogenic programme. We performed an injury study in Dlk1 transgenic mice, ectopically expressing ovine Dlk1 (membrane bound C2 variant under control of the myosin light chain promotor, and detected an early, enhanced formation of myotubes in Dlk1 transgenic mice. We then stably transfected the mouse myoblast cell line, C2C12, with full-length Dlk1 (soluble A variant and detected an inhibition of myotube formation, which could be reversed by adding Dlk1 antibody to the culture supernatant. These results suggest that Dlk1 is involved in controlling the myogenic programme and that the various splice forms may exert different effects. Interestingly, both in the Dlk1 transgenic mice and the DLK1-C2C12 cells, we detected reduced myostatin expression, suggesting that the effect of Dlk1 on the myogenic programme might involve the myostatin signaling pathway. In support of a relationship between Dlk1 and myostatin we detected reciprocal expression of these two transcripts during different cell cycle stages of human myoblasts. Together our results suggest that Dlk1 is a candidate marker for skeletal muscle tumors and might be involved directly in skeletal muscle tumor formation through a modulatory effect on the

  4. DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts.

    Science.gov (United States)

    Kanomata, Kazuhiro; Kokabu, Shoichiro; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2009-06-01

    Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

  5. The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts.

    Directory of Open Access Journals (Sweden)

    Shy Cian Khor

    Full Text Available Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF and α-tocopherol (ATF in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal expression, myogenic differentiation and myogenic regulatory factors (MRFs expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.

  6. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

    Directory of Open Access Journals (Sweden)

    Sebastian Frese

    Full Text Available Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human

  7. Clone-derived human AF-amniotic fluid stem cells are capable of skeletal myogenic differentiation in vitro and in vivo.

    Science.gov (United States)

    Ma, Xiaorong; Zhang, Shengli; Zhou, Junmei; Chen, Baisong; Shang, Yafeng; Gao, Tongbing; Wang, Xue; Xie, Hua; Chen, Fang

    2012-08-01

    Stem cell-based therapy may be the most promising method to cure skeletal muscle degenerative diseases such as Duchenne muscular dystrophy (DMD) and trauma in the future. Human amniotic fluid is enriched with early-stage stem cells from developing fetuses and these cells have cardiomyogenic potential both in vitro and in vivo. In the present study, we investigated the characteristics of human amniotic fluid-derived AF-type stem (HAF-AFS) cells by flow cytometry, immunofluorescence staining, reverse-transcription polymerase chain reaction, and osteogenic and adipogenic differentiation analysis. After confirming the stemness of HAF-AFS cells, we tested whether HAF-AFS cells could differentiate into skeletal myogenic cells in vitro and incorporate into regenerating skeletal muscle in vivo. By temporary exposure to the DNA demethylation agent 5-aza-2'-deoxycytidine (5-Aza dC) or co-cultured with C2C12 myoblasts, HAF-AFS cells differentiated into skeletal myogenic cells, expressing skeletal myogenic cell-specific markers such as Desmin, Troponin I (Tn I) and α-Actinin. Four weeks after transplantation into cardiotoxin-injured and X-ray-irradiated tibialis anterior (TA) muscles of NOD/SCID mice, HAF-AFS cells survived, differentiated into myogenic precursor cells and fused with host myofibres. The findings that HAF-AFS cells differentiate into myogenic cells in vitro and incorporate in skeletal muscle regeneration in vivo hold the promise of HAF-AFS cell-based therapy for skeletal muscle degenerative diseases. Copyright © 2012 John Wiley & Sons, Ltd.

  8. Biodistribution studies of 99mTc-labeled myoblasts in a murine model of muscular dystrophy

    International Nuclear Information System (INIS)

    Colombo, F.R.; Torrente, Y.; Casati, R.; Benti, R.; Corti, S.; Salani, S.; D'Angelo, M.G.; DeLiso, A.; Scarlato, G.; Bresolin, N.; Gerundini, P.

    2001-01-01

    The purpose of this study was twofold: first, to evaluate the myoblast labeling of various 99m Tc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding 99m Tc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [ 99m Tc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin

  9. Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.

    Science.gov (United States)

    Kubis, Hans-Peter; Scheibe, Renate J; Decker, Brigitte; Hufendiek, Karsten; Hanke, Nina; Gros, Gerolf; Meissner, Joachim D

    2016-04-01

    A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation. © 2015 International Federation for Cell Biology.

  10. Decellularized Human Skeletal Muscle as Biologic Scaffold for Reconstructive Surgery

    Directory of Open Access Journals (Sweden)

    Andrea Porzionato

    2015-07-01

    Full Text Available Engineered skeletal muscle tissues have been proposed as potential solutions for volumetric muscle losses, and biologic scaffolds have been obtained by decellularization of animal skeletal muscles. The aim of the present work was to analyse the characteristics of a biologic scaffold obtained by decellularization of human skeletal muscles (also through comparison with rats and rabbits and to evaluate its integration capability in a rabbit model with an abdominal wall defect. Rat, rabbit and human muscle samples were alternatively decellularized with two protocols: n.1, involving sodium deoxycholate and DNase I; n.2, trypsin-EDTA and Triton X-NH4OH. Protocol 2 proved more effective, removing all cellular material and maintaining the three-dimensional networks of collagen and elastic fibers. Ultrastructural analyses with transmission and scanning electron microscopy confirmed the preservation of collagen, elastic fibres, glycosaminoglycans and proteoglycans. Implantation of human scaffolds in rabbits gave good results in terms of integration, although recellularization by muscle cells was not completely achieved. In conclusion, human skeletal muscles may be effectively decellularized to obtain scaffolds preserving the architecture of the extracellular matrix and showing mechanical properties suitable for implantation/integration. Further analyses will be necessary to verify the suitability of these scaffolds for in vitro recolonization by autologous cells before in vivo implantation.

  11. Injectable skeletal muscle matrix hydrogel promotes neovascularization and muscle cell infiltration in a hindlimb ischemia model

    Directory of Open Access Journals (Sweden)

    JA DeQuach

    2012-06-01

    Full Text Available Peripheral artery disease (PAD currently affects approximately 27 million patients in Europe and North America, and if untreated, may progress to the stage of critical limb ischemia (CLI, which has implications for amputation and potential mortality. Unfortunately, few therapies exist for treating the ischemic skeletal muscle in these conditions. Biomaterials have been used to increase cell transplant survival as well as deliver growth factors to treat limb ischemia; however, existing materials do not mimic the native skeletal muscle microenvironment they are intended to treat. Furthermore, no therapies involving biomaterials alone have been examined. The goal of this study was to develop a clinically relevant injectable hydrogel derived from decellularized skeletal muscle extracellular matrix and examine its potential for treating PAD as a stand-alone therapy by studying the material in a rat hindlimb ischemia model. We tested the mitogenic activity of the scaffold’s degradation products using an in vitro assay and measured increased proliferation rates of smooth muscle cells and skeletal myoblasts compared to collagen. In a rat hindlimb ischemia model, the femoral artery was ligated and resected, followed by injection of 150 µL of skeletal muscle matrix or collagen 1 week post-injury. We demonstrate that the skeletal muscle matrix increased arteriole and capillary density, as well as recruited more desmin-positive and MyoD-positive cells compared to collagen. Our results indicate that this tissue-specific injectable hydrogel may be a potential therapy for treating ischemia related to PAD, as well as have potential beneficial effects on restoring muscle mass that is typically lost in CLI.

  12. Primary skeletal muscle myoblasts from chronic heart failure patients exhibit loss of anti-inflammatory and proliferative activity

    NARCIS (Netherlands)

    Sente, T.; Berendoncks, A.M. Van; Jonckheere, A.I.; Rodenburg, R.J.T.; Lauwers, P.; Hoof, V. Van; Wouters, A.; Lardon, F.; Hoymans, V.Y.; Vrints, C.J.

    2016-01-01

    BACKGROUND: Peripheral skeletal muscle wasting is a common finding with adverse effects in chronic heart failure (HF). Whereas its clinical relevance is beyond doubt, the underlying pathophysiological mechanisms are not yet fully elucidated. We aimed to introduce and characterize the primary culture

  13. Proliferation of Human Primary Myoblasts Is Associated with Altered Energy Metabolism in Dependence on Ageing In Vivo and In Vitro

    Directory of Open Access Journals (Sweden)

    Reedik Pääsuke

    2016-01-01

    Full Text Available Background. Ageing is associated with suppressed regenerative potential of muscle precursor cells due to decrease of satellite cells and suppressive intramuscular milieu on their activation, associated with ageing-related low-grade inflammation. The aim of the study was to characterize the function of oxidative phosphorylation (OXPHOS, glycolysis, adenylate kinase (AK, and creatine kinase (CK mediated systems in young and older individuals. Materials and Methods. Myoblasts were cultivated from biopsies taken by transcutaneous conchotomy from vastus lateralis muscle in young (20–29 yrs, n=7 and older (70–79 yrs, n=7 subjects. Energy metabolism was assessed in passages 2 to 6 by oxygraphy and enzyme analysis. Results. In myoblasts of young and older subjects the rate of OXPHOS decreased during proliferation from passages 2 to 6. The total activities of CK and AK decreased. Myoblasts of passage 2 cultivated from young muscle showed higher rate of OXPHOS and activities of CK and AK compared to myoblasts from older subjects while hexokinase and pyruvate kinase were not affected by ageing. Conclusions. Proliferation of myoblasts in vitro is associated with downregulation of OXPHOS and energy storage and transfer systems. Ageing in vivo exerts an impact on satellite cells which results in altered metabolic profile in favour of the prevalence of glycolytic pathways over mitochondrial OXPHOS of myoblasts.

  14. Inhibition of Na+ channel currents in rat myoblasts by 4-aminopyridine

    International Nuclear Information System (INIS)

    Lu Boxun; Liu Linyun; Liao Lei; Zhang Zhihong; Mei Yanai

    2005-01-01

    Our previous study revealed that 4-aminopyridine (4-AP), a specific blocker of A-type current, could also inhibit inward Na + currents (I Na ) with a state-independent mechanism in rat cerebellar granule cells. In the present study, we report an inhibitory effect of 4-AP on voltage-gated and tetrodotoxin (TTX)-sensitive I Na recorded from cultured rat myoblasts. 4-AP inhibited I Na amplitude in a dose-dependent manner between the concentrations of 0.5 and 10 mM without significant alteration in the activation or inactivation kinetics of the channel. By comparison to the 4-AP-induced inhibitory effect on cerebellum neurons, the inhibitory effect on myoblasts was enhanced through repetitive pulse and inflected by changing frequency. Specifically, the lower the frequency of pulse, the higher the inhibition observed, suggesting that block manner is inversely use-dependent. Moreover, experiments adding 4-AP to the intracellular solution indicate that the inhibitory effects are localized inside the cell. Additionally, 4-AP significantly modifies the properties of steady-state activation and inactivation kinetics of the channel. Our data suggest that the K + channel blocker 4-AP inhibits both neuron and myoblast Na + channels via different mechanisms. These findings may also provide information regarding 4-AP-induced pharmacological and toxicological effects in clinical use and experimental research

  15. Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

    Science.gov (United States)

    Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

    2016-07-01

    Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.

  16. An Antibody Blocking Activin Type II Receptors Induces Strong Skeletal Muscle Hypertrophy and Protects from Atrophy

    Science.gov (United States)

    Minetti, Giulia C.; Sheppard, KellyAnn; Ibebunjo, Chikwendu; Feige, Jerome N.; Hartmann, Steffen; Brachat, Sophie; Rivet, Helene; Koelbing, Claudia; Morvan, Frederic; Hatakeyama, Shinji

    2014-01-01

    The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings. PMID:24298022

  17. Bone morphogenetic protein-2 functions as a negative regulator in the differentiation of myoblasts, but not as an inducer for the formations of cartilage and bone in mouse embryonic tongue

    Directory of Open Access Journals (Sweden)

    Suzuki Erika

    2011-07-01

    Full Text Available Abstract Background In vitro studies using the myogenic cell line C2C12 demonstrate that bone morphogenetic protein-2 (BMP-2 converts the developmental pathway of C2C12 from a myogenic cell lineage to an osteoblastic cell lineage. Further, in vivo studies using null mutation mice demonstrate that BMPs inhibit the specification of the developmental fate of myogenic progenitor cells. However, the roles of BMPs in the phases of differentiation and maturation in skeletal muscles have yet to be determined. The present study attempts to define the function of BMP-2 in the final stage of differentiation of mouse tongue myoblast. Results Recombinant BMP-2 inhibited the expressions of markers for the differentiation of skeletal muscle cells, such as myogenin, muscle creatine kinase (MCK, and fast myosin heavy chain (fMyHC, whereas BMP-2 siRNA stimulated such markers. Neither the recombinant BMP-2 nor BMP-2 siRNA altered the expressions of markers for the formation of cartilage and bone, such as osteocalcin, alkaline phosphatase (ALP, collagen II, and collagen X. Further, no formation of cartilage and bone was observed in the recombinant BMP-2-treated tongues based on Alizarin red and Alcian blue stainings. Neither recombinant BMP-2 nor BMP-2 siRNA affected the expression of inhibitor of DNA binding/differentiation 1 (Id1. The ratios of chondrogenic and osteogenic markers relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a house keeping gene were approximately 1000-fold lower than those of myogenic markers in the cultured tongue. Conclusions BMP-2 functions as a negative regulator for the final differentiation of tongue myoblasts, but not as an inducer for the formation of cartilage and bone in cultured tongue, probably because the genes related to myogenesis are in an activation mode, while the genes related to chondrogenesis and osteogenesis are in a silencing mode.

  18. Transplantation of Allogeneic PW1pos/Pax7neg Interstitial Cells Enhance Endogenous Repair of Injured Porcine Skeletal Muscle

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    Fiona C. Lewis, BSc, PhD

    2017-12-01

    Full Text Available Skeletal muscle-derived PW1pos/Pax7neg interstitial cells (PICs express and secrete a multitude of proregenerative growth factors and cytokines. Utilizing a porcine preclinical skeletal muscle injury model, delivery of allogeneic porcine PICs (pPICs significantly improved and accelerated myofiber regeneration and neocapillarization, compared with saline vehicle control-treated muscles. Allogeneic pPICs did not contribute to new myofibers or capillaries and were eliminated by the host immune system. In conclusion, allogeneic pPIC transplantation stimulated the endogenous stem cell pool to bring about enhanced autologous skeletal muscle repair and regeneration. This allogeneic cell approach is considered a cost-effective, easy to apply, and readily available regenerative therapeutic strategy.

  19. Biodistribution studies of {sup 99m}Tc-labeled myoblasts in a murine model of muscular dystrophy

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    Colombo, F.R. E-mail: colombof@policlinico.mi.it; Torrente, Y.; Casati, R.; Benti, R.; Corti, S.; Salani, S.; D' Angelo, M.G.; DeLiso, A.; Scarlato, G.; Bresolin, N.; Gerundini, P

    2001-11-01

    The purpose of this study was twofold: first, to evaluate the myoblast labeling of various {sup 99m}Tc complexes and to select the complex that best accomplishes this labeling, and second to evaluate the biodistribution of myoblasts labeled with this complex using mice with MDX muscular dystrophy (the murine homologue of Duchenne's muscular dystrophy). The following ligands were used to prepare the corresponding {sup 99m}Tc complexes: hexakis-methoxy-isobutyl-isonitrile (MIBI), bis(2-ethoxyethyl)diphosphinoethane (Tf), (RR,SS)-4,8-diaza-3,6,6,9-tetramethyl-undecane-2,10-dione-bisoxime (HM-PAO), bis(N-ethyl)dithiocarbamate (NEt), and bis(N-ethoxy, N-ethyl)dithiocarbamate (NOEt). One million murine myoblasts were incubated for 30-60 minutes with 5 mCi of each of the 99mTc complexes prepared from the above ligands. Viability was assessed by microscopic counting after trypan blue staining, and the radioactivity absorbed in the cells was measured after centrifugation. The compound with the highest uptake in cellular pellets was [{sup 99m}Tc]N-NOEt. The biodistribution of myoblasts labeled with this complex was evaluated after intraaortic injection in dystrophic mice. Such an approach has the potential of effecting widespread gene transfer through the bloodstream to muscles lacking dystrophin.

  20. Adipose Tissue-Derived Stem Cell Secreted IGF-1 Protects Myoblasts from the Negative Effect of Myostatin

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    Sebastian Gehmert

    2014-01-01

    Full Text Available Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs, these cells (ASCs provide a therapeutic option for Duchenne Muscular Dystrophy (DMD. But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases.

  1. Specific association of growth-associated protein 43 with calcium release units in skeletal muscles of lower vertebrates

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    G.A. Caprara

    2014-10-01

    Full Text Available Growth-associated protein 43 (GAP43, is a strictly conserved protein among vertebrates implicated in neuronal development and neurite branching. Since GAP43 structure contains a calmodulin-binding domain, this protein is able to bind calmodulin and gather it nearby membrane network, thus regulating cytosolic calcium and consequently calcium-dependent intracellular events. Even if for many years GAP43 has been considered a neuronal-specific protein, evidence from different laboratories described its presence in myoblasts, myotubes and adult skeletal muscle fibers. Data from our laboratory showed that GAP43 is localized between calcium release units (CRUs and mitochondria in mammalian skeletal muscle suggesting that, also in skeletal muscle, this protein can be a key player in calcium/calmodulin homeostasis. However, the previous studies could not clearly distinguish between a mitochondrion- or a triad-related positioning of GAP43. To solve this question, the expression and localization of GAP43 was studied in skeletal muscle of Xenopus and Zebrafish known to have triads located at the level of the Z-lines and mitochondria not closely associated with them. Western blotting and immunostaining experiments revealed the expression of GAP43 also in skeletal muscle of lower vertebrates (like amphibians and fishes, and that the protein is localized closely to the triad junction. Once more, these results and GAP43 structural features, support an involvement of the protein in the dynamic intracellular Ca2+ homeostasis, a common conserved role among the different species.

  2. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Dunham, Ian [EMBL-European Bioinformatics Institute (EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom); Murai, Kasumi [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Jones, Philip H., E-mail: phj20@cam.ac.uk [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom)

    2011-07-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  3. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    International Nuclear Information System (INIS)

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko; Dunham, Ian; Murai, Kasumi; Jones, Philip H.

    2011-01-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  4. Subglottic stenosis in short-statured children: a case for further investigation of airway symptoms in patients with skeletal dysplasias.

    Science.gov (United States)

    Lee, Jonathan H; Ellison, Jay W; Schears, Gregory J; Thompson, Dana M

    2006-01-01

    Clinical evaluation of children with skeletal dysplasias is often concentrated on morphologic and radiographic assessments, but many of these patients also have disease processes of the ear, nose, and throat. We report a case of an 11-month-old girl with an unknown short-limbed dwarfism, similar to acromicric dysplasia, with grade II subglottic stenosis. Laryngotracheoplasty with anterior autologous costal cartilage graft and posterior cricoid split was performed at age 13 months, with subsequent improvement of her airway status. In cases of children with skeletal dysplasias and obstructive airway symptoms, formal otolaryngologic evaluation is warranted for definitive diagnosis and treatment.

  5. [Molecular mechanism for ET-1-induced insulin resistance in skeletal muscle cells].

    Science.gov (United States)

    Horinouchi, Takahiro; Mazaki, Yuichi; Terada, Koji; Miwa, Soichi

    2018-01-01

    Insulin resistance is a condition where the sensitivity to insulin of the tissues expressing insulin receptor (InsR) is decreased due to a functional disturbance of InsR-mediated intracellular signaling. Insulin promotes the entry of glucose into the tissues and skeletal muscle is the most important tissue responsible for the insulin's action of decreasing blood glucose levels. Endothelin-1 (ET-1), a potent vasoconstrictor and pro-inflammatory peptide, induces insulin resistance through a direct action on skeletal muscle. However, the signaling pathways of ET-1-induced insulin resistance in skeletal muscle remain unclear. Here we show molecular mechanism underlying the inhibitory effect of ET-1 on insulin-stimulated Akt phosphorylation and glucose uptake in myotubes of rat L6 skeletal muscle cell line. mRNA expression levels of differentiation marker genes, MyoD and myogenin, were increased during L6 myoblasts differentiation into myotubes. Some of myotubes possessed the ability to spontaneously contract. In myotubes, insulin promoted Akt phosphorylation at Thr 308 and Ser 473 , and [ 3 H]-labelled 2-deoxy-D-glucose ([ 3 H]2-DG) uptake. The insulin-facilitated Akt phosphorylation and [ 3 H]2-DG uptake were inhibited by ET-1. The inhibitory effect of ET-1 was counteracted by blockade of ET type A receptor (ET A R), inhibition of G q/11 protein, and siRNA knockdown of G protein-coupled receptor kinase 2 (GRK2). The exogenously overexpressed GRK2 directly bound to endogenous Akt and their association was facilitated by ET-1. In summary, activation of ET A R with ET-1 inhibits insulin-induced Akt phosphorylation and [ 3 H]2-DG uptake in a G q/11 protein- and GRK2-dependent manner in skeletal muscle. These findings indicate that ET A R and GRK2 are potential targets for insulin resistance.

  6. A preclinical evaluation of an autologous living hyaline-like cartilaginous graft for articular cartilage repair: a pilot study.

    Science.gov (United States)

    Peck, Yvonne; He, Pengfei; Chilla, Geetha Soujanya V N; Poh, Chueh Loo; Wang, Dong-An

    2015-11-09

    In this pilot study, an autologous synthetic scaffold-free construct with hyaline quality, termed living hyaline cartilaginous graft (LhCG), was applied for treating cartilage lesions. Implantation of autologous LhCG was done at load-bearing regions of the knees in skeletally mature mini-pigs for 6 months. Over the course of this study, significant radiographical improvement in LhCG treated sites was observed via magnetic resonance imaging. Furthermore, macroscopic repair was effected by LhCG at endpoint. Microscopic inspection revealed that LhCG engraftment restored cartilage thickness, promoted integration with surrounding native cartilage, produced abundant cartilage-specific matrix molecules, and re-established an intact superficial tangential zone. Importantly, the repair efficacy of LhCG was quantitatively shown to be comparable to native, unaffected cartilage in terms of biochemical composition and biomechanical properties. There were no complications related to the donor site of cartilage biopsy. Collectively, these results imply that LhCG engraftment may be a viable approach for articular cartilage repair.

  7. G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

    Science.gov (United States)

    Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

    2013-12-01

    Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities. © 2013 FEBS.

  8. Comparative Analyses between Skeletal Muscle miRNAomes from Large White and Min Pigs Revealed MicroRNAs Associated with Postnatal Muscle Hypertrophy.

    Science.gov (United States)

    Sheng, Xihui; Wang, Ligang; Ni, Hemin; Wang, Lixian; Qi, Xiaolong; Xing, Shuhan; Guo, Yong

    2016-01-01

    The molecular mechanism regulated by microRNAs (miRNAs) that underlies postnatal hypertrophy of skeletal muscle is complex and remains unclear. Here, the miRNAomes of longissimus dorsi muscle collected at five postnatal stages (60, 120, 150, 180, and 210 days after birth) from Large White (commercial breed) and Min pigs (indigenous breed of China) were analyzed by Illumina sequencing. We identified 734 miRNAs comprising 308 annotated miRNAs and 426 novel miRNAs, of which 307 could be considered pig-specific. Comparative analysis between two breeds suggested that 60 and 120 days after birth were important stages for skeletal muscle hypertrophy and intramuscular fat accumulation. A total of 263 miRNAs were significantly differentially expressed between two breeds at one or more developmental stages. In addition, the differentially expressed miRNAs between every two adjacent developmental stages in each breed were determined. Notably, ssc-miR-204 was significantly more highly expressed in Min pig skeletal muscle at all postnatal stages compared with its expression in Large White pig skeletal muscle. Based on gene ontology and KEGG pathway analyses of its predicted target genes, we concluded that ssc-miR-204 may exert an impact on postnatal hypertrophy of skeletal muscle by regulating myoblast proliferation. The results of this study will help in elucidating the mechanism underlying postnatal hypertrophy of skeletal muscle modulated by miRNAs, which could provide valuable information for improvement of pork quality and human myopathy.

  9. Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

    Directory of Open Access Journals (Sweden)

    João A Paredes

    Full Text Available Loss of thymidine kinase 2 (TK2 causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA depletion syndrome (MDS in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

  10. Gene expression deregulation in postnatal skeletal muscle of TK2 deficient mice reveals a lower pool of proliferating myogenic progenitor cells.

    Science.gov (United States)

    Paredes, João A; Zhou, Xiaoshan; Höglund, Stefan; Karlsson, Anna

    2013-01-01

    Loss of thymidine kinase 2 (TK2) causes a heterogeneous myopathic form of mitochondrial DNA (mtDNA) depletion syndrome (MDS) in humans that predominantly affects skeletal muscle tissue. In mice, TK2 deficiency also affects several tissues in addition to skeletal muscle, including brain, heart, adipose tissue, kidneys and causes death about 3 weeks after birth. We analysed skeletal muscle and heart muscle tissues of Tk2 knockout mice at postnatal development phase and observed that TK2 deficient pups grew slower and their skeletal muscles appeared significantly underdeveloped, whereas heart was close to normal in size. Both tissues showed mtDNA depletion and mitochondria with altered ultrastructure, as revealed by transmission electron microscopy. Gene expression microarray analysis showed a strong down-regulation of genes involved in cell cycle and cell proliferation in both tissues, suggesting a lower pool of undifferentiated proliferating cells. Analysis of isolated primary myoblasts from Tk2 knockout mice showed slow proliferation, less ability to differentiate and signs of premature senescence, even in absence of mtDNA depletion. Our data demonstrate that TK2 deficiency disturbs myogenic progenitor cells function in postnatal skeletal muscle and we propose this as one of the causes of underdeveloped phenotype and myopathic characteristic of the TK2 deficient mice, in addition to the progressive mtDNA depletion, mitochondrial damage and respiratory chain deficiency in post-mitotic differentiated tissue.

  11. A New In Vitro Co-Culture Model Using Magnetic Force-Based Nanotechnology

    NARCIS (Netherlands)

    Takanari, Hiroki; Miwa, Keiko; Fu, Xianming; Nakai, Junichi; Ito, Akira; Ino, Kousuke; Honda, Hiroyuki; Tonomura, Wataru; Konishi, Satoshi; Opthof, Tobias; van der Heyden, Marcel Ag; Kodama, Itsuo; Lee, Jong-Kook

    2016-01-01

    Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with "patterned" or "randomly-mixed" co-culture of SkMBs

  12. Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model

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    In Young Choi

    2016-06-01

    Full Text Available Duchenne muscular dystrophy (DMD remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs. Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our “chemical-compound-based” strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological “dual-SMAD” inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form “rescued” multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human “DMD-in-a-dish” model using hiPSC-based disease modeling.

  13. Effect of heat stress on contractility of tissue-engineered artificial skeletal muscle.

    Science.gov (United States)

    Takagi, Shunya; Nakamura, Tomohiro; Fujisato, Toshia

    2018-01-23

    The effects of heat stress on tissue like skeletal muscle have been widely studied. However, the mechanism responsible for the effect of heat stress is still unclear. A useful experimental tissue model is necessary because muscle function in cell culture may differ from native muscle and measuring its contractility is difficult. We previously reported three-dimensional tissue-engineered artificial skeletal muscle (TEM) that can be easily set in a measurement apparatus for quantitative evaluation of contractility. We have now applied TEM to the investigation of heat stress. We analyzed contractility immediately after thermal exposure at 39 °C for 24 or 48 h to evaluate the acute effects and after thermal exposure followed by normal culture to evaluate the aftereffects. Peak twitch contractile force and time-to-peak twitch were used as contractile parameters. Heat stress increased the TCF in the early stage (1 week) after normal culture; the TCF decreased temporarily in the middle to late stages (2-3 weeks). These results suggest that heat stress may affect both myoblast fusion and myotube differentiation in the early stage of TEM culture, but not myotube maturation in the late stage. The TCF increase rate with thermal exposure was significantly higher than that without thermal exposure. Although detailed analysis at the molecular level is necessary for further investigation, our artificial skeletal muscle may be a promising tool for heat stress investigation.

  14. Microfluidic-enhanced 3D bioprinting of aligned myoblast-laden hydrogels leads to functionally organized myofibers in vitro and in vivo.

    Science.gov (United States)

    Costantini, Marco; Testa, Stefano; Mozetic, Pamela; Barbetta, Andrea; Fuoco, Claudia; Fornetti, Ersilia; Tamiro, Francesco; Bernardini, Sergio; Jaroszewicz, Jakub; Święszkowski, Wojciech; Trombetta, Marcella; Castagnoli, Luisa; Seliktar, Dror; Garstecki, Piotr; Cesareni, Gianni; Cannata, Stefano; Rainer, Alberto; Gargioli, Cesare

    2017-07-01

    We present a new strategy for the fabrication of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is based on a microfluidic printing head coupled to a co-axial needle extruder for high-resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we formulated a tailored bioink with a photocurable semi-synthetic biopolymer (PEG-Fibrinogen) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 3-5 days of culture, the encapsulated myoblasts started migrating and fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation, sarcomerogenesis, and functionality. Following subcutaneous implantation in the back of immunocompromised mice, bioprinted constructs generated organized artificial muscle tissue in vivo. Finally, we demonstrate that our microfluidic printing head allows to design three dimensional multi-cellular assemblies with an exquisite compartmentalization of the encapsulated cells. Our results demonstrate an enhanced myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid candidate for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for human clinical application. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  15. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

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    Aiko Amagai

    2012-01-01

    Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

  16. FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1.

    Directory of Open Access Journals (Sweden)

    Sandra J Feeney

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

  17. Transcriptional profiling identifies differentially expressed genes in developing turkey skeletal muscle

    Directory of Open Access Journals (Sweden)

    Velleman Sandra G

    2011-03-01

    Full Text Available Abstract Background Skeletal muscle growth and development from embryo to adult consists of a series of carefully regulated changes in gene expression. Understanding these developmental changes in agriculturally important species is essential to the production of high quality meat products. For example, consumer demand for lean, inexpensive meat products has driven the turkey industry to unprecedented production through intensive genetic selection. However, achievements of increased body weight and muscle mass have been countered by an increased incidence of myopathies and meat quality defects. In a previous study, we developed and validated a turkey skeletal muscle-specific microarray as a tool for functional genomics studies. The goals of the current study were to utilize this microarray to elucidate functional pathways of genes responsible for key events in turkey skeletal muscle development and to compare differences in gene expression between two genetic lines of turkeys. To achieve these goals, skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia, 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy, and 16wk (market age from two genetic lines: a randombred control line (RBC2 maintained without selection pressure, and a line (F selected from the RBC2 line for increased 16wk body weight. Array hybridizations were performed in two experiments: Experiment 1 directly compared the developmental stages within genetic line, while Experiment 2 directly compared the two lines within each developmental stage. Results A total of 3474 genes were differentially expressed (false discovery rate; FDR Conclusions The current study identified gene pathways and uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of

  18. TGF-β's delay skeletal muscle progenitor cell differentiation in an isoform-independent manner

    International Nuclear Information System (INIS)

    Schabort, Elske J.; Merwe, Mathilde van der; Loos, Benjamin; Moore, Frances P.; Niesler, Carola U.

    2009-01-01

    Satellite cells are a quiescent heterogenous population of mononuclear stem and progenitor cells which, once activated, differentiate into myotubes and facilitate skeletal muscle repair or growth. The Transforming Growth Factor-β (TGF-β) superfamily members are elevated post-injury and their importance in the regulation of myogenesis and wound healing has been demonstrated both in vitro and in vivo. Most studies suggest a negative role for TGF-β on satellite cell differentiation. However, none have compared the effect of these three isoforms on myogenesis in vitro. This is despite known isoform-specific effects of TGF-β1, -β2 and -β3 on wound repair in other tissues. In the current study we compared the effect of TGF-β1, -β2 and -β3 on proliferation and differentiation of the C2C12 myoblast cell-line. We found that, irrespective of the isoform, TGF-β increased proliferation of C2C12 cells by changing the cellular localisation of PCNA to promote cell division and prevent cell cycle exit. Concomitantly, TGF-β1, -β2 and -β3 delayed myogenic commitment by increasing MyoD degradation and decreasing myogenin expression. Terminal differentiation, as measured by a decrease in myosin heavy chain (MHC) expression, was also delayed. These results demonstrate that TGF-β promotes proliferation and delays differentiation of C2C12 myoblasts in an isoform-independent manner

  19. A New In Vitro Co-Culture Model Using Magnetic Force-Based Nanotechnology

    NARCIS (Netherlands)

    Takanari, Hiroki; Miwa, Keiko; Fu, Xian Ming; Nakai, Junichi; Ito, Akira; Ino, Kousuke; Honda, Hiroyuki; Tonomura, Wataru; Konishi, Satoshi; Opthof, Tobias; van der Heyden, Marcel A G; Kodama, Itsuo; Lee, Jong Kook

    2016-01-01

    Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with “patterned” or “randomly-mixed” co-culture of SkMBs

  20. Autologous Fat Injection for Augmented Mammoplasty

    International Nuclear Information System (INIS)

    Yoon, Eul Sik; Seo, Bo Kyoung; Yi, Ann; Cho, Kyu Ran

    2008-01-01

    Autologous fat injection is one of the methods utilized for augmented mammoplasty methods. In this surgical procedure, the fat for transfer is obtained from the donor site of the patient's own body by liposuction and the fat is then injected into the breast. We report here cases of three patients who underwent autologous fat injection. Two of the patients had palpable masses that were present after surgery. The serial imaging findings and surgical method of autologous fat transfer are demonstrated

  1. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  2. Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model.

    Science.gov (United States)

    Choi, In Young; Lim, HoTae; Estrellas, Kenneth; Mula, Jyothi; Cohen, Tatiana V; Zhang, Yuanfan; Donnelly, Christopher J; Richard, Jean-Philippe; Kim, Yong Jun; Kim, Hyesoo; Kazuki, Yasuhiro; Oshimura, Mitsuo; Li, Hongmei Lisa; Hotta, Akitsu; Rothstein, Jeffrey; Maragakis, Nicholas; Wagner, Kathryn R; Lee, Gabsang

    2016-06-07

    Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. Biocompatible, Biodegradable, and Electroactive Polyurethane-Urea Elastomers with Tunable Hydrophilicity for Skeletal Muscle Tissue Engineering.

    Science.gov (United States)

    Chen, Jing; Dong, Ruonan; Ge, Juan; Guo, Baolin; Ma, Peter X

    2015-12-30

    It remains a challenge to develop electroactive and elastic biomaterials to mimic the elasticity of soft tissue and to regulate the cell behavior during tissue regeneration. We designed and synthesized a series of novel electroactive and biodegradable polyurethane-urea (PUU) copolymers with elastomeric property by combining the properties of polyurethanes and conducting polymers. The electroactive PUU copolymers were synthesized from amine capped aniline trimer (ACAT), dimethylol propionic acid (DMPA), polylactide, and hexamethylene diisocyanate. The electroactivity of the PUU copolymers were studied by UV-vis spectroscopy and cyclic voltammetry. Elasticity and Young's modulus were tailored by the polylactide segment length and ACAT content. Hydrophilicity of the copolymer films was tuned by changing DMPA content and doping of the copolymer. Cytotoxicity of the PUU copolymers was evaluated by mouse C2C12 myoblast cells. The myogenic differentiation of C2C12 myoblasts on copolymer films was also studied by analyzing the morphology of myotubes and relative gene expression during myogenic differentiation. The chemical structure, thermal properties, surface morphology, and processability of the PUU copolymers were characterized by NMR, FT-IR, gel permeation chromatography (GPC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), and solubility testing, respectively. Those biodegradable electroactive elastic PUU copolymers are promising materials for repair of soft tissues such as skeletal muscle, cardiac muscle, and nerve.

  4. Angiotensin II Evokes Angiogenic Signals within Skeletal Muscle through Co-ordinated Effects on Skeletal Myocytes and Endothelial Cells

    Science.gov (United States)

    Gorman, Jennifer L.; Liu, Sammy T. K.; Slopack, Dara; Shariati, Khashayar; Hasanee, Adam; Olenich, Sara; Olfert, I. Mark; Haas, Tara L.

    2014-01-01

    Skeletal muscle overload induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2, leading to new capillary growth. We found that the overload-induced increase in angiogenesis, as well as increases in VEGF, MMP-2 and MT1-MMP transcripts were abrogated in muscle VEGF KO mice, highlighting the critical role of myocyte-derived VEGF in controlling this process. The upstream mediators that contribute to overload-induced expression of VEGF have yet to be ascertained. We found that muscle overload increased angiotensinogen expression, a precursor of angiotensin (Ang) II, and that Ang II signaling played an important role in basal VEGF production in C2C12 cells. Furthermore, matrix-bound VEGF released from myoblasts induced the activation of endothelial cells, as evidenced by elevated endothelial cell phospho-p38 levels. We also found that exogenous Ang II elevates VEGF expression, as well as MMP-2 transcript levels in C2C12 myotubes. Interestingly, these responses also were observed in skeletal muscle endothelial cells in response to Ang II treatment, indicating that these cells also can respond directly to the stimulus. The involvement of Ang II in muscle overload-induced angiogenesis was assessed. We found that blockade of AT1R-dependent Ang II signaling using losartan did not attenuate capillary growth. Surprisingly, increased levels of VEGF protein were detected in overloaded muscle from losartan-treated rats. Similarly, we observed elevated VEGF production in cultured endothelial cells treated with losartan alone or in combination with Ang II. These studies conclusively establish the requirement for muscle derived VEGF in overload-induced angiogenesis and highlight a role for Ang II in basal VEGF production in skeletal muscle. However, while Ang II signaling is activated following overload and plays a role in muscle VEGF production, inhibition of this pathway is not sufficient to halt overload

  5. SPARC Interacts with Actin in Skeletal Muscle in Vitro and in Vivo

    DEFF Research Database (Denmark)

    Jørgensen, Louise H; Jepsen, Pia Lørup; Boysen, Anders

    2017-01-01

    to actin. This interaction is present in regenerating myofibers of patients with Duchenne muscular dystrophy, polymyositis, and compartment syndrome. Analysis of the α-, β-, and γ-actin isoforms in SPARC knockout myoblasts reveals a changed expression pattern with dominance of γ-actin. In SPARC knockout......The cytoskeleton is an integral part of skeletal muscle structure, and reorganization of the cytoskeleton occurs during various modes of remodeling. We previously found that the extracellular matrix protein secreted protein acidic and rich in cysteine (SPARC) is up-regulated and expressed...... intracellularly in developing muscle, during regeneration and in myopathies, which together suggests that SPARC might serve a specific role within muscle cells. Using co-immunoprecipitation combined with mass spectrometry and verified by staining for direct protein-protein interaction, we find that SPARC binds...

  6. Black ginseng activates Akt signaling, thereby enhancing myoblast differentiation and myotube growth

    Directory of Open Access Journals (Sweden)

    Soo-Yeon Lee

    2018-01-01

    Conclusion: BG enhances myoblast differentiation and myotube hypertrophy by activating Akt/mTOR/p70S6k axis. Thus, our study demonstrates that BG has promising potential to treat or prevent muscle loss related to aging or other pathological conditions, such as diabetes.

  7. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    Directory of Open Access Journals (Sweden)

    Haohan Wang

    2016-03-01

    Full Text Available Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

  8. Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System.

    Science.gov (United States)

    Smith, Alec S T; Passey, Samantha L; Martin, Neil R W; Player, Darren J; Mudera, Vivek; Greensmith, Linda; Lewis, Mark P

    2016-01-01

    Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field. © 2016 S. Karger AG, Basel.

  9. Inflammatory effects of autologous, genetically modified autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses.

    Science.gov (United States)

    Pigott, J H; Ishihara, A; Wellman, M L; Russell, D S; Bertone, A L

    2013-01-01

    To compare the clinical and inflammatory joint responses to intra-articular injection of bone marrow-derived mesenchymal stem cells (MSC) including autologous, genetically modified autologous, allogeneic, or xenogeneic cells in horses. Six five-year-old Thoroughbred mares had one fetlock joint injected with Gey's balanced salt solution as the vehicle control. Each fetlock joint of each horse was subsequently injected with 15 million MSC from the described MSC groups, and were assessed for 28 days for clinical and inflammatory parameters representing synovitis, joint swelling, and pain. There were not any significant differences between autologous and genetically modified autologous MSC for synovial fluid total nucleated cell count, total protein, interleukin (IL)-6, IL-10, fetlock circumference, oedema score, pain-free range-of-motion, and soluble gene products that were detected for at least two days. Allogeneic and xenogeneic MSC produced a greater increase in peak of inflammation at 24 hours than either autologous MSC group. Genetically engineered MSC can act as vehicles to deliver gene products to the joint; further investigation into the therapeutic potential of this cell therapy is warranted. Intra-articular MSC injection resulted in a moderate acute inflammatory joint response that was greater for allogeneic and xenogeneic MSC than autologous MSC. Clinical management of this response may minimize this effect.

  10. Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation.

    Science.gov (United States)

    Nisr, Raid B; Affourtit, Charles

    2014-02-01

    Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. © 2013.

  11. Insulin acutely improves mitochondrial function of rat and human skeletal muscle by increasing coupling efficiency of oxidative phosphorylation☆

    Science.gov (United States)

    Nisr, Raid B.; Affourtit, Charles

    2014-01-01

    Insulin is essential for the regulation of fuel metabolism and triggers the uptake of glucose by skeletal muscle. The imported glucose is either stored or broken down, as insulin stimulates glycogenesis and ATP synthesis. The mechanism by which ATP production is increased is incompletely understood at present and, generally, relatively little functional information is available on the effect of insulin on mitochondrial function. In this paper we have exploited extracellular flux technology to investigate insulin effects on the bioenergetics of rat (L6) and human skeletal muscle myoblasts and myotubes. We demonstrate that a 20-min insulin exposure significantly increases (i) the cell respiratory control ratio, (ii) the coupling efficiency of oxidative phosphorylation, and (iii) the glucose sensitivity of anaerobic glycolysis. The improvement of mitochondrial function is explained by an insulin-induced immediate decrease of mitochondrial proton leak. Palmitate exposure annuls the beneficial mitochondrial effects of insulin. Our data improve the mechanistic understanding of insulin-stimulated ATP synthesis, and reveal a hitherto undisclosed insulin sensitivity of cellular bioenergetics that suggests a novel way of detecting insulin responsiveness of cells. PMID:24212054

  12. Type II iodothyronine deiodinase provides intracellular 3,5,3′-triiodothyronine to normal and regenerating mouse skeletal muscle

    Science.gov (United States)

    Marsili, Alessandro; Tang, Dan; Harney, John W.; Singh, Prabhat; Zavacki, Ann Marie; Dentice, Monica; Salvatore, Domenico

    2011-01-01

    The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T4) to 3,5,3′-triiodothyronine (T3), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T3 under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T4-to-T3 conversion increases during differentiation in C2C12 myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given 125I-T4 and 131I-T3, the intracellular 125I-T3/131I-T3 ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the 125I-T3/131I-T3 ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in 125I-T3/131I-T3 ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of 125I-T3 is doubled after skeletal muscle injury. Thus, D2-mediated T4-to-T3 conversion generates significant intracellular T3 in normal mouse skeletal muscle, with the increased T3 required for muscle regeneration being provided by increased D2 synthesis, not by T3 from the circulation. PMID:21771965

  13. Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts.

    Science.gov (United States)

    Ohira, Yoshinobu; Matsuoka, Yoshikazu; Kawano, Fuminori; Ogura, Akihiko; Higo, Yoko; Ohira, Takashi; Terada, Masahiro; Oke, Yoshihiko; Nakai, Naoya

    2011-01-01

    The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.

  14. Satellite cell proliferation in adult skeletal muscle

    Science.gov (United States)

    Booth, Frank W. (Inventor); Thomason, Donald B. (Inventor); Morrison, Paul R. (Inventor); Stancel, George M. (Inventor)

    1995-01-01

    Novel methods of retroviral-mediated gene transfer for the in vivo corporation and stable expression of eukaryotic or prokaryotic foreign genes in tissues of living animals is described. More specifically, methods of incorporating foreign genes into mitotically active cells are disclosed. The constitutive and stable expression of E. coli .beta.-galactosidase gene under the promoter control of the Moloney murine leukemia virus long terminal repeat is employed as a particularly preferred embodiment, by way of example, establishes the model upon which the incorporation of a foreign gene into a mitotically-active living eukaryotic tissue is based. Use of the described methods in therapeutic treatments for genetic diseases, such as those muscular degenerative diseases, is also presented. In muscle tissue, the described processes result in genetically-altered satellite cells which proliferate daughter myoblasts which preferentially fuse to form a single undamaged muscle fiber replacing damaged muscle tissue in a treated animal. The retroviral vector, by way of example, includes a dystrophin gene construct for use in treating muscular dystrophy. The present invention also comprises an experimental model utilizable in the study of the physiological regulation of skeletal muscle gene expression in intact animals.

  15. Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy.

    Science.gov (United States)

    Goh, Qingnian; Millay, Douglas P

    2017-02-10

    Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy.

  16. Biphasic regulation of intracellular calcium by gemfibrozil contributes to inhibiting L6 myoblast differentiation: implications for clinical myotoxicity.

    Science.gov (United States)

    Liu, Aiming; Yang, Julin; Gonzalez, Frank J; Cheng, Gary Q; Dai, Renke

    2011-02-18

    Gemfibrozil is the most myotoxic fibrate drug commonly used for dyslipidemia, but the mechanism is poorly understood. The current study revealed that gemfibrozil inhibits myoblast differentiation through the regulation of intracellular calcium ([Ca(2+)]i) as revealed in L6 myoblasts by use of laser scan confocal microscopy and flow cytometry using Fluo-4 AM as a probe. Gemfibrozil at 20-400 μM, could regulate [Ca(2+)]i in L6 cells in a biphasic manner, and sustained reduction was observed when the concentration reached 200 μM. Inhibition of L6 differentiation by gemfibrozil was concentration-dependent with maximal effect noted between 200 and 400 μM, as indicated by creatine kinase activities and the differentiation index, respectively. In differentiating L6 myoblasts, gemfibrozil at concentrations below 400 μM led to no significant signs of apoptosis or cytotoxicity, whereas differentiation, inhibited by 200 μM gemfibrozil, was only partially recovered. A good correlation was noted between gemfibrozil concentrations that regulate [Ca(2+)]i and inhibit L6 myoblasts differentiation, and both are within the range of total serum concentrations found in the clinic. These data suggest a potential pharmacodynamic effect of gemfibrozil on myogenesis as a warning sign, in addition to the complex pharmacokinetic interactions. It is also noteworthy that mobilization of [Ca(2+)]i by gemfibrozil may trigger complex biological responses besides myocyte differentiation. Information revealed in this study explores the mechanism of gemfibrozil-induced myotoxicity through the regulation of intracellular calcium.

  17. Antifibrotic effects of Smad4 small interfering RNAs in injured skeletal muscle after acute contusion.

    Science.gov (United States)

    Li, H; Chen, J; Chen, S; Zhang, Q; Chen, S

    2011-10-01

    Muscle injuries are common musculoskeletal problems encountered in sports medicine clinics. In this study, we examined the effect of lentivirus-mediated small interfering RNA (siRNA) targeting Smad4 on the suppression of the fibrosis in injured skeletal muscles. We found that Smad4-siRNA could efficiently knock down the expression of Smad4 in the C2C12 myoblast cells and in the contunded mice gastrocnemius muscle. The expression of mRNA level of Smad4 decreased to 11% and 49% compared to the control group, respectively, and the expression of protein level decreased to 13% and 57% respectively. Moreover, the lentivirus-mediated siRNA was stably transfected only into the skeletal muscle and not into the liver of the animals. In contunded mice gastrocnemius, the collagenous and vimentin-positive area in the Smad4 siRNA group reduced to 36% and 37% compared to the control group, respectively. Furthermore, compared to the scrambled Smad4 siRNA-injected mice and PBS control-injected mice, the muscle function of the mice injected with lentivirus-mediated Smad4 siRNA improved in terms of both fast-twitch and tetanic strength (P<0.05). The results suggest that the gene therapy of inhibiting Smad4 by lentivirus-mediated siRNA could be a useful approach to prevent scar tissue formation and improve the function of injured skeletal muscle. © Georg Thieme Verlag KG Stuttgart · New York.

  18. Topologically Micropatterned Collagen and Poly(ε-caprolactone) Struts Fabricated Using the Poly(vinyl alcohol) Fibrillation/Leaching Process To Develop Efficiently Engineered Skeletal Muscle Tissue.

    Science.gov (United States)

    Kim, Minseong; Kim, WonJin; Kim, GeunHyung

    2017-12-20

    Optimally designed three-dimensional (3D) biomedical scaffolds for skeletal muscle tissue regeneration pose significant research challenges. Currently, most studies on scaffolds focus on the two-dimensional (2D) surface structures that are patterned in the micro-/nanoscales with various repeating sizes and shapes to induce the alignment of myoblasts and myotube formation. The 2D patterned surface clearly provides effective analytical results of pattern size and shape of the myoblast alignment and differentiation. However, it is inconvenient in terms of the direct application for clinical usage due to the limited thickness and 3D shapeability. Hence, the present study suggests an innovative hydrogel or synthetic structure that consists of uniaxially surface-patterned cylindrical struts for skeleton muscle regeneration. The alignment of the pattern on the hydrogel (collagen) and poly(ε-caprolactone) struts was attained with the fibrillation of poly(vinyl alcohol) and the leaching process. Various cell culture results indicate that the C2C12 cells on the micropatterned collagen structure were fully aligned, and that a significantly high level of myotube formation was achieved when compared to the collagen structures that were not treated with the micropatterning process.

  19. Lactate dehydrogenase regulation in aged skeletal muscle: Regulation by anabolic steroids and functional overload.

    Science.gov (United States)

    Washington, Tyrone A; Healey, Julie M; Thompson, Raymond W; Lowe, Larry L; Carson, James A

    2014-09-01

    Aging alters the skeletal muscle response to overload-induced growth. The onset of functional overload is characterized by increased myoblast proliferation and an altered muscle metabolic profile. The onset of functional overload is associated with increased energy demands that are met through the interconversion of lactate and pyruvate via the activity of lactate dehydrogenase (LDH). Testosterone targets many of the processes activated at the onset of functional overload. However, the effect of aging on this metabolic plasticity at the onset of functional overload and how anabolic steroid administration modulates this response is not well understood. The purpose of this study was to determine if aging would alter overload-induced LDH activity and expression at the onset of functional overload and whether anabolic steroid administration would modulate this response. Five-month and 25-month male Fischer 344xF1 BRN were given nandrolone decanoate (ND) or sham injections for 14days and then the plantaris was functionally overloaded (OV) for 3days by synergist ablation. Aging reduced muscle LDH-A & LDH-B activity 70% (pyoung muscle. Our study provides evidence that aging alters aspects of skeletal muscle metabolic plasticity normally induced by overload and anabolic steroid administration. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. A systems-based investigation into vitamin D and skeletal muscle repair, regeneration, and hypertrophy.

    Science.gov (United States)

    Owens, Daniel J; Sharples, Adam P; Polydorou, Ioanna; Alwan, Nura; Donovan, Timothy; Tang, Jonathan; Fraser, William D; Cooper, Robert G; Morton, James P; Stewart, Claire; Close, Graeme L

    2015-12-15

    Skeletal muscle is a direct target for vitamin D. Observational studies suggest that low 25[OH]D correlates with functional recovery of skeletal muscle following eccentric contractions in humans and crush injury in rats. However, a definitive association is yet to be established. To address this gap in knowledge in relation to damage repair, a randomised, placebo-controlled trial was performed in 20 males with insufficient concentrations of serum 25(OH)D (45 ± 25 nmol/l). Prior to and following 6 wk of supplemental vitamin D3 (4,000 IU/day) or placebo (50 mg of cellulose), participants performed 20 × 10 damaging eccentric contractions of the knee extensors, with peak torque measured over the following 7 days of recovery. Parallel experimentation using isolated human skeletal muscle-derived myoblast cells from biopsies of 14 males with low serum 25(OH)D (37 ± 11 nmol/l) were subjected to mechanical wound injury, which enabled corresponding in vitro studies of muscle repair, regeneration, and hypertrophy in the presence and absence of 10 or 100 nmol 1α,25(OH)2D3. Supplemental vitamin D3 increased serum 25(OH)D and improved recovery of peak torque at 48 h and 7 days postexercise. In vitro, 10 nmol 1α,25(OH)2D3 improved muscle cell migration dynamics and resulted in improved myotube fusion/differentiation at the biochemical, morphological, and molecular level together with increased myotube hypertrophy at 7 and 10 days postdamage. Together, these preliminary data are the first to characterize a role for vitamin D in human skeletal muscle regeneration and suggest that maintaining serum 25(OH)D may be beneficial for enhancing reparative processes and potentially for facilitating subsequent hypertrophy. Copyright © 2015 the American Physiological Society.

  1. Patterning Muscles Using Organizers: Larval Muscle Templates and Adult Myoblasts Actively Interact to Pattern the Dorsal Longitudinal Flight Muscles of Drosophila

    Science.gov (United States)

    Roy, Sudipto; VijayRaghavan, K.

    1998-01-01

    Pattern formation in muscle development is often mediated by special cells called muscle organizers. During metamorphosis in Drosophila, a set of larval muscles function as organizers and provide scaffolding for the development of the dorsal longitudinal flight muscles. These organizers undergo defined morphological changes and dramatically split into templates as adult fibers differentiate during pupation. We have investigated the cellular mechanisms involved in the use of larval fibers as templates. Using molecular markers that label myoblasts and the larval muscles themselves, we show that splitting of the larval muscles is concomitant with invasion by imaginal myoblasts and the onset of differentiation. We show that the Erect wing protein, an early marker of muscle differentiation, is not only expressed in myoblasts just before and after fusion, but also in remnant larval nuclei during muscle differentiation. We also show that interaction between imaginal myoblasts and larval muscles is necessary for transformation of the larval fibers. In the absence of imaginal myoblasts, the earliest steps in metamorphosis, such as the escape of larval muscles from histolysis and changes in their innervation, are normal. However, subsequent events, such as the splitting of these muscles, fail to progress. Finally, we show that in a mutant combination, null for Erect wing function in the mesoderm, the splitting of the larval muscles is aborted. These studies provide a genetic and molecular handle for the understanding of mechanisms underlying the use of muscle organizers in muscle patterning. Since the use of such organizers is a common theme in myogenesis in several organisms, it is likely that many of the processes that we describe are conserved. PMID:9606206

  2. Proliferation and skeletal myotube formation capability of C2C12 and H9c2 cells on isotropic and anisotropic electrospun nanofibrous PHB scaffolds

    International Nuclear Information System (INIS)

    Ricotti, Leonardo; Genchi, Giada G; Menciassi, Arianna; Polini, Alessandro; Iandolo, Donata; Pisignano, Dario; Ciofani, Gianni; Mattoli, Virgilio; Vazão, Helena; Ferreira, Lino

    2012-01-01

    This study aims at investigating the behavior in terms of the proliferation and skeletal muscle differentiation capability of two myoblastic cell lines, C2C12 and H9c2, on both isotropic and anisotropic electrospun nanofibrous poly(hydroxybutyrate) (PHB) scaffolds, as well as on PHB films and polystyrene controls. After a careful characterization of the matrices in terms of surface morphology, surface roughness and mechanical properties, the proliferation rate and the capability of the two cell lines to form skeletal myotubes were evaluated. Genetic analyses were also performed in order to assess the differentiation level of the cells on the different substrates. We demonstrated that the aligned nanofibrous mesh decreases the proliferation activity and provides a higher differentiative stimulus. We also clarified how the nanofibrous substrate influences myotube formation, and quantified a series of myotube-related parameters for both C2C12 and H9c2 cells. (paper)

  3. Fabrication of myogenic engineered tissue constructs.

    Science.gov (United States)

    Pacak, Christina A; Cowan, Douglas B

    2009-05-01

    Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel, and NaHCO(3). The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures. Once the tissue has solidified at 37 degrees C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

  4. Stem Cell Antigen-1 in Skeletal Muscle Function

    OpenAIRE

    Bernstein, Harold S.; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J.; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

    2013-01-01

    Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1...

  5. Shikonin increases glucose uptake in skeletal muscle cells and improves plasma glucose levels in diabetic Goto-Kakizaki rats.

    Directory of Open Access Journals (Sweden)

    Anette I Öberg

    Full Text Available BACKGROUND: There is considerable interest in identifying compounds that can improve glucose homeostasis. Skeletal muscle, due to its large mass, is the principal organ for glucose disposal in the body and we have investigated here if shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increases glucose uptake in skeletal muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: Shikonin increases glucose uptake in L6 skeletal muscle myotubes, but does not phosphorylate Akt, indicating that in skeletal muscle cells its effect is medaited via a pathway distinct from that used for insulin-stimulated uptake. Furthermore we find no evidence for the involvement of AMP-activated protein kinase in shikonin induced glucose uptake. Shikonin increases the intracellular levels of calcium in these cells and this increase is necessary for shikonin-mediated glucose uptake. Furthermore, we found that shikonin stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myoblasts. The beneficial effect of shikonin on glucose uptake was investigated in vivo by measuring plasma glucose levels and insulin sensitivity in spontaneously diabetic Goto-Kakizaki rats. Treatment with shikonin (10 mg/kg intraperitoneally once daily for 4 days significantly decreased plasma glucose levels. In an insulin sensitivity test (s.c. injection of 0.5 U/kg insulin, plasma glucose levels were significantly lower in the shikonin-treated rats. In conclusion, shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium. CONCLUSIONS/SIGNIFICANCE: Shikonin increases glucose uptake in skeletal muscle cells via an insulin-independent pathway dependent on calcium. The beneficial effects of shikonin on glucose metabolism, both in vitro and in vivo, show that the compound possesses properties that make it of considerable interest for developing novel treatment of type 2 diabetes.

  6. Chimeric autologous/allogeneic constructs for skin regeneration.

    Science.gov (United States)

    Rasmussen, Cathy Ann; Tam, Joshua; Steiglitz, Barry M; Bauer, Rebecca L; Peters, Noel R; Wang, Ying; Anderson, R Rox; Allen-Hoffmann, B Lynn

    2014-08-01

    The ideal treatment for severe cutaneous injuries would eliminate the need for autografts and promote fully functional, aesthetically pleasing autologous skin regeneration. NIKS progenitor cell-based skin tissues have been developed to promote healing by providing barrier function and delivering wound healing factors. Independently, a device has recently been created to "copy" skin by harvesting full-thickness microscopic tissue columns (MTCs) in lieu of autografts traditionally harvested as sheets. We evaluated the feasibility of combining these two technologies by embedding MTCs in NIKS-based skin tissues to generate chimeric autologous/allogeneic constructs. Chimeric constructs have the potential to provide immediate wound coverage, eliminate painful donor site wounds, and promote restoration of a pigmented skin tissue possessing hair follicles, sweat glands, and sebaceous glands. After MTC insertion, chimeric constructs and controls were reintroduced into air-interface culture and maintained in vitro for several weeks. Tissue viability, proliferative capacity, and morphology were evaluated after long-term culture. Our results confirmed successful MTC insertion and integration, and demonstrated the feasibility of generating chimeric autologous/allogeneic constructs that preserved the viability, proliferative capacity, and structure of autologous pigmented skin. These feasibility studies established the proof-of-principle necessary to further develop chimeric autologous/allogeneic constructs for the treatment of complex skin defects. Reprint & Copyright © 2014 Association of Military Surgeons of the U.S.

  7. Analysis of Mitochondrial Network Morphology in Cultured Myoblasts from Patients with Mitochondrial Disorders

    Czech Academy of Sciences Publication Activity Database

    Sládková, J.; Spáčilová, J.; Čapek, Martin; Tesařová, M.; Hansíková, H.; Honzík, T.; Martínek, J.; Zámečník, J.; Kostková, O.; Zeman, J.

    2015-01-01

    Roč. 39, č. 5 (2015), s. 340-350 ISSN 0191-3123 R&D Projects: GA ČR(CZ) GB14-36804G; GA MŠk(CZ) LH13028 Institutional support: RVO:67985823 Keywords : cristae * Fiji * image analysis * mitochondrial disorders * myoblasts * ultrastructure Subject RIV: EA - Cell Biology Impact factor: 0.828, year: 2015

  8. Signals of Ezh2, Src, and Akt Involve in Myostatin-Pax7 Pathways Regulating the Myogenic Fate Determination during the Sheep Myoblast Proliferation and Differentiation

    Science.gov (United States)

    Li, Li; Liu, Ruizao; Zhang, Li; Zhao, Fuping; Lu, Jian; Zhang, Xiaoning; Du, Lixin

    2015-01-01

    Myostatin and Pax7 have been well documented individually, however, the mechanism by which Myostatin regulates Pax7 is seldom reported. Here, based on muscle transcriptome analysis in Texel (Myostatin mutant) and Ujumqin (wild type) sheep across the five fetal stages, we constructed and examined the Myostatin-Pax7 pathways in muscle. Then we validated the signals by RNAi in the proliferating and differentiating sheep myoblasts in vitro at mRNA, protein, and cell morphological levels. We reveal that Myostatin signals to Pax7 at least through Ezh2, Src, and Akt during the sheep myoblast proliferation and differentiation. Other signals such as p38MAPK, mTOR, Erk1/2, Wnt, Bmp2, Smad, Tgfb1, and p21 are most probably involved in the Myostatin-affected myogenic events. Myostatin knockdown significantly reduces the counts of nucleus and myotube, but not the fusion index of myoblasts during cell differentiation. In addition, findings also indicate that Myostatin is required for normal myogenic differentiation of the sheep myoblasts, which is different from the C2C12 myoblasts. We expand the regulatory network of Myostatin-Pax7 pathways and first illustrate that Myostatin as a global regulator participates in the epigenetic events involved in myogenesis, which contributes to understand the molecular mechanism of Myostatin in regulation of myogenesis. PMID:25811841

  9. Exercise training and return to a well-balanced diet activate the neuregulin 1/ErbB pathway in skeletal muscle of obese rats

    Science.gov (United States)

    Ennequin, Gaël; Boisseau, Nathalie; Caillaud, Kevin; Chavanelle, Vivien; Gerbaix, Maude; Metz, Lore; Etienne, Monique; Walrand, Stéphane; Masgrau, Aurélie; Guillet, Christelle; Courteix, Daniel; Niu, Airu; Li, Yi-Ping; Capel, Fréderic; Sirvent, Pascal

    2015-01-01

    Some studies suggest that the signalling pathway of neuregulin 1 (NRG1), a protein involved in the regulation of skeletal muscle metabolism, could be altered by nutritional and exercise interventions. We hypothesized that diet-induced obesity could lead to alterations of the NRG1 signalling pathway and that chronic exercise could improve NRG1 signalling in rat skeletal muscle. To test this hypothesis, male Wistar rats received a high fat/high sucrose (HF/HS) diet for 16 weeks. At the end of this period, NRG1 and ErbB expression/activity in skeletal muscle was assessed. The obese rats then continued the HF/HS diet or were switched to a well-balanced diet. Moreover, in both groups, half of the animals also performed low intensity treadmill exercise training. After another 8 weeks, NRG1 and ErbB expression/activity in skeletal muscle were tested again. The 16 week HF/HS diet induced obesity, but did not significantly affect the NRG1/ErbB signalling pathway in rat skeletal muscle. Conversely, after the switch to a well-balanced diet, NRG1 cleavage ratio and ErbB4 amount were increased. Chronic exercise training also promoted NRG1 cleavage, resulting in increased ErbB4 phosphorylation. This result was associated with increased protein expression and phosphorylation ratio of the metalloprotease ADAM17, which is involved in NRG1 shedding. Similarly, in vitro stretch-induced activation of ADAM17 in rat myoblasts induced NRG1 cleavage and ErbB4 activation. These results show that low intensity endurance training and well-balanced diet activate the NRG1-ErbB4 pathway, possibly via the metalloprotease ADAM17, in skeletal muscle of diet-induced obese rats. PMID:25820551

  10. Ionizing radiation increases primary cilia incidence and induces multiciliation in C2C12 myoblasts

    Czech Academy of Sciences Publication Activity Database

    Filipová, A.; Diaz-Garcia, D.; Bezrouk, A.; Čížková, D.; Havelek, R.; Vávrová, J.; Dayanithi, Govindan; Řezáčová, M.

    2015-01-01

    Roč. 39, č. 8 (2015), s. 943-953 ISSN 1065-6995 Institutional support: RVO:68378041 Keywords : cell line * ionizing radiation * multiple cilia * myoblast * primary cilium * serum starvation stress Subject RIV: FP - Other Medical Disciplines Impact factor: 1.663, year: 2015

  11. Biomimetic elastomeric, conductive and biodegradable polycitrate-based nanocomposites for guiding myogenic differentiation and skeletal muscle regeneration.

    Science.gov (United States)

    Du, Yuzhang; Ge, Juan; Li, Yannan; Ma, Peter X; Lei, Bo

    2018-03-01

    Artificial muscle-like biomaterials have gained tremendous interests owing to their broad applications in regenerative medicine, wearable devices, bioelectronics and artificial intelligence. Unfortunately, key challenges are still existed for current materials, including biomimetic viscoelasticity, biocompatibility and biodegradation, multifunctionality. Herein, for the first time, we develop highly elastomeric, conductive and biodegradable poly (citric acid-octanediol-polyethylene glycol)(PCE)-graphene (PCEG) nanocomposites, and demonstrate their applications in myogenic differentiation and guiding skeletal muscle tissue regeneration. In PCEG nanocomposites, PCE provides the biomimetic elastomeric behavior, and the addition of reduced graphene oxide (RGO) endows the enhanced mechanical strength and conductivity. The highly elastomeric behavior, significantly enhanced modulus (400%-800%), strength (200%-300%) of PCEG nanocomposites with controlled biodegradability and electrochemical conductivity were achieved. The myoblasts proliferation and myogenic differentiation were significantly improved by PCEG nanocomposite. Significantly high in vivo biocompatibility of PCEG nanocomposites was observed when implanted in the subcutaneous tissue for 4 weeks in rats. PCEG nanocomposites could significantly enhance the muscle fibers and blood vessels formation in vivo in a skeletal muscle lesion model of rat. This study may provide a novel strategy to develop multifunctional elastomeric nanocomposites with high biocompatibility for potential soft tissue regeneration and stretchable bioelectronic devices. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Predeposit autologous blood transfusion: Do we require to promote it?

    Directory of Open Access Journals (Sweden)

    Gurjit Singh

    2015-01-01

    Full Text Available Introduction: Safest blood a patient can receive is his own. Quest for safe blood transfusion has remained of prime concern. To meet this aspiration, various forms of autologous blood transfusions can be practiced. It is especially suitable for patients with rare blood groups and religious sects such as Jehovah′s witness autologous transfusion is extremely safe. Cross matching is not required; iso-immunization to a foreign body is excluded. Fear of transfusion transmissible disease can be ignored. Therefore, autologous blood transfusion is required to be revisited. Materials and Methods: This is a prospective study carried out at Padmashree Dr. D. Y. Patil Medical College, Hospital and Research Centre, Pune between July 2010 and May 2012. Study comprised of 100 patients divided into two groups, autologous and homologous. Benefits of autologous transfusion were studied. Results: There was no significant change in hematocrit and blood parameters after blood donation. That is mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration (P < 0.001 after blood donation. Only one complication of vasovagal syncope was observed at the time of blood donation. Conclusion: Autologous blood transfusion is safe. Easy alternative to be practiced in elective surgeries, especially in patients with rare blood group or believers of Jehovah′s witness faith. It helps to reduce the shortfall in national blood inventory. Autologous blood donation should be practiced whenever possible.

  13. Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats.

    Science.gov (United States)

    Zargar, Sana; Moreira, Tracy S; Samimi-Seisan, Helena; Jeganathan, Senthure; Kakade, Dhanshri; Islam, Nushaba; Campbell, Jonathan; Adegoke, Olasunkanmi A J

    2011-06-01

    Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.

  14. Water-Hydrogel Binding Affinity Modulates Freeze-Drying-Induced Micropore Architecture and Skeletal Myotube Formation.

    Science.gov (United States)

    Rich, Max H; Lee, Min Kyung; Marshall, Nicholas; Clay, Nicholas; Chen, Jinrong; Mahmassani, Ziad; Boppart, Marni; Kong, Hyunjoon

    2015-08-10

    Freeze-dried hydrogels are increasingly used to create 3D interconnected micropores that facilitate biomolecular and cellular transports. However, freeze-drying is often plagued by variance in micropore architecture based on polymer choice. We hypothesized that water-polymer binding affinity plays a significant role in sizes and numbers of micropores formed through freeze-drying, influencing cell-derived tissue quality. Poly(ethylene glycol)diacrylate (PEGDA) hydrogels with alginate methacrylate (AM) were used due to AM's higher binding affinity for water than PEGDA. PEGDA-AM hydrogels with larger AM concentrations resulted in larger sizes and numbers of micropores than pure PEGDA hydrogels, attributed to the increased mass of water binding to the PEGDA-AM gel. Skeletal myoblasts loaded in microporous PEGDA-AM hydrogels were active to produce 3D muscle-like tissue, while those loaded in pure PEGDA gels were localized on the gel surface. We propose that this study will be broadly useful in designing and improving the performance of various microporous gels.

  15. Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

    International Nuclear Information System (INIS)

    Park, In-Hyun; Erbay, Ebru; Nuzzi, Paul; Chen Jie

    2005-01-01

    The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector

  16. Skeletal Muscle Differentiation on a Chip Shows Human Donor Mesoangioblasts' Efficiency in Restoring Dystrophin in a Duchenne Muscular Dystrophy Model.

    Science.gov (United States)

    Serena, Elena; Zatti, Susi; Zoso, Alice; Lo Verso, Francesca; Tedesco, F Saverio; Cossu, Giulio; Elvassore, Nicola

    2016-12-01

    : Restoration of the protein dystrophin on muscle membrane is the goal of many research lines aimed at curing Duchenne muscular dystrophy (DMD). Results of ongoing preclinical and clinical trials suggest that partial restoration of dystrophin might be sufficient to significantly reduce muscle damage. Different myogenic progenitors are candidates for cell therapy of muscular dystrophies, but only satellite cells and pericytes have already entered clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from DMD patients, using a microengineered model. We designed an ad hoc experimental strategy to miniaturize on a chip the standard process of muscle regeneration independent of variables such as inflammation and fibrosis. It is based on the coculture, at different ratios, of human dystrophin-positive myogenic progenitors and dystrophin-negative myoblasts in a substrate with muscle-like physiological stiffness and cell micropatterns. Results showed that both healthy myoblasts and mesoangioblasts restored dystrophin expression in DMD myotubes. However, mesoangioblasts showed unexpected efficiency with respect to myoblasts in dystrophin production in terms of the amount of protein produced (40% vs. 15%) and length of the dystrophin membrane domain (210-240 µm vs. 40-70 µm). These results show that our microscaled in vitro model of human DMD skeletal muscle validated previous in vivo preclinical work and may be used to predict efficacy of new methods aimed at enhancing dystrophin accumulation and distribution before they are tested in vivo, reducing time, costs, and variability of clinical experimentation. This study aimed to provide in vitro quantitative evidence of the ability of human mesoangioblasts to restore dystrophin, in terms of protein accumulation and distribution, within myotubes derived from

  17. L6E9 Myoblasts Are Deficient of Myostatin and Additional TGF- Members Are Candidates to Developmentally Control Their Fiber Formation

    Directory of Open Access Journals (Sweden)

    Stefania Rossi

    2010-01-01

    Full Text Available This work provides evidence that the robust myoblast differentiation observed in L6E9 cells is causally linked to deficiency of myostatin, which, conversely, has been found to be expressed in C2C12 cells. However, despite the absence of endogenous myostatin, L6E9 myoblasts expressed functional Activin receptors type II (ActRIIs and follistatin as well as the highly related TGF- members Activins and GDF11, suggesting that in this cell line the regulation of fiber size might be under the control of multiple regulators regardless of myostatin. In line with this hypothesis, delivery of a dominant-negative ActRIIb form or the increase of follistatin, as obtained via Trichostatin treatment or stable transfection of a short human follistatin form, enhanced the L6E9 cell differentiation and further increased the size of myotubes, suggesting that L6E9 myoblasts provide a spontaneous myostatin knock-out in vitro model to study TGF- ligands involved in developmental regulation of fiber size.

  18. Preoperative autologous plateletpheresis in patients undergoing open heart surgery.

    Directory of Open Access Journals (Sweden)

    Tomar Akhlesh

    2003-01-01

    Full Text Available Blood conservation is an important aspect of care provided to the patients undergoing cardiac operations with cardiopulmonary bypass (CPB. It is even more important in patients with anticipated prolonged CPB, redo cardiac surgery, patients having negative blood group and in patients undergoing emergency cardiac surgery. In prolonged CPB the blood is subjected to more destruction of important coagulation factors, in redo surgery the separation of adhesions leads to increased bleeding and difficulty in achieving the haemostasis and in patients with negative blood group and emergency operations, the availability of sufficient blood can be a problem. Harvesting the autologous platelet rich plasma (PRP can be a useful method of blood conservation in these patients. The above four categories of patients were prospectively studied, using either autologous whole blood donation or autologous platelet rich plasma (PRP harvest in the immediate pre-bypass period. Forty two patients were included in the study and randomly divided into two equal groups of 21 each, control group (Group I in which one unit of whole blood was withdrawn, and PRP group (Group II where autologous plateletpheresis was utilised. After reversal of heparin, autologous whole blood was transfused in the control group and autologous PRP was transfused in the PRP group. The chest tube drainage and the requirement of homologous blood and blood products were recorded. Average PRP harvest was 643.33 +/- 133.51 mL in PRP group and the mean whole blood donation was 333.75 +/- 79.58 mL in the control group. Demographic, preoperative and intra operative data showed no statistically significant differences between the two groups. The PRP group patients drained 26.44% less (p<0.001 and required 38.5% less homologous blood and blood products (p<0.05, in the postoperative period. Haemoglobin levels on day zero (day of operation and day three were statistically not different between the two groups. We

  19. Autologous fat transplantation for labia majora reconstruction.

    Science.gov (United States)

    Vogt, P M; Herold, C; Rennekampff, H O

    2011-10-01

    A case of autologous fat transplantation for labia majora augmentation after ablative surgery is presented. The patient reported pain and deformity of the left labium majus after resection for Bowen's disease. The symptoms had not been solved by classic plastic surgical reconstructions including a pudendal thigh fasciocutaneous flap. Use of autologous fat transplantation facilitated an improved aesthetic result while preserving residual sensation to the external genitalia and improving symptoms of mucosal exposure and dryness.

  20. Autologous bone marrow purging with LAK cells.

    Science.gov (United States)

    Giuliodori, L; Moretti, L; Stramigioli, S; Luchetti, F; Annibali, G M; Baldi, A

    1993-12-01

    In this study we will demonstrate that LAK cells, in vitro, can lyse hematologic neoplastic cells with a minor toxicity of the staminal autologous marrow cells. In fact, after bone marrow and LAK co-culture at a ratio of 1/1 for 8 hours, the inhibition on the GEMM colonies resulted to be 20% less compared to the untreated marrow. These data made LAK an inviting agent for marrow purging in autologous bone marrow transplantation.

  1. Myoblast sensitivity and fibroblast insensitivity to osteogenic conversion by BMP-2 correlates with the expression of Bmpr-1a

    Directory of Open Access Journals (Sweden)

    North Kathryn N

    2009-05-01

    Full Text Available Abstract Background Osteoblasts are considered to primarily arise from osseous progenitors within the periosteum or bone marrow. We have speculated that cells from local soft tissues may also take on an osteogenic phenotype. Myoblasts are known to adopt a bone gene program upon treatment with the osteogenic bone morphogenetic proteins (BMP-2,-4,-6,-7,-9, but their osteogenic capacity relative to other progenitor types is unclear. We further hypothesized that the sensitivity of cells to BMP-2 would correlate with BMP receptor expression. Methods We directly compared the BMP-2 sensitivity of myoblastic murine cell lines and primary cells with osteoprogenitors from osseous tissues and fibroblasts. Fibroblasts forced to undergo myogenic conversion by transduction with a MyoD-expressing lentiviral vector (LV-MyoD were also examined. Outcome measures included alkaline phosphatase expression, matrix mineralization, and expression of osteogenic genes (alkaline phosphatase, osteocalcin and bone morphogenetic protein receptor-1A as measured by quantitative PCR. Results BMP-2 induced a rapid and robust osteogenic response in myoblasts and osteoprogenitors, but not in fibroblasts. Myoblasts and osteoprogenitors grown in osteogenic media rapidly upregulated Bmpr-1a expression. Chronic BMP-2 treatment resulted in peak Bmpr-1a expression at day 6 before declining, suggestive of a negative feedback mechanism. In contrast, fibroblasts expressed low levels of Bmpr-1a that was only weakly up-regulated by BMP-2 treatment. Bioinformatics analysis confirmed the presence of myogenic responsive elements in the proximal promoter region of human and murine BMPR-1A/Bmpr-1a. Forced myogenic gene expression in fibroblasts was associated with a significant increase in Bmpr-1a expression and a synergistic increase in the osteogenic response to BMP-2. Conclusion These data demonstrate the osteogenic sensitivity of muscle progenitors and provide a mechanistic insight into the

  2. Heavy ion irradiation induces autophagy in irradiated C2C12 myoblasts and their bystander cells

    International Nuclear Information System (INIS)

    Hino, Mizuki; Tajika, Yuki; Hamada, Nobuyuki

    2010-01-01

    Autophagy is one of the major processes involved in the degradation of intracellular materials. Here, we examined the potential impact of heavy ion irradiation on the induction of autophagy in irradiated C2C12 mouse myoblasts and their non-targeted bystander cells. In irradiated cells, ultrastructural analysis revealed the accumulation of autophagic structures at various stages of autophagy (id est (i.e.) phagophores, autophagosomes and autolysosomes) within 20 min after irradiation. Multivesicular bodies (MVBs) and autolysosomes containing MVBs (amphisomes) were also observed. Heavy ion irradiation increased the staining of microtubule-associated protein 1 light chain 3 and LysoTracker Red (LTR). Such enhanced staining was suppressed by an autophagy inhibitor 3-methyladenine. In addition to irradiated cells, bystander cells were also positive with LTR staining. Altogether, these results suggest that heavy ion irradiation induces autophagy not only in irradiated myoblasts but also in their bystander cells. (author)

  3. Complex interactions between human myoblasts and the surrounding 3D fibrin-based matrix.

    Directory of Open Access Journals (Sweden)

    Stéphane Chiron

    Full Text Available Anchorage of muscle cells to the extracellular matrix is crucial for a range of fundamental biological processes including migration, survival and differentiation. Three-dimensional (3D culture has been proposed to provide a more physiological in vitro model of muscle growth and differentiation than routine 2D cultures. However, muscle cell adhesion and cell-matrix interplay of engineered muscle tissue remain to be determined. We have characterized cell-matrix interactions in 3D muscle culture and analyzed their consequences on cell differentiation. Human myoblasts were embedded in a fibrin matrix cast between two posts, cultured until confluence, and then induced to differentiate. Myoblasts in 3D aligned along the longitudinal axis of the gel. They displayed actin stress fibers evenly distributed around the nucleus and a cortical mesh of thin actin filaments. Adhesion sites in 3D were smaller in size than in rigid 2D culture but expression of adhesion site proteins, including α5 integrin and vinculin, was higher in 3D compared with 2D (p<0.05. Myoblasts and myotubes in 3D exhibited thicker and ellipsoid nuclei instead of the thin disk-like shape of the nuclei in 2D (p<0.001. Differentiation kinetics were faster in 3D as demonstrated by higher mRNA concentrations of α-actinin and myosin. More important, the elastic modulus of engineered muscle tissues increased significantly from 3.5 ± 0.8 to 7.4 ± 4.7 kPa during proliferation (p<0.05 and reached 12.2 ± 6.0 kPa during differentiation (p<0.05, thus attesting the increase of matrix stiffness during proliferation and differentiation of the myocytes. In conclusion, we reported modulations of the adhesion complexes, the actin cytoskeleton and nuclear shape in 3D compared with routine 2D muscle culture. These findings point to complex interactions between muscle cells and the surrounding matrix with dynamic regulation of the cell-matrix stiffness.

  4. Autologous fat grafting: use of closed syringe microcannula system for enhanced autologous structural grafting

    Directory of Open Access Journals (Sweden)

    Alexander RW

    2013-04-01

    Full Text Available Robert W Alexander,1 David Harrell2 1Department of Surgery, School of Medicine and Dentistry, University of Washington, Seattle, WA, USA; 2Harvest-Terumo Inc, Plymouth, MA, USA Objectives: Provide background for use of acquiring autologous adipose tissue as a tissue graft and source of adult progenitor cells for use in cosmetic plastic surgery. Discuss the background and mechanisms of action of closed syringe vacuum lipoaspiration, with emphasis on accessing adipose-derived mesenchymal/stromal cells and the stromal vascular fraction (SVF for use in aesthetic, structural reconstruction and regenerative applications. Explain a proven protocol for acquiring high-quality autologous fat grafts (AFG with use of disposable, microcannula systems. Design: Explain the components and advantage of use of the patented super luer-lock and microcannulas system for use with the closed-syringe system. A sequential explanation of equipment selection for minimally traumatic lipoaspiration in small volumes is presented, including use of blunt injection cannulas to reduce risk of embolism. Results: Thousands of AFG have proven safe and efficacious for lipoaspiration techniques for large and small structural fat grafting procedures. The importance and advantages of gentle harvesting of the adipose tissue complex has become very clear in the past 5 years. The closed-syringe system offers a minimally invasive, gentle system with which to mobilize subdermal fat tissues in a suspension form. Resulting total nuclear counting of undifferentiated cells of the adipose-derived -SVF suggests that the yield achieved is better than use of always-on, constant mechanical pump applied vacuum systems. Conclusion: Use of a closed-syringe lipoaspiration system featuring disposable microcannulas offers a safe and effective means of harvesting small volumes of nonmanipulated adipose tissues and its accompanying progenitor cells within the SVF. Closed syringes and microcannulas are

  5. Dual roles of palladin protein in in vitro myogenesis: inhibition of early induction but promotion of myotube maturation.

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    Ngoc-Uyen-Nhi Nguyen

    Full Text Available Palladin is a microfilament-associated phosphoprotein whose function in skeletal muscle has rarely been studied. Therefore, we investigate whether myogenesis is influenced by the depletion of palladin expression known to interfere with the actin cytoskeleton dynamic required for skeletal muscle differentiation. The inhibition of palladin in C2C12 myoblasts leads to precocious myogenic differentiation with a concomitant reduction in cell apoptosis. This premature myogenesis is caused, in part, by an accelerated induction of p21, myogenin, and myosin heavy chain, suggesting that palladin acts as a negative regulator in early differentiation phases. Paradoxically, palladin-knockdown myoblasts are unable to differentiate terminally, despite their ability to perform some initial steps of differentiation. Cells with attenuated palladin expression form thinner myotubes with fewer myonuclei compared to those of the control. It is noteworthy that a negative regulator of myogenesis, myostatin, is activated in palladin-deficient myotubes, suggesting the palladin-mediated impairment of late-stage myogenesis. Additionally, overexpression of 140-kDa palladin inhibits myoblast differentiation while 200-kDa and 90-kDa palladin-overexpressed cells display an enhanced differentiation rate. Together, our data suggest that palladin might have both positive and negative roles in maintaining the proper skeletal myogenic differentiation in vitro.

  6. Heterokaryon analysis of muscle differentiation: regulation of the postmitotic state.

    Science.gov (United States)

    Clegg, C H; Hauschka, S D

    1987-08-01

    MM14 mouse myoblasts withdraw irreversibly from the cell cycle and become postmitotic within a few hours of being deprived of fibroblast growth factor (Clegg, C. H., T. A. Linkhart, B. B. Olwin, and S. D. Hauschka, 1987, J. Cell Biol., 105:949-956). To examine the mechanisms that may regulate this developmental state of skeletal muscle, we tested the mitogen responsiveness of various cell types after their polyethylene glycol-mediated fusion with post-mitotic myocytes. Heterokaryons containing myocytes and quiescent nonmyogenic cells such as 3T3, L cell, and a differentiation-defective myoblast line (DD-1) responded to mitogen-rich medium by initiating DNA synthesis. Myonuclei replicated DNA and reexpressed thymidine kinase. In contrast, (myocyte x G1 myoblast) heterokaryons failed to replicate DNA in mitogen-rich medium and became postmitotic. This included cells with a nuclear ratio of three myoblasts to one myocyte. Proliferation dominance in (myocyte x 3T3 cell) and (myocyte x DD-1) heterokaryons was conditionally regulated by the timing of mitogen treatment; such cells became postmitotic when mitogen exposure was delayed for as little as 6 h after cell fusion. In addition, (myocyte x DD-1) heterokaryons expressed a muscle-specific trait and lost epidermal growth factor receptors when they became postmitotic. These results demonstrate that DNA synthesis is not irreversibly blocked in skeletal muscle; myonuclei readily express proliferation-related functions when provided with a mitogenic signal. Rather, myocyte-specific repression of DNA synthesis in heterokaryons argues that the postmitotic state of skeletal muscle is regulated by diffusible factors that inhibit processes of cellular mitogenesis.

  7. Preoperative autologous plateletpheresis in patients undergoing open heart surgery.

    Science.gov (United States)

    Tomar, Akhlesh S; Tempe, Deepak K; Banerjee, Amit; Hegde, Radhesh; Cooper, Andrea; Khanna, S K

    2003-07-01

    Blood conservation is an important aspect of care provided to the patients undergoing cardiac operations with cardiopulmonary bypass (CPB). It is even more important in patients with anticipated prolonged CPB, redo cardiac surgery, patients having negative blood group and in patients undergoing emergency cardiac surgery. In prolonged CPB the blood is subjected to more destruction of important coagulation factors, in redo surgery the separation of adhesions leads to increased bleeding and difficulty in achieving the haemostasis and in patients with negative blood group and emergency operations, the availability of sufficient blood can be a problem. Harvesting the autologous platelet rich plasma (PRP) can be a useful method of blood conservation in these patients. The above four categories of patients were prospectively studied, using either autologous whole blood donation or autologous platelet rich plasma (PRP) harvest in the immediate pre-bypass period. Forty two patients were included in the study and randomly divided into two equal groups of 21 each, control group (Group I) in which one unit of whole blood was withdrawn, and PRP group (Group II) where autologous plateletpheresis was utilised. After reversal of heparin, autologous whole blood was transfused in the control group and autologous PRP was transfused in the PRP group. The chest tube drainage and the requirement of homologous blood and blood products were recorded. Average PRP harvest was 643.33 +/- 133.51 mL in PRP group and the mean whole blood donation was 333.75 +/- 79.58 mL in the control group. Demographic, preoperative and intra operative data showed no statistically significant differences between the two groups. The PRP group patients drained 26.44% less (pblood products (pconservation in terms of better haemostasis, and less requirement of blood and blood products in the postoperative period as compared with the autologous whole blood donation. This technique can be especially useful in the

  8. Cost effectiveness of autologous blood transfusion – A developing ...

    African Journals Online (AJOL)

    An autologous blood donation program was set up at National Orthopaedic Hospital, Igbobi Lagos in 1992 in response to the rising sero prevalence of HIV observed in our “relative replacement” donors. A retrospective batch analysis of patients who received autologous transfusion and those who received homologous ...

  9. Autologous patch graft in tube shunt surgery.

    Science.gov (United States)

    Aslanides, I M; Spaeth, G L; Schmidt, C M; Lanzl, I M; Gandham, S B

    1999-10-01

    To evaluate an alternate method of covering the subconjunctival portion of the tube in aqueous shunt surgery. Evidence of tube erosion, graft-related infection, graft melting, or other associated intraocular complications were evaluated. A retrospective study of 16 patients (17 eyes) who underwent tube shunt surgery at Wills Eye Hospital between July 1991 and October 1996 was conducted. An autologous either "free" or "rotating" scleral lamellar graft was created to cover the subconjunctival portion of the tube shunt. All patients were evaluated for at least 6 months, with a mean follow-up of 14.8 months (range 6-62 months). All eyes tolerated the autologous graft well, with no clinical evidence of tube erosion, or graft-related or intraocular complications. Autologous patch graft in tube shunt surgery appears--in selected cases--to be an effective, safe and inexpensive surgical alternative to allogenic graft materials. It also offers ease of availability, and eliminates the risk of transmitting infectious disease.

  10. Inhibiting c-Jun N-terminal kinase partially attenuates caffeine-dependent cell death without alleviating the caffeine-induced reduction in mitochondrial respiration in C2C12 skeletal myotubes

    International Nuclear Information System (INIS)

    Downs, R.M.; Hughes, M.A.; Kinsey, S.T.; Johnson, M.C.; Baumgarner, B.L.

    2016-01-01

    Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release. - Highlights: • Caffeine

  11. Detection of satellite cells during skeletal muscle wound healing in rats: time-dependent expressions of Pax7 and MyoD in relation to wound age.

    Science.gov (United States)

    Tian, Zhi-Ling; Jiang, Shu-Kun; Zhang, Miao; Wang, Meng; Li, Jiao-Yong; Zhao, Rui; Wang, Lin-Lin; Li, Shan-Shan; Liu, Min; Zhang, Meng-Zhou; Guan, Da-Wei

    2016-01-01

    The study was focused on time-dependent expressions of paired-box transcription factor 7 (Pax7) and myoblast determination protein (MyoD) during skeletal muscle wound healing. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17, and 21 days after injury, respectively (five rats in each posttraumatic interval). Five rats were employed as control. By morphometric analysis, the data based on the number of Pax7(+)/MyoD(-), Pax7(+)/MyoD(+), and Pax7(-)/MyoD(+) cells were highly correlated with the wound age. Pax7 and MyoD expressions were upregulated after injury by Western blot and quantitative real-time PCR assays. The relative quantity of Pax7 protein peaked at 5 days after injury, which was >1.13, and decreased thereafter. Similarly, the relative quantity of MyoD mRNA expression peaked at 3 days after injury, which was >2.59. The relative quantity of Pax7 protein >0.73 or mRNA expression >2.38 or the relative quantity of MyoD protein >1.33 suggested a wound age of 3 to 7 days. The relative quantity of MyoD mRNA expression >2.02 suggested a wound age of 1 to 7 days post-injury. In conclusion, the expressions of Pax7 and MyoD are upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting that Pax7 and MyoD may be potential markers for wound age estimation in skeletal muscle.

  12. Cord Blood Banking Standards: Autologous Versus Altruistic.

    Science.gov (United States)

    Armitage, Sue

    2015-01-01

    Cord blood (CB) is either donated to public CB banks for use by any patient worldwide for whom it is a match or stored in a private bank for potential autologous or family use. It is a unique cell product that has potential for treating life-threatening diseases. The majority of CB products used today are for hematopoietic stem cell transplantation and are accessed from public banks. CB is still evolving as a hematopoietic stem cell source, developing as a source for cellular immunotherapy products, such as natural killer, dendritic, and T-cells, and fast emerging as a non-hematopoietic stem cell source in the field of regenerative medicine. This review explores the regulations, standards, and accreditation schemes that are currently available nationally and internationally for public and private CB banking. Currently, most of private banking is under regulated as compared to public banking. Regulations and standards were initially developed to address the public arena. Early responses from the medical field regarding private CB banking was that at the present time, because of insufficient scientific data to support autologous banking and given the difficulty of making an accurate estimate of the need for autologous transplantation, private storage of CB as "biological insurance" should be discouraged (1, 2, 3). To ensure success and the true realization of the full potential of CB, whether for autologous or allogeneic use, it is essential that each and every product provided for current and future treatments meets high-quality, international standards.

  13. Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7.

    Science.gov (United States)

    Darabi, Radbod; Perlingeiro, Rita C R

    2016-01-01

    Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition, patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless, directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis, which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here, we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes, enabling researchers to use these cells for disease modeling as well as therapeutic purposes.

  14. Autologous chondrocyte implantation: superior biologic properties of hyaline cartilage repairs.

    Science.gov (United States)

    Henderson, Ian; Lavigne, Patrick; Valenzuela, Herminio; Oakes, Barry

    2007-02-01

    Information regarding the quality of autologous chondrocyte implantation repair is needed to determine whether the current autologous chondrocyte implantation surgical technology and the subsequent biologic repair processes are capable of reliably forming durable hyaline or hyaline-like cartilage in vivo. We report and analyze the properties and qualities of autologous chondrocyte implantation repairs. We evaluated 66 autologous chondrocyte implantation repairs in 57 patients, 55 of whom had histology, indentometry, and International Cartilage Repair Society repair scoring at reoperation for mechanical symptoms or pain. International Knee Documentation Committee scores were used to address clinical outcome. Maximum stiffness, normalized stiffness, and International Cartilage Repair Society repair scoring were higher for hyaline articular cartilage repairs compared with fibrocartilage, with no difference in clinical outcome. Reoperations revealed 32 macroscopically abnormal repairs (Group B) and 23 knees with normal-looking repairs in which symptoms leading to arthroscopy were accounted for by other joint disorders (Group A). In Group A, 65% of repairs were either hyaline or hyaline-like cartilage compared with 28% in Group B. Autologous chondrocyte repairs composed of fibrocartilage showed more morphologic abnormalities and became symptomatic earlier than hyaline or hyaline-like cartilage repairs. The hyaline articular cartilage repairs had biomechanical properties comparable to surrounding cartilage and superior to those associated with fibrocartilage repairs.

  15. What is the role of autologous blood transfusion in major spine surgery?

    Science.gov (United States)

    Kumar, Naresh; Chen, Yongsheng; Nath, Chinmoy; Liu, Eugene Hern Choon

    2012-06-01

    Major spine surgery is associated with significant blood loss, which has numerous complications. Blood loss is therefore an important concern when undertaking any major spine surgery. Blood loss can be addressed by reducing intraoperative blood loss and replenishing perioperative blood loss. Reducing intraoperative blood loss helps maintain hemodynamic equilibrium and provides a clearer operative field during surgery. Homologous blood transfusion is still the mainstay for replenishing blood loss in major spine surgery across the world, despite its known adverse effects. These significant adverse effects can be seen in up to 20% of patients. Autologous blood transfusion avoids the risks associated with homologous blood transfusion and has been shown to be cost-effective. This article reviews the different methods of autologous transfusion and focuses on the use of intraoperative cell salvage in major spine surgery. Autologous blood transfusion is a proven alternative to homologous transfusion in major spine surgery, avoiding most, if not all of these adverse effects. However, autologous blood transfusion rates in major spine surgery remain low across the world. Autologous blood transfusion may obviate the need for homologous transfusion completely. We encourage spine surgeons to consider autologous blood transfusion wherever feasible.

  16. Regeneration of Tissues and Organs Using Autologous Cells

    Energy Technology Data Exchange (ETDEWEB)

    Anthony Atala

    2010-04-28

    The Joint Commission for Health Care Organizations recently declared the shortage of transplantable organs and tissues a public health crisis. As such, there is about one death every 30 seconds due to organ failure. Complications and rejection are still significant albeit underappreciated problems. It is often overlooked that organ transplantation results in the patient being placed on an immune suppression regimen that will ultimate shorten their life span. Patients facing reconstruction often find that surgery is difficult or impossible due to the shortage of healthy autologous tissue. In many cases, autografting is a compromise between the condition and the cure that can result in substantial diminution of quality of life. The national cost of caring for persons who might benefit from engineered tissues or organs has reached $600 billion annually. Autologous tissue technologies have been developed as an alternative to transplantation or reconstructive surgery. Autologous tissues derived from the patient's own cells are capable of correcting numerous pathologies and injuries. The use of autologous cells eliminates the risks of rejection and immunological reactions, drastically reduces the time that patients must wait for lifesaving surgery, and negates the need for autologous tissue harvest, thereby eliminating the associated morbidities. In fact, the use of autologous tissues to create functional organs is one of the most important and groundbreaking steps ever taken in medicine. Although the basic premise of creating tissues in the laboratory has progressed dramatically, only a limited number of tissue developments have reached the patients to date. This is due, in part, to the several major technological challenges that require solutions. To that end, we have been in pursuit of more efficient ways to expand cells in vitro, methods to improve vascular support so that relevant volumes of engineered tissues can be grown, and constructs that can mimic the

  17. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    Energy Technology Data Exchange (ETDEWEB)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany); Renkawitz-Pohl, Renate, E-mail: renkawit@biologie.uni-marburg.de [Developmental Biology, Department of Biology, Philipps-Universität Marburg, Karl-von-Frisch-Strasse 8, 35037 Marburg (Germany)

    2013-02-15

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity.

  18. Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7

    International Nuclear Information System (INIS)

    Bonn, Bettina R.; Rudolf, Anja; Hornbruch-Freitag, Christina; Daum, Gabor; Kuckwa, Jessica; Kastl, Lena; Buttgereit, Detlev; Renkawitz-Pohl, Renate

    2013-01-01

    Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell–cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles. - Highlights: ► The class XVIII myosin encoding gene Mhcl is transcribed in the mesoderm. ► Mhcl localization at contact sites of fusing myoblasts depends on Rols7. ► Mhcl interacts in vitro with Rols7 which is essential for myogenesis. ► Functional redundancy with other myosins is likely as mutants show no muscle defects. ► Mhcl localizes adjacent to Z-discs of sarcomeres and might support muscle integrity

  19. The CDM Superfamily Protein MBC Directs Myoblast Fusion through a Mechanism That Requires Phosphatidylinositol 3,4,5-Triphosphate Binding but Is Independent of Direct Interaction with DCrk▿§

    Science.gov (United States)

    Balagopalan, Lakshmi; Chen, Mei-Hui; Geisbrecht, Erika R.; Abmayr, Susan M.

    2006-01-01

    myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila melanogaster embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, we established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or “Docker”), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. We demonstrate that the SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding. PMID:17030600

  20. DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Benjamin D Pope

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is linked to contraction of an array of tandem 3.3-kb repeats (D4Z4 at 4q35.2 from 11-100 copies to 1-10 copies. The extent to which D4Z4 contraction at 4q35.2 affects overall 4q35.2 chromatin organization remains unclear. Because DNA replication timing is highly predictive of long-range chromatin interactions, we generated genome-wide replication-timing profiles for FSHD and control myogenic precursor cells. We compared non-immortalized myoblasts from four FSHD patients and three control individuals to each other and to a variety of other human cell types. This study also represents the first genome-wide comparison of replication timing profiles in non-immortalized human cell cultures. Myoblasts from both control and FSHD individuals all shared a myoblast-specific replication profile. In contrast, male and female individuals were readily distinguished by monoallelic differences in replication timing at DXZ4 and other regions across the X chromosome affected by X inactivation. We conclude that replication timing is a robust cell-type specific feature that is unaffected by FSHD-related D4Z4 contraction.

  1. Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

    DEFF Research Database (Denmark)

    Sellathurai, Jeeva; Nielsen, Joachim; Hejbøl, Eva Kildall

    2016-01-01

    -PCR, immunocytochemistry and western blot. RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET......, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch...... and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β...

  2. TNF inhibits Notch-1 in skeletal muscle cells by Ezh2 and DNA methylation mediated repression: implications in duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Swarnali Acharyya

    2010-08-01

    Full Text Available Classical NF-kappaB signaling functions as a negative regulator of skeletal myogenesis through potentially multiple mechanisms. The inhibitory actions of TNFalpha on skeletal muscle differentiation are mediated in part through sustained NF-kappaB activity. In dystrophic muscles, NF-kappaB activity is compartmentalized to myofibers to inhibit regeneration by limiting the number of myogenic progenitor cells. This regulation coincides with elevated levels of muscle derived TNFalpha that is also under IKKbeta and NF-kappaB control.Based on these findings we speculated that in DMD, TNFalpha secreted from myotubes inhibits regeneration by directly acting on satellite cells. Analysis of several satellite cell regulators revealed that TNFalpha is capable of inhibiting Notch-1 in satellite cells and C2C12 myoblasts, which was also found to be dependent on NF-kappaB. Notch-1 inhibition occurred at the mRNA level suggesting a transcriptional repression mechanism. Unlike its classical mode of action, TNFalpha stimulated the recruitment of Ezh2 and Dnmt-3b to coordinate histone and DNA methylation, respectively. Dnmt-3b recruitment was dependent on Ezh2.We propose that in dystrophic muscles, elevated levels of TNFalpha and NF-kappaB inhibit the regenerative potential of satellite cells via epigenetic silencing of the Notch-1 gene.

  3. A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

    Directory of Open Access Journals (Sweden)

    Knight James DR

    2012-03-01

    Full Text Available Abstract Background The p38α mitogen-activated protein kinase (MAPK is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear. Methods To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms. Results Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation. Conclusion Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.

  4. α-Syntrophin stabilizes catalase to reduce endogenous reactive oxygen species levels during myoblast differentiation.

    Science.gov (United States)

    Moon, Jae Yun; Choi, Su Jin; Heo, Cheol Ho; Kim, Hwan Myung; Kim, Hye Sun

    2017-07-01

    α-Syntrophin is a component of the dystrophin-glycoprotein complex that interacts with various intracellular signaling proteins in muscle cells. The α-syntrophin knock-down C2 cell line (SNKD), established by infecting lentivirus particles with α-syntrophin shRNA, is characterized by a defect in terminal differentiation and increase in cell death. Since myoblast differentiation is accompanied by intensive mitochondrial biogenesis, the generation of intracellular reactive oxygen species (ROS) is also increased during myogenesis. Two-photon microscopy imaging showed that excessive intracellular ROS accumulated during the differentiation of SNKD cells as compared with control cells. The formation of 4-hydroxynonenal adduct, a byproduct of lipid peroxidation during oxidative stress, significantly increased in differentiated SNKD myotubes and was dramatically reduced by epigallocatechin-3-gallate, a well-known ROS scavenger. Among antioxidant enzymes, catalase was significantly decreased during differentiation of SNKD cells without changes at the mRNA level. Of interest was the finding that the degradation of catalase was rescued by MG132, a proteasome inhibitor, in the SNKD cells. This study demonstrates a novel function of α-syntrophin. This protein plays an important role in the regulation of oxidative stress from endogenously generated ROS during myoblast differentiation by modulating the protein stability of catalase. © 2017 Federation of European Biochemical Societies.

  5. Efficacy of autologous platelets in macular hole surgery.

    Science.gov (United States)

    Konstantinidis, Aristeidis; Hero, Mark; Nanos, Panagiotis; Panos, Georgios D

    2013-01-01

    The introduction of optical coherence tomography has allowed accurate measurement of the size of macular holes. A retrospective consecutive review was performed of 21 patients undergoing macular hole repair with vitrectomy, gas tamponade, and autologous platelet injection and we assessed the effect of macular hole parameters on anatomic and functional outcomes. We looked at the demographic features, final visual outcome, and anatomical closure. Twenty-one patients were included in the study. They underwent routine vitrectomy with gas tamponade (C3F8) and injection of autologous platelets. All patients were advised to maintain a facedown posture for 2 weeks. Anatomical closure was confirmed in all cases and 20 out of 21 of patients had improved postoperative visual acuity by two or more lines. In our series, the macular hole dimensions did not have much effect on the final results. The use of autologous platelets and strict facedown posture seems to be the deciding factor in good anatomical and visual outcome irrespective of macular hole dimensions.

  6. Oxidative stress induces caveolin 1 degradation and impairs caveolae functions in skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Alexis Mougeolle

    Full Text Available Increased level of oxidative stress, a major actor of cellular aging, impairs the regenerative capacity of skeletal muscle and leads to the reduction in the number and size of muscle fibers causing sarcopenia. Caveolin 1 is the major component of caveolae, small membrane invaginations involved in signaling and endocytic trafficking. Their role has recently expanded to mechanosensing and to the regulation of oxidative stress-induced pathways. Here, we increased the amount of reactive oxidative species in myoblasts by addition of hydrogen peroxide (H2O2 at non-toxic concentrations. The expression level of caveolin 1 was significantly decreased as early as 10 min after 500 μM H2O2 treatment. This reduction was not observed in the presence of a proteasome inhibitor, suggesting that caveolin 1 was rapidly degraded by the proteasome. In spite of caveolin 1 decrease, caveolae were still able to assemble at the plasma membrane. Their functions however were significantly perturbed by oxidative stress. Endocytosis of a ceramide analog monitored by flow cytometry was significantly diminished after H2O2 treatment, indicating that oxidative stress impaired its selective internalization via caveolae. The contribution of caveolae to the plasma membrane reservoir has been monitored after osmotic cell swelling. H2O2 treatment increased membrane fragility revealing that treated cells were more sensitive to an acute mechanical stress. Altogether, our results indicate that H2O2 decreased caveolin 1 expression and impaired caveolae functions. These data give new insights on age-related deficiencies in skeletal muscle.

  7. Exercise training and return to a well-balanced diet activate the neuregulin 1/ErbB pathway in skeletal muscle of obese rats.

    Science.gov (United States)

    Ennequin, Gaël; Boisseau, Nathalie; Caillaud, Kevin; Chavanelle, Vivien; Gerbaix, Maude; Metz, Lore; Etienne, Monique; Walrand, Stéphane; Masgrau, Aurélie; Guillet, Christelle; Courteix, Daniel; Niu, Airu; Li, Yi-Ping; Capel, Fréderic; Sirvent, Pascal

    2015-06-15

    associated with increased protein expression and phosphorylation ratio of the metalloprotease ADAM17, which is involved in NRG1 shedding. Similarly, in vitro stretch-induced activation of ADAM17 in rat myoblasts induced NRG1 cleavage and ErbB4 activation. These results show that low intensity endurance training and well-balanced diet activate the NRG1-ErbB4 pathway, possibly via the metalloprotease ADAM17, in skeletal muscle of diet-induced obese rats. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  8. NF-κB–YY1–miR-29 Regulatory Circuitry in Skeletal Myogenesis and Rhabdomyosarcoma

    Science.gov (United States)

    Wang, Huating; Garzon, Ramiro; Sun, Hao; Ladner, Katherine J.; Singh, Ravi; Dahlman, Jason; Cheng, Alfred; Hall, Brett M.; Qualman, Stephen J.; Chandler, Dawn S.; Croce, Carlo M.; Guttridge, Denis C.

    2008-01-01

    SUMMARY Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and Rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts miR-29 is repressed by NF-κB acting through YY1 and the Polycomb. During myogenesis, NF-κB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-κB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its pro-myogenic function. Together, results identify a NF-κB–YY1–miR-29 regulatory circuit whose disruption may contribute to RMS. SIGNIFICANCE MicroRNAs regulate skeletal myogenesis, but their impact in muscle diseases is not well understood. Here we describe miR-29 as an enhancer of myogenic differentiation and a suppressor of RMS. We find that miR-29 exists in a regulatory circuit involving NF-κB and YY1. In myoblasts NF-B acts through YY1 to epigenetically suppress miR-29, while during differentiation miR-29 is induced to facilitate myogenesis by a negative feedback on YY1. Significantly, RMS tumors lose miR-29 due to an elevation in NF-B and YY1, and readjustment of miR-29 levels in RMS stimulates differentiation. Thus, myogenesis is dependent on NF-κB–YY1–miR-29 circuitry whose dysfunction may contribute to RMS pathogenesis. Such findings offer potential avenues for the diagnosis and treatment of muscle relevant cancers. PMID:18977326

  9. Tumescent mastectomy technique in autologous breast reconstruction.

    Science.gov (United States)

    Vargas, Christina R; Koolen, Pieter G L; Ho, Olivia A; Ricci, Joseph A; Tobias, Adam M; Lin, Samuel J; Lee, Bernard T

    2015-10-01

    Use of the tumescent mastectomy technique has been reported to facilitate development of a hydrodissection plane, reduce blood loss, and provide adjunct analgesia. Previous studies suggest that tumescent dissection may contribute to adverse outcomes after immediate implant reconstruction; however, its effect on autologous microsurgical reconstruction has not been established. A retrospective review was conducted of all immediate microsurgical breast reconstruction procedures at a single academic center between January 2004 and December 2013. Records were queried for age, body mass index, mastectomy weight, diabetes, hypertension, smoking, preoperative radiation, reconstruction flap type, and autologous flap weight. Outcomes of interest were mastectomy skin necrosis, complete and partial flap loss, return to the operating room, breast hematoma, seroma, and infection. There were 730 immediate autologous breast reconstructions performed during the study period; 46% with the tumescent dissection technique. Groups were similar with respect to baseline patient and procedural characteristics. Univariate analysis revealed no significant difference in the incidence of mastectomy skin necrosis, complete or partial flap loss, return to the operating room, operative time, estimated blood loss, recurrence, breast hematoma, seroma, or infection in patients undergoing tumescent mastectomy. Multivariate analysis also demonstrated no significant association between the use of tumescent technique and postoperative breast mastectomy skin necrosis (P = 0.980), hematoma (P = 0.759), or seroma (P = 0.340). Use of the tumescent dissection technique during mastectomy is not significantly associated with adverse outcomes after microsurgical breast reconstruction. Despite concern for its impact on implant reconstruction, our findings suggest that this method can be used safely preceding autologous procedures. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Stem cell antigen-1 in skeletal muscle function.

    Science.gov (United States)

    Bernstein, Harold S; Samad, Tahmina; Cholsiripunlert, Sompob; Khalifian, Saami; Gong, Wenhui; Ritner, Carissa; Aurigui, Julian; Ling, Vivian; Wilschut, Karlijn J; Bennett, Stephen; Hoffman, Julien; Oishi, Peter

    2013-08-15

    Stem cell antigen-1 (Sca-1) is a member of the Ly-6 multigene family encoding highly homologous, glycosyl-phosphatidylinositol-anchored membrane proteins. Sca-1 is expressed on muscle-derived stem cells and myogenic precursors recruited to sites of muscle injury. We previously reported that inhibition of Sca-1 expression stimulated myoblast proliferation in vitro and regulated the tempo of muscle repair in vivo. Despite its function in myoblast expansion during muscle repair, a role for Sca-1 in normal, post-natal muscle has not been thoroughly investigated. We systematically compared Sca-1-/- (KO) and Sca-1+/+ (WT) mice and hindlimb muscles to elucidate the tissue, contractile, and functional effects of Sca-1 in young and aging animals. Comparison of muscle volume, fibrosis, myofiber cross-sectional area, and Pax7+ myoblast number showed little differences between ages or genotypes. Exercise protocols, however, demonstrated decreased stamina in KO versus WT mice, with young KO mice achieving results similar to aging WT animals. In addition, KO mice did not improve with practice, while WT animals demonstrated conditioning over time. Surprisingly, myomechanical analysis of isolated muscles showed that KO young muscle generated more force and experienced less fatigue. However, KO muscle also demonstrated incomplete relaxation with fatigue. These findings suggest that Sca-1 is necessary for muscle conditioning with exercise, and that deficient conditioning in Sca-1 KO animals becomes more pronounced with age.

  11. Perinatal outcomes after gestational surrogacy versus autologous IVF: analysis of national data.

    Science.gov (United States)

    Sunkara, Sesh Kamal; Antonisamy, Belavendra; Selliah, Hepsy Y; Kamath, Mohan S

    2017-12-01

    Anonymized data were obtained from the Human Fertilization and Embryology Authority to determine whether gestational surrogacy influences perinatal outcomes compared with pregnancies after autologous IVF. A total of 103,160 singleton live births, including 244 after gestational surrogacy, 87,571 after autologous fresh IVF and intractyoplasmic sperm injection (ICSI) and 15,345 after autologous frozen embryo transfers were analysed. Perinatal outcomes of pretern birth (PTB), low birth weight (LBW) and high birth weight (HBW) were compared. No difference was found in the risk of PTB and LBW after gestational surrogacy compared with autologous fresh IVF-ICSI: PTB (adjusted OR 0.90, 95% CI 0.56 to 1.42), LBW (adjusted OR 0.90, 95% CI 0.57 to 1.43) and gestational surrogacy compared with autologous frozen embryo transfers: PTB (adjusted OR 0.96, 95% CI 0.58 to 1.60), LBW (adjusted OR 1.16, 95% CI 0.69 to 1.96). The incidence of HBW was significantly higher after gestational surrogacy compared with fresh IVF-ICSI (adjusted OR 1.94, 95% CI 1.38 to 2.75); no difference was found in HBW between gestational surrogacy and autologous frozen embryo transfers. The dataset is limited by lack of information on confounders, i.e. ethnicity, body mass index, underlying medical history, which could result in residual confounding. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  12. Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts

    International Nuclear Information System (INIS)

    Watanabe, Tomonobu M.; Higuchi, Sayaka; Kawauchi, Keiko; Tsukasaki, Yoshikazu; Ichimura, Taro; Fujita, Hideaki

    2012-01-01

    Highlights: ► Change in the epigenetic landscape during myogenesis was optically investigated. ► Mobility of nuclear proteins was used to state the epigenetic status of the cell. ► Mobility of nuclear proteins decreased as myogenesis progressed in C2C12. ► Differentiation state diagram was developed using parameters obtained. -- Abstract: Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extent of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation stage during myogenesis using the state diagram developed with the parameters obtained in this study.

  13. Autologous tenocyte therapy for experimental Achilles tendinopathy in a rabbit model.

    Science.gov (United States)

    Chen, Jimin; Yu, Qian; Wu, Bing; Lin, Zhen; Pavlos, Nathan J; Xu, Jiake; Ouyang, Hongwei; Wang, Allan; Zheng, Ming H

    2011-08-01

    Tendinopathy of the Achilles tendon is a chronic degenerative condition that frequently does not respond to treatment. In the current study, we propose that autologous tenocytes therapy (ATT) is effective in treating tendon degeneration in a collagenase-induced rabbit Achilles tendinopathy model. Chronic tendinopathy was created in the left Achilles tendon of 44 rabbits by an intratendonous injection of type I collagenase. Forty-two rabbits were randomly allocated into three groups of 14 and received control treatment; autologous tenocytes digested from tendon tissue; and autologous tenocytes digested from epitendineum tissue. For cell tracking in vivo, the remaining two animals were injected with autologous tenocytes labeled with a nano-scale super-paramagnetic iron oxide (Feridex). Rabbits were sacrificed at 4 and 8 weeks after the therapeutic injection, and tendon tissue was analyzed by histology, immunostaining, and biomechanical testing to evaluate tissue repair. Autologous tenocyte treatment improved tendon remodeling, histological outcomes, collagen content, and tensile strength of tendinopathic Achilles tendons. Injected tenocytes were integrated into tendon matrix and could be tracked up to 8 weeks in vivo. Immunohistochemistry showed that ATT improved type I collagen expression in repaired tendon but did not affect type III collagen and secreted protein, acidic and rich in cysteine expression. ATT may be a useful treatment of chronic Achilles tendinopathy.

  14. LOCAL CORTICOSTEROID VS. AUTOLOGOUS BLOOD FOR PLANTAR FASCIITIS

    Directory of Open Access Journals (Sweden)

    Syam Sunder B

    2017-01-01

    Full Text Available BACKGROUND Plantar fasciitis is the most common cause of heel pain for which professional care is sought. Initially thought of as an inflammatory process, plantar fasciitis is a disorder of degenerative changes in the fascia and maybe more accurately termed plantar fasciosis. Traditional therapeutic efforts have been directed at decreasing the presumed inflammation. These treatments include icing, Nonsteroidal Anti-inflammatory Drugs (NSAIDs, rest and activity modification, corticosteroids, botulinum toxin type A, splinting, shoe modifications and orthosis. Other treatment techniques have been directed at resolving the degeneration caused by the disease process. In general, these techniques are designed to create an acute inflammatory reaction with the goal of restarting the healing process. These techniques include autologous blood injection, Platelet-Rich Plasma (PRP injection, nitroglycerin patches, Extracorporeal Shock Wave Therapy (ESWT and surgical procedures. Recently, research has focused on regenerative therapies with high expectations of success. The use of autologous growth factors is thought to heal through collagen regeneration and the stimulation of a well-ordered angiogenesis. These growth factors are administered in the form of autologous whole blood or Platelet-Rich Plasma (PRP. Platelets can be isolated using simple cell-separating systems. The degranulation of the alpha granules in the platelets releases many different growth factors that play a role in tissue regeneration processes. Platelet-derived growth factor, transforming growth factor-P, vascular-derived endothelial growth factor, epithelial growth factor, hepatocyte growth factor and insulin-like growth factor are examples of such growth factors. Injections with autologous growth factors are becoming common in clinical practice. The present study was an attempt to compare the efficacy of autologous blood injection in plantar fasciitis by comparing it with the local

  15. Quantitative skeletal scintiscanning

    International Nuclear Information System (INIS)

    Haushofer, R.

    1982-01-01

    330 patients were examined by skeletal scintiscanning with sup(99m)Tc pyrophosphate and sup(99m)methylene diphosphonate in the years between 1977 and 1979. Course control examinations were carried out in 12 patients. The collective of patients presented with primary skeletal tumours, metastases, inflammatory and degenerative skeletal diseases. Bone scintiscanning combined with the ''region of interest'' technique was found to be an objective and reproducible technique for quantitative measurement of skeletal radioactivity concentrations. The validity of nuclear skeletal examinations can thus be enhanced as far as diagnosis, course control, and differential diagnosis are concerned. Quantitative skeletal scintiscanning by means of the ''region of interest'' technique has opened up a new era in skeletal diagnosis by nuclear methods. (orig./MG) [de

  16. Expression profiling of skeletal muscle following acute and chronic β2-adrenergic stimulation: implications for hypertrophy, metabolism and circadian rhythm

    Directory of Open Access Journals (Sweden)

    Lynch Gordon S

    2009-09-01

    Full Text Available Abstract Background Systemic administration of β-adrenoceptor (β-AR agonists has been found to induce skeletal muscle hypertrophy and significant metabolic changes. In the context of energy homeostasis, the importance of β-AR signaling has been highlighted by the inability of β1-3-AR-deficient mice to regulate energy expenditure and susceptibility to diet induced obesity. However, the molecular pathways and gene expression changes that initiate and maintain these phenotypic modulations are poorly understood. Therefore, the aim of this study was to identify differential changes in gene expression in murine skeletal muscle associated with systemic (acute and chronic administration of the β2-AR agonist formoterol. Results Skeletal muscle gene expression (from murine tibialis anterior was profiled at both 1 and 4 hours following systemic administration of the β2-AR agonist formoterol, using Illumina 46K mouse BeadArrays. Illumina expression profiling revealed significant expression changes in genes associated with skeletal muscle hypertrophy, myoblast differentiation, metabolism, circadian rhythm, transcription, histones, and oxidative stress. Differentially expressed genes relevant to the regulation of muscle mass and metabolism (in the context of the hypertrophic phenotype were further validated by quantitative RT-PCR to examine gene expression in response to both acute (1-24 h and chronic administration (1-28 days of formoterol at multiple timepoints. In terms of skeletal muscle hypertrophy, attenuation of myostatin signaling (including differential expression of myostatin, activin receptor IIB, phospho-Smad3 etc was observed following acute and chronic administration of formoterol. Acute (but not chronic administration of formoterol also significantly induced the expression of genes involved in oxidative metabolism, including hexokinase 2, sorbin and SH3 domain containing 1, and uncoupling protein 3. Interestingly, formoterol

  17. Effectiveness of autologous transfusion system in primary total hip and knee arthroplasty.

    LENUS (Irish Health Repository)

    Schneider, Marco M

    2014-01-01

    Autologous transfusion has become a cost-efficient and useful option in the treatment of patients with high blood loss following major orthopaedic surgery. However, the effectiveness of autologous transfusion in total joint replacement remains controversial.

  18. Early de novo DNA methylation and prolonged demethylation in the muscle lineage.

    Science.gov (United States)

    Tsumagari, Koji; Baribault, Carl; Terragni, Jolyon; Varley, Katherine E; Gertz, Jason; Pradhan, Sirharsa; Badoo, Melody; Crain, Charlene M; Song, Lingyun; Crawford, Gregory E; Myers, Richard M; Lacey, Michelle; Ehrlich, Melanie

    2013-03-01

    Myogenic cell cultures derived from muscle biopsies are excellent models for human cell differentiation. We report the first comprehensive analysis of myogenesis-specific DNA hyper- and hypo-methylation throughout the genome for human muscle progenitor cells (both myoblasts and myotubes) and skeletal muscle tissue vs. 30 non-muscle samples using reduced representation bisulfite sequencing. We also focused on four genes with extensive hyper- or hypo-methylation in the muscle lineage (PAX3, TBX1, MYH7B/MIR499 and OBSCN) to compare DNA methylation, DNaseI hypersensitivity, histone modification, and CTCF binding profiles. We found that myogenic hypermethylation was strongly associated with homeobox or T-box genes and muscle hypomethylation with contractile fiber genes. Nonetheless, there was no simple relationship between differential gene expression and myogenic differential methylation, rather only for subsets of these genes, such as contractile fiber genes. Skeletal muscle retained ~30% of the hypomethylated sites but only ~3% of hypermethylated sites seen in myogenic progenitor cells. By enzymatic assays, skeletal muscle was 2-fold enriched globally in genomic 5-hydroxymethylcytosine (5-hmC) vs. myoblasts or myotubes and was the only sample type enriched in 5-hmC at tested myogenic hypermethylated sites in PAX3/CCDC140 andTBX1. TET1 and TET2 RNAs, which are involved in generation of 5-hmC and DNA demethylation, were strongly upregulated in myoblasts and myotubes. Our findings implicate de novo methylation predominantly before the myoblast stage and demethylation before and after the myotube stage in control of transcription and co-transcriptional RNA processing. They also suggest that, in muscle, TET1 or TET2 are involved in active demethylation and in formation of stable 5-hmC residues.

  19. Autologous serum therapy in chronic urticaria

    Directory of Open Access Journals (Sweden)

    Sharmila Patil

    2013-01-01

    Full Text Available Autologous serum therapy is a promising therapy for treatment resistant urticaria. This is useful in developing countries as this is economical option. Minimum instruments like centrifuge, syringe and needles are required for the procedure.

  20. Induced skeletal mutations

    International Nuclear Information System (INIS)

    Selby, P.B.

    1979-01-01

    This paper describes a large-scale experiment that, by means of breeding tests, confirmed that many dominant skeletal mutations are induced by large-dose radiation exposure. The author also discusses: (1) the major advantages and disadvantages of the skeletal method in improving estimates of genetic hazard to man; (2) future uses of the skeletal method; (3) direct estimation of risk beyond the first generation using the skeletal method; and (4) the possibility of using the skeletal method as a quick and easy screen for chemical mutagens

  1. Autologous Hematopoietic Stem Cell Transplantation to Prevent Antibody Mediated Rejection After Vascularized Composite Allotransplantation

    Science.gov (United States)

    2017-10-01

    Award Number: W81XWH-16-1-0664 TITLE: Autologous Hematopoietic Stem Cell Transplantation to Prevent Antibody-Mediated Rejection after...Annual 3. DATES COVERED 15 Sep 2016 – 14 Sep 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Autologous Hematopoietic Stem Cell Transplantation to...sensitization, autologous hematopoietic stem cell transplantation, antibody mediated rejection, donor specific antibodies 16. SECURITY CLASSIFICATION OF

  2. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    International Nuclear Information System (INIS)

    Takegahara, Yuki; Yamanouchi, Keitaro; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-01-01

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies

  3. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    Energy Technology Data Exchange (ETDEWEB)

    Takegahara, Yuki; Yamanouchi, Keitaro, E-mail: akeita@mail.ecc.u-tokyo.ac.jp; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.

  4. Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Jaewoo Pak

    2016-08-01

    Full Text Available This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs and homogenized extracellular matrix (ECM in the form of adipose stromal vascular fraction (SVF, along with hyaluronic acid (HA and platelet-rich plasma (PRP activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI data, functional rating index, range of motion (ROM, and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees.

  5. MicroRNA-27a Is Induced by Leucine and Contributes to Leucine-Induced Proliferation Promotion in C2C12 Cells

    Directory of Open Access Journals (Sweden)

    Guangmang Liu

    2013-07-01

    Full Text Available Leucine, a branched chain amino acid, is well known to stimulate protein synthesis in skeletal muscle. However, the role of leucine in myoblast proliferation remains unclear. In this study, we found that leucine could promote proliferation of C2C12 cells. Moreover, expressions of miR-27a and myostatin (a bona fide target of miR-27a were upregulated and downregulated, respectively, following leucine treatment. We also found that miR-27a loss-of-function by transfection of a miR-27a inhibitor suppressed the promotion of myoblast proliferation caused by leucine. Our results suggest that miR-27a is induced by leucine and contributes to leucine-induced proliferation promotion of myoblast.

  6. Treatment of Refractory Filamentary Keratitis With Autologous Serum Tears.

    Science.gov (United States)

    Read, Sarah P; Rodriguez, Marianeli; Dubovy, Sander; Karp, Carol L; Galor, Anat

    2017-09-01

    To report a case of filamentary keratitis (FK) successfully treated with autologous serum tears and to review the pathogenesis and management of FK. Case report including high-resolution anterior segment optical coherence tomography and filament histopathology. A 61-year-old Hispanic man presented with pain and photophobia of the right eye. He was found to have a corneal epithelial defect and a small peripheral infiltrate 4 months after Laser Assisted in situ Keratomileusis. After resolution of the epithelial defect, he developed FK. Over a 4-month period, conservative management with aggressive lubrication, lid hygiene, topical corticosteroids, topical cyclosporine, bandage contact lenses, and oral doxycycline failed to resolve the corneal filaments. Notably, treatment with 20% autologous serum tears, four times daily, led to a sustained resolution of the FK within 1 week. This case demonstrates the complexity of FK management and introduces autologous serum tears as a viable management option when conservative approaches to this condition fail.

  7. TNF Inhibits Notch-1 in Skeletal Muscle Cells by Ezh2 and DNA Methylation Mediated Repression: Implications in Duchenne Muscular Dystrophy

    Science.gov (United States)

    Acharyya, Swarnali; Sharma, Sudarshana M.; Cheng, Alfred S.; Ladner, Katherine J.; He, Wei; Kline, William; Wang, Huating; Ostrowski, Michael C.; Huang, Tim H.; Guttridge, Denis C.

    2010-01-01

    Background Classical NF-κB signaling functions as a negative regulator of skeletal myogenesis through potentially multiple mechanisms. The inhibitory actions of TNFα on skeletal muscle differentiation are mediated in part through sustained NF-κB activity. In dystrophic muscles, NF-κB activity is compartmentalized to myofibers to inhibit regeneration by limiting the number of myogenic progenitor cells. This regulation coincides with elevated levels of muscle derived TNFα that is also under IKKβ and NF-κB control. Methodology/Principal Findings Based on these findings we speculated that in DMD, TNFα secreted from myotubes inhibits regeneration by directly acting on satellite cells. Analysis of several satellite cell regulators revealed that TNFα is capable of inhibiting Notch-1 in satellite cells and C2C12 myoblasts, which was also found to be dependent on NF-κB. Notch-1 inhibition occurred at the mRNA level suggesting a transcriptional repression mechanism. Unlike its classical mode of action, TNFα stimulated the recruitment of Ezh2 and Dnmt-3b to coordinate histone and DNA methylation, respectively. Dnmt-3b recruitment was dependent on Ezh2. Conclusions/Significance We propose that in dystrophic muscles, elevated levels of TNFα and NF-κB inhibit the regenerative potential of satellite cells via epigenetic silencing of the Notch-1 gene. PMID:20814569

  8. Role of skeletal muscle in ear development.

    Science.gov (United States)

    Rot, Irena; Baguma-Nibasheka, Mark; Costain, Willard J; Hong, Paul; Tafra, Robert; Mardesic-Brakus, Snjezana; Mrduljas-Djujic, Natasa; Saraga-Babic, Mirna; Kablar, Boris

    2017-10-01

    The current paper is a continuation of our work described in Rot and Kablar, 2010. Here, we show lists of 10 up- and 87 down-regulated genes obtained by a cDNA microarray analysis that compared developing Myf5-/-:Myod-/- (and Mrf4-/-) petrous part of the temporal bone, containing middle and inner ear, to the control, at embryonic day 18.5. Myf5-/-:Myod-/- fetuses entirely lack skeletal myoblasts and muscles. They are unable to move their head, which interferes with the perception of angular acceleration. Previously, we showed that the inner ear areas most affected in Myf5-/-:Myod-/- fetuses were the vestibular cristae ampullaris, sensitive to angular acceleration. Our finding that the type I hair cells were absent in the mutants' cristae was further used here to identify a profile of genes specific to the lacking cell type. Microarrays followed by a detailed consultation of web-accessible mouse databases allowed us to identify 6 candidate genes with a possible role in the development of the inner ear sensory organs: Actc1, Pgam2, Ldb3, Eno3, Hspb7 and Smpx. Additionally, we searched for human homologues of the candidate genes since a number of syndromes in humans have associated inner ear abnormalities. Mutations in one of our candidate genes, Smpx, have been reported as the cause of X-linked deafness in humans. Our current study suggests an epigenetic role that mechanical, and potentially other, stimuli originating from muscle, play in organogenesis, and offers an approach to finding novel genes responsible for altered inner ear phenotypes.

  9. Telmisartan ameliorates insulin sensitivity by activating the AMPK/SIRT1 pathway in skeletal muscle of obese db/db mice

    Directory of Open Access Journals (Sweden)

    Shiota Asuka

    2012-11-01

    Full Text Available Abstract Background Telmisartan is a well-established angiotensin II type 1 receptor blocker that improves insulin sensitivity in animal models of obesity and insulin resistance, as well as in humans. Telmisartan has been reported to function as a partial agonist of the peroxisome proliferator-activated receptor (PPAR γ, which is also targeted by the nicotinamide adenine dinucleotide (NAD-dependent deacetylase (SIRT1. Here, we investigated the pathways through which telmisartan acts on skeletal muscle, in vitro as well as in vivo. Methods Nine-week-old male db/db mice were fed a 60% high-fat diet, with orally administrated either vehicle (carboxymethyl-cellulose, CMC, 5 mg/kg telmisartan, or 5 mg/kg telmisartan and 1 mg/kg GW9662, a selective irreversible antagonist of PPARγ, for 5 weeks. Effects of telmisartan on Sirt1 mRNA, AMPK phosphorylation, and NAD+/NADH ratio were determined in C2C12 cultured myocytes. Results and discussion Telmisartan treatment improved insulin sensitivity in obese db/db mice fed a high-fat diet and led to reduction in the size of hypertrophic pancreatic islets in these mice. Moreover, in vitro treatment with telmisartan led to increased expression of Sirt1 mRNA in C2C12 skeletal muscle cells; the increase in Sirt1 mRNA in telmisartan-treated C2C12 myoblasts occurred concomitantly with an increase in AMPK phosphorylation, an increase in NAD+/NADH ratio, and increases in the mRNA levels of PGC1α, FATP1, ACO, and GLUT4. Conclusions Our results indicate that telmisartan acts through a PPARγ-independent pathway, but at least partially exerts its effects by acting directly on skeletal muscle AMPK/SIRT1 pathways.

  10. p38 MAPK activation and H3K4 trimethylation is decreased by lactate in vitro and high intensity resistance training in human skeletal muscle.

    Directory of Open Access Journals (Sweden)

    Lena Willkomm

    Full Text Available Exercise induces adaptation of skeletal muscle by acutely modulating intracellular signaling, gene expression, protein turnover and myogenic activation of skeletal muscle stem cells (Satellite cells, SCs. Lactate (La-induced metabolic stimulation alone has been shown to modify SC proliferation and differentiation. Although the mechanistic basis remains elusive, it was demonstrated that La affects signaling via p38 mitogen activated protein kinase (p38 MAPK which might contribute to trimethylation of histone 3 lysine 4 (H3K4me3 known to regulate satellite cell proliferation and differentiation. We investigated the effects of La on p38 MAPK and H3K4me3 in a model of activated SCs. Differentiating C2C12 myoblasts were treated with La (20 mM and samples analysed using qRT-PCR, immunofluorescence, and western blotting. We determined a reduction of p38 MAPK phosphorylation, decreased H3K4me3 and reduced expression of Myf5, myogenin, and myosin heavy chain (MHC leading to decreased differentiation of La-treated C2C12 cells after 5 days of repeated La treatment. We further investigated whether this regulatory pathway would be affected in human skeletal muscle by the application of two different resistance exercise regimes (RE associated with distinct metabolic demands and blood La accumulation. Muscle biopsies were obtained 15, 30 min, 1, 4, and 24 h post exercise after moderate intensity RE (STD vs. high intensity RE (HIT. Consistent with in vitro results, reduced p38 phosphorylation and blunted H3K4me3 were also observed upon metabolically demanding HIT RE in human skeletal muscle. Our data provide evidence that La-accumulation acutely affects p38 MAPK signaling, gene expression and thereby cell differentiation and adaptation in vitro, and likely in vivo.

  11. p38 MAPK activation and H3K4 trimethylation is decreased by lactate in vitro and high intensity resistance training in human skeletal muscle.

    Science.gov (United States)

    Willkomm, Lena; Gehlert, Sebastian; Jacko, Daniel; Schiffer, Thorsten; Bloch, Wilhelm

    2017-01-01

    Exercise induces adaptation of skeletal muscle by acutely modulating intracellular signaling, gene expression, protein turnover and myogenic activation of skeletal muscle stem cells (Satellite cells, SCs). Lactate (La)-induced metabolic stimulation alone has been shown to modify SC proliferation and differentiation. Although the mechanistic basis remains elusive, it was demonstrated that La affects signaling via p38 mitogen activated protein kinase (p38 MAPK) which might contribute to trimethylation of histone 3 lysine 4 (H3K4me3) known to regulate satellite cell proliferation and differentiation. We investigated the effects of La on p38 MAPK and H3K4me3 in a model of activated SCs. Differentiating C2C12 myoblasts were treated with La (20 mM) and samples analysed using qRT-PCR, immunofluorescence, and western blotting. We determined a reduction of p38 MAPK phosphorylation, decreased H3K4me3 and reduced expression of Myf5, myogenin, and myosin heavy chain (MHC) leading to decreased differentiation of La-treated C2C12 cells after 5 days of repeated La treatment. We further investigated whether this regulatory pathway would be affected in human skeletal muscle by the application of two different resistance exercise regimes (RE) associated with distinct metabolic demands and blood La accumulation. Muscle biopsies were obtained 15, 30 min, 1, 4, and 24 h post exercise after moderate intensity RE (STD) vs. high intensity RE (HIT). Consistent with in vitro results, reduced p38 phosphorylation and blunted H3K4me3 were also observed upon metabolically demanding HIT RE in human skeletal muscle. Our data provide evidence that La-accumulation acutely affects p38 MAPK signaling, gene expression and thereby cell differentiation and adaptation in vitro, and likely in vivo.

  12. Use of 5-Bromodeoxyuridine and irradiation for the estimation of the myoblast and myocyte content of primary rat heart cell cultures

    International Nuclear Information System (INIS)

    Masse, M.J.O.; Harary, I.

    1980-01-01

    A method for killing dividing cells was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo-natal rat heart. Cells were cultivated in BUdR (5-bromodeoxyuridine) 10 -4 M for 3 days and then irradiated with long uv light. The selective elimination of dividing cells led to a loss of myosin Ca 2+ -activated ATPase in the cultures. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1-day postnatal rats and 48% in cultures from 4-day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages

  13. Screening for autologous blood transfusions

    DEFF Research Database (Denmark)

    Mørkeberg, J; Belhage, B; Ashenden, M

    2009-01-01

    parameter in the screening for autologous blood doping. Three bags of blood (approximately 201+/-11 g of Hb) were withdrawn from 16 males and stored at either -80 degrees C (-80 T, n=8) or +4 degrees C (+4 T, n=8) and reinfused 10 weeks or 4 weeks later, respectively. Seven subjects served as controls...

  14. Autologous blood transfusion in open heart surgeries under cardio-pulmonary bypass - Clinical appraisal

    Directory of Open Access Journals (Sweden)

    B. Sartaj Hussain

    2017-01-01

    Full Text Available Autologous blood withdrawal before instituting cardiopulmonary bypass (CPB protects the platelets, preserve red cell mass and reduce allogeneic transfusion requirements. Ideal condition for autologous blood donation is elective cardiac surgery where there is a high probability of blood transfusion. The purpose of this study was to assess the role of preoperative autologous blood donation in cardiac surgeries. Out of 150 patients registered, 50 cases were excluded on the basis of hemoglobin content ( [J Med Allied Sci 2017; 7(1.000: 48-54

  15. ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin-Hua [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Bauman, William A. [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Cardozo, Christopher, E-mail: chris.cardozo@va.gov [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States)

    2015-08-14

    Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle. - Highlights: • AnkrD1 is upregulated by TNFα and represses NF-κB-induced transcriptional activity. • AnkrD1 binds to p50 subunit of NF-κB and is recruited to NF-κB bound to chromatin. • AnkrD1 mediates a feed-back inhibitory loop

  16. Eradication of breast cancer with bone metastasis by autologous formalin-fixed tumor vaccine (AFTV) combined with palliative radiation therapy and adjuvant chemotherapy: a case report.

    Science.gov (United States)

    Kuranishi, Fumito; Ohno, Tadao

    2013-06-04

    Skeletal metastasis of breast carcinoma is refractory to intensive chemo-radiation therapy and therefore is assumed impossible to cure. Here, we report an advanced case of breast cancer with vertebra-Th7 metastasis that showed complete response to combined treatments with formalin-fixed autologous tumor vaccine (AFTV), palliative radiation therapy with 36 Gy, and adjuvant chemotherapy with standardized CEF (cyclophosphamide, epirubicin, and 5FU), zoledronic acid, and aromatase inhibitors following mastectomy for the breast tumor. The patient has been disease-free for more than 4 years after the mammary surgery and remains well with no evidence of metastasis or local recurrence. Thus, a combination of AFTV, palliative radiation therapy, and adjuvant chemotherapy may be an effective treatment for this devastating disease.

  17. Hemifacial atrophy treated with autologous fat transplantation

    Directory of Open Access Journals (Sweden)

    Gandhi Vijay

    2005-01-01

    Full Text Available A 23-year-old male developed right hemifacial atrophy following marphea profunda. Facial asymmetry due to residual atrophy was treated with autologous fat harvested from buttocks with marked cosmetic improvement.

  18. Prospective randomized comparison of scar appearances between cograft of acellular dermal matrix with autologous split-thickness skin and autologous split-thickness skin graft alone for full-thickness skin defects of the extremities.

    Science.gov (United States)

    Yi, Ju Won; Kim, Jae Kwang

    2015-03-01

    The purpose of this study was to evaluate the clinical outcomes of cografting of acellular dermal matrix with autologous split-thickness skin and autologous split-thickness skin graft alone for full-thickness skin defects on the extremities. In this prospective randomized study, 19 consecutive patients with full-thickness skin defects on the extremities following trauma underwent grafting using either cograft of acellular dermal matrix with autologous split-thickness skin graft (nine patients, group A) or autologous split-thickness skin graft alone (10 patients, group B) from June of 2011 to December of 2012. The postoperative evaluations included observation of complications (including graft necrosis, graft detachment, or seroma formation) and Vancouver Scar Scale score. No statistically significant difference was found regarding complications, including graft necrosis, graft detachment, or seroma formation. At week 8, significantly lower Vancouver Scar Scale scores for vascularity, pliability, height, and total score were found in group A compared with group B. At week 12, lower scores for pliability and height and total scores were identified in group A compared with group B. For cases with traumatic full-thickness skin defects on the extremities, a statistically significant better result was achieved with cograft of acellular dermal matrix with autologous split-thickness skin graft than with autologous split-thickness skin graft alone in terms of Vancouver Scar Scale score. Therapeutic, II.

  19. Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors

    Science.gov (United States)

    2013-01-01

    Background In sea urchin larvae the circumesophageal fibers form a prominent muscle system of mesodermal origin. Although the morphology and later development of this muscle system has been well-described, little is known about the molecular signature of these cells or their precise origin in the early embryo. As an invertebrate deuterostome that is more closely related to the vertebrates than other commonly used model systems in myogenesis, the sea urchin fills an important phylogenetic gap and provides a unique perspective on the evolution of muscle cell development. Results Here, we present a comprehensive description of the development of the sea urchin larval circumesophageal muscle lineage beginning with its mesodermal origin using high-resolution localization of the expression of several myogenic transcriptional regulators and differentiation genes. A few myoblasts are bilaterally distributed at the oral vegetal side of the tip of the archenteron and first appear at the late gastrula stage. The expression of the differentiation genes Myosin Heavy Chain, Tropomyosin I and II, as well as the regulatory genes MyoD2, FoxF, FoxC, FoxL1, Myocardin, Twist, and Tbx6 uniquely identify these cells. Interestingly, evolutionarily conserved myogenic factors such as Mef2, MyoR and Six1/2 are not expressed in sea urchin myoblasts but are found in other mesodermal domains of the tip of the archenteron. The regulatory states of these domains were characterized in detail. Moreover, using a combinatorial analysis of gene expression we followed the development of the FoxF/FoxC positive cells from the onset of expression to the end of gastrulation. Our data allowed us to build a complete map of the Non-Skeletogenic Mesoderm at the very early gastrula stage, in which specific molecular signatures identify the precursors of different cell types. Among them, a small group of cells within the FoxY domain, which also express FoxC and SoxE, have been identified as plausible myoblast

  20. The MSX1 homeoprotein recruits G9a methyltransferase to repressed target genes in myoblast cells.

    Directory of Open Access Journals (Sweden)

    Jingqiang Wang

    Full Text Available Although the significance of lysine modifications of core histones for regulating gene expression is widely appreciated, the mechanisms by which these modifications are incorporated at specific regulatory elements during cellular differentiation remains largely unknown. In our previous studies, we have shown that in developing myoblasts the Msx1 homeoprotein represses gene expression by influencing the modification status of chromatin at its target genes. We now show that genomic binding by Msx1 promotes enrichment of the H3K9me2 mark on repressed target genes via recruitment of G9a histone methyltransferase, the enzyme responsible for catalyzing this histone mark. Interaction of Msx1 with G9a is mediated via the homeodomain and is required for transcriptional repression and regulation of cellular differentiation, as well as enrichment of the H3K9me2 mark in proximity to Msx1 binding sites on repressed target genes in myoblast cells as well as the developing limb. We propose that regulation of chromatin status by Msx1 recruitment of G9a and other histone modifying enzymes to regulatory regions of target genes represents an important means of regulating the gene expression during development.

  1. Autologous fat transplantation for depressed linear scleroderma-induced facial atrophic scars.

    Science.gov (United States)

    Roh, Mi Ryung; Jung, Jin Young; Chung, Kee Yang

    2008-12-01

    Facial linear scleroderma results in depressed atrophic scars. Autologous fat transplantation has been widely used, and fat appears to be an ideal material for filling depressed atrophic scars and contour deformities, but long-term results for autologous fat transplantation are controversial. To review the short- and long-term results of 20 patients who underwent multiple autologous fat transplantations for depressed atrophic scar correction. Twenty patients with clinically inactive facial linear scleroderma were included. They received at least two transplantations and had a 12-month follow-up evaluation. On the forehead, 51% to 75% improvement (average grading scale: 2.4) was achieved when observed at least 12 months after the last treatment. For the chin, correction was poor (average grading scale: 0.7) with less than 25% improvement. The infraorbital area showed fair correction, but the nose showed poor correction. Two of three patients with scalp reduction surgery showed excellent results, showing only slight scar widening. Autologous fat transplantation is an effective method for long-term correction of depressed atrophic scars left by linear scleroderma on the forehead but is less effective for corrections on the nose, infraorbital area, and chin.

  2. Use of containers with sterilizing filter in autologous serum eyedrops.

    Science.gov (United States)

    López-García, José S; García-Lozano, Isabel

    2012-11-01

    To assess the effect of the use of containers with an adapted sterilizing filter on the contamination of autologous serum eyedrops. Prospective, consecutive, comparative, and randomized study. Thirty patients with Sjögren syndrome. One hundred seventy-six autologous serum containers used in home therapy were studied; 48 of them included an adapted filter (Hyabak; Thea, Clermont-Ferrand, France), and the other 128 were conventional containers. Containers equipped with a filter were tested at 7, 14, 21, and 28 days of use, whereas conventional containers were studied after 7 days of use. In addition, testing for contamination was carried out in 14 conventional containers used during in-patient therapy every week for 4 weeks. In all cases, the preparation of the autologous serum was similar. Blood agar and chocolate agar were used as regular culture media for the microbiologic studies, whereas Sabouraud agar with chloramphenicol was the medium for fungal studies. Microbiologic contamination of containers with autologous serum eyedrops. Only one of the containers with an adapted sterilizing filter (2.1%) became contaminated with Staphylococcus epidermidis after 1 month of treatment, whereas the contamination rate among conventional containers reached 28.9% after 7 days of treatment. The most frequent germs found in the samples were coagulase-negative Staphylococcus (48.6%). With regard the containers used in the in-patient setting, 2 (14.3%) became contaminated after 2 weeks, 5 (35.7%) became contaminated after 3 weeks, and 5 (50%) became contaminated after 4 weeks, leaving 7 (50%) that did not become contaminated after 1 month of treatment. Using containers with an adapted filter significantly reduces the contamination rates in autologous serum eyedrops, thus extending the use of such container by the patients for up to 4 weeks with virtually no contamination risks. Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

  3. Effect of neurturin on multipotent cells isolated from the adult skeletal muscle

    International Nuclear Information System (INIS)

    Vourc'h, Patrick; Lacar, Benjamin; Mignon, Laurence; Lucas, Paul A.; Young, Henry E.; Chesselet, Marie-Francoise

    2005-01-01

    Ligands of the glial cell line-derived neurotrophic factors (GDNF)-family are trophic factors for the development and survival of multiple cell types, however their effects on non-neuronal stem cells are unknown. We examined the action of neurturin on a candidate stem cell population isolated from adult skeletal muscles. When grown as spheres, these cells expressed mRNAs for GDNF, persephin, GFR-α2, GFR-α4 (neurturin receptor), and Ret. Exposure of these cells to neurturin significantly augmented cell numbers via increased cell proliferation. After addition of retinoic acid, the cells exited the cell cycle, developed thin processes, and became immunoreactive for βIII-tubulin, while Ret mRNA expression decreased, without changes in the level of GFR-α2 mRNA. Neurturin induced an outgrowth of processes on these βIII-tubulin positive cells. Neurturin may therefore be beneficial in the use of these multipotent cells isolated from adult muscles for autologous transplants in neurological applications

  4. A clinical study on the feasibility of autologous cord blood transfusion for anemia of prematurity.

    Science.gov (United States)

    Khodabux, Chantal M; von Lindern, Jeannette S; van Hilten, Joost A; Scherjon, Sicco; Walther, Frans J; Brand, Anneke

    2008-08-01

    The objective was to investigate the use of autologous red blood cells (RBCs) derived from umbilical cord blood (UCB), as an alternative for allogeneic transfusions in premature infants admitted to a tertiary neonatal center. UCB collection was performed at deliveries of less than 32 weeks of gestation and processed into autologous RBC products. Premature infants requiring a RBC transfusion were randomly assigned to an autologous or allogeneic product. The primary endpoint was an at least 50 percent reduction in allogeneic transfusion needs. Fifty-seven percent of the collections harvested enough volume (> or =15 mL) for processing. After being processed, autologous products (> or =10 mL/kg) were available for 36 percent of the total study population and for 27 percent of the transfused infants and could cover 58 percent (range, 25%-100%) of the transfusion needs within the 21-day product shelf life. Availability of autologous products depended most on the gestational age. Infants born between 24 and 28 weeks had the lowest availability (17%). All products, however, would be useful in view of their high (87%) transfusion needs. Availability was highest (48%) for the infants born between 28 and 30 weeks. For 42 percent of the infants with transfusion needs in this group, autologous products were available. For the infants born between 30 and 32 weeks, autologous products were available for 36 percent of the infants. Transfusion needs in this group were, however, much lower (19%) compared to the other gestational groups. Autologous RBCs derived from UCB could not replace 50 percent of allogeneic transfusions due to the low UCB volumes collected and subsequent low product availability.

  5. Chimeric Human Skin Substitute Tissue: A Novel Treatment Option for the Delivery of Autologous Keratinocytes.

    Science.gov (United States)

    Rasmussen, Cathy A; Allen-Hoffmann, B Lynn

    2012-04-01

    For patients suffering from catastrophic burns, few treatment options are available. Chimeric coculture of patient-derived autologous cells with a "carrier" cell source of allogeneic keratinocytes has been proposed as a means to address the complex clinical problem of severe skin loss. Currently, autologous keratinocytes are harvested, cultured, and expanded to form graftable epidermal sheets. However, epidermal sheets are thin, are extremely fragile, and do not possess barrier function, which only develops as skin stratifies and matures. Grafting is typically delayed for up to 4 weeks to propagate a sufficient quantity of the patient's cells for application to wound sites. Fully stratified chimeric bioengineered skin substitutes could not only provide immediate wound coverage and restore barrier function, but would simultaneously deliver autologous keratinocytes to wounds. The ideal allogeneic cell source for this application would be an abundant supply of clinically evaluated, nontumorigenic, pathogen-free, human keratinocytes. To evaluate this potential cell-based therapy, mixed populations of a green fluorescent protein-labeled neonatal human keratinocyte cell line (NIKS) and unlabeled primary keratinocytes were used to model the allogeneic and autologous components of chimeric monolayer and organotypic cultures. Relatively few autologous keratinocytes may be required to produce fully stratified chimeric skin substitute tissue substantially composed of autologous keratinocyte-derived regions. The need for few autologous cells interspersed within an allogeneic "carrier" cell population may decrease cell expansion time, reducing the time to patient application. This study provides proof of concept for utilizing NIKS keratinocytes as the allogeneic carrier for the generation of bioengineered chimeric skin substitute tissues capable of providing immediate wound coverage while simultaneously supplying autologous human cells for tissue regeneration.

  6. Mechanical stimuli activation of calpain is required for myoblast differentiation and occurs via an ERK/MAP kinase signaling pathway

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    2006-01-01

    a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have shown that mechanical signals transmitted through the C2C12 cells interaction with laminin cause an increase in cellular differentiation. This signaling results in an increase in the number of myotubes formed in the cultures...

  7. Adaptive Immune Response Impairs the Efficacy of Autologous Transplantation of Engineered Stem Cells in Dystrophic Dogs

    Science.gov (United States)

    Sitzia, Clementina; Farini, Andrea; Jardim, Luciana; Razini, Paola; Belicchi, Marzia; Cassinelli, Letizia; Villa, Chiara; Erratico, Silvia; Parolini, Daniele; Bella, Pamela; da Silva Bizario, Joao Carlos; Garcia, Luis; Dias-Baruffi, Marcelo; Meregalli, Mirella; Torrente, Yvan

    2016-01-01

    Duchenne muscular dystrophy is the most common genetic muscular dystrophy. It is caused by mutations in the dystrophin gene, leading to absence of muscular dystrophin and to progressive degeneration of skeletal muscle. We have demonstrated that the exon skipping method safely and efficiently brings to the expression of a functional dystrophin in dystrophic CD133+ cells injected scid/mdx mice. Golden Retriever muscular dystrophic (GRMD) dogs represent the best preclinical model of Duchenne muscular dystrophy, mimicking the human pathology in genotypic and phenotypic aspects. Here, we assess the capacity of intra-arterial delivered autologous engineered canine CD133+ cells of restoring dystrophin expression in Golden Retriever muscular dystrophy. This is the first demonstration of five-year follow up study, showing initial clinical amelioration followed by stabilization in mild and severe affected Golden Retriever muscular dystrophy dogs. The occurrence of T-cell response in three Golden Retriever muscular dystrophy dogs, consistent with a memory response boosted by the exon skipped-dystrophin protein, suggests an adaptive immune response against dystrophin. PMID:27506452

  8. Anti-skeletal muscle atrophy effect of Oenothera odorata root extract via reactive oxygen species-dependent signaling pathways in cellular and mouse model.

    Science.gov (United States)

    Lee, Yong-Hyeon; Kim, Wan-Joong; Lee, Myung-Hun; Kim, Sun-Young; Seo, Dong-Hyun; Kim, Han-Sung; Gelinsky, Michael; Kim, Tack-Joong

    2016-01-01

    Skeletal muscle atrophy can be defined as a decrease of muscle volume caused by injury or lack of use. This condition is associated with reactive oxygen species (ROS), resulting in various muscular disorders. We acquired 2D and 3D images using micro-computed tomography in gastrocnemius and soleus muscles of sciatic-denervated mice. We confirmed that sciatic denervation-small animal model reduced muscle volume. However, the intraperitoneal injection of Oenothera odorata root extract (EVP) delayed muscle atrophy compared to a control group. We also investigated the mechanism of muscle atrophy's relationship with ROS. EVP suppressed expression of SOD1, and increased expression of HSP70, in both H2O2-treated C2C12 myoblasts and sciatic-denervated mice. Moreover, EVP regulated apoptotic signals, including caspase-3, Bax, Bcl-2, and ceramide. These results indicate that EVP has a positive effect on reducing the effect of ROS on muscle atrophy.

  9. Autologous blood transfusion in total knee replacement surgery.

    Science.gov (United States)

    Sarkanović, Mirka Lukić; Gvozdenović, Ljiljana; Savić, Dragan; Ilić, Miroslav P; Jovanović, Gordana

    2013-03-01

    Total knee replacement (TKR) surgery is one of the most frequent and the most extensive procedures in orthopedic surgery, accompanied with some serious complications. Perioperative blood loss is one of the most serious losses, so it is vital to recognize and treat such losses properly. Autologous blood transfusion is the only true alternative for the allogeneic blood. The aim of this study was to to examine if autologous blood transfusion reduces usage of allogenic blood in total knee replacement surgery, as well as to examine possible effect of autologous blood transfusion on postoperative complications, recovery and hospital stay of patients after total knee replacement surgery. During the controlled, prospective, randomised study we compared two groups of patients (n = 112) with total prosthesis implanted in their knee. The group I consisted of the patients who received the transfusion of other people's (allogeneic) blood (n = 57) and the group II of the patients whose blood was collected postoperatively and then given them [their own (autologous) blood] (n = 55). The transfusion trigger for both groups was hemoglobin level of 85 g/L. In the group of patients whose blood was collected perioperatively only 9 (0.9%) of the patients received transfusion of allogeneic blood, as opposed to the control group in which 98.24% of the patients received the transfusion of allogeneic blood (p blood was collected stayed in hospital for 6.18 days, while the patients of the control group stayed 7.67 days (p blood transfusion is a very effective method for reducing consumption of allogenic blood and thus, indirectly for reducing all complications related to allogenic blood transfusion. There is also a positive influence on postoperative recovery after total knee replacement surgery due to the reduction of hospital stay, and indirectly on the reduction of hospital costs.

  10. Serotonin and Histamine Therapy Increases Tetanic Forces of Myoblasts, Reduces Muscle Injury, and Improves Grip Strength Performance of Dmdmdx Mice

    Directory of Open Access Journals (Sweden)

    Volkan Gurel

    2015-11-01

    Full Text Available Duchenne muscular dystrophy (DMD is a recessive X-linked fatal disorder caused by a mutation in the dystrophin gene. Although several therapeutic approaches have been studied, none has led to substantial long-term effects in patients. The aim of this study was to test a serotonin and histamine (S&H combination on human skeletal myoblasts and Dmdmdx mice for its effects on muscle strength and injury. Normal human bioartificial muscles (BAMs were treated, and muscle tetanic forces and muscle injury tests were performed using the MyoForce Analysis System. Dmdmdx mice, the murine model of DMD, were administered serotonin, histamine, or S&H combination twice daily for 6 weeks, and functional performance tests were conducted once a week. The S&H combination treatment caused significant increases in tetanic forces at all time points and concentrations tested as compared to the saline controls. Dose response of the BAMs to the treatment demonstrated a significant increase in force generation at all concentrations compared to the controls after 3 to 4 days of drug treatment. The highest 3 concentrations had a significant effect on lowering contractile-induced injury as measured by a reduction in the release of adenylate kinase. Histamine-only and S&H treatments improved grip strength of Dmdmdx mice, whereas serotonin-only treatment resulted in no significant improvement in muscle strength. The results of this study indicate that S&H therapy might be a promising new strategy for muscular dystrophies and that the mechanism should be further investigated.

  11. Quality of intraoperative autologous blood withdrawal used for retransfusion after cardiopulmonary bypass.

    Science.gov (United States)

    Flom-Halvorsen, Hanne I; Øvrum, Eivind; Øystese, Rolf; Brosstad, Frank

    2003-09-01

    Intraoperative autologous blood withdrawal protects the pooled blood from the deleterious effects of cardiopulmonary bypass. Following reinfusion after cardiopulmonary bypass, the fresh autologous blood contributes to less coagulation abnormalities and reduces postoperative bleeding and the need for allogeneic blood products. However, few data have been available concerning the quality and potential activation of fresh blood stored at room temperature in the operating room. Forty coronary artery bypass grafting patients undergoing a consistent intraoperative and postoperative autotransfusion protocol had a median of 1,000 mL of autologous blood withdrawn before cardiopulmonary bypass. After heparinization the blood was drained from the venous catheter via venous cannula into standard blood bags and stored in the operating room until termination of cardiopulmonary bypass. Samples for hemostatic and inflammatory markers were taken from the pooled blood immediately before it was returned to the patient. There was some activation of platelets in the stored autologous blood, as measured by an increase of beta-thromboglobulin. Indications of thrombin formation, as assessed by plasma levels of thrombin-antithrombin complex and prothrombin fragment 1.2 were not seen, and there was no fibrinolytic activity. The red blood cells remained intact, indicated by the absence of plasma free hemoglobin. As for the inflammatory response, the levels of the terminal complement complex remained stable, and the cytokines tumor necrosis factor-alpha and interleukin 6 levels were not increased during storage. The complement activation products increased minimally, but remained within normal ranges. Except for slight activation of platelets, there was no indication of coagulation, hemolysis, fibrinolysis, or immunologic activity in the autologous blood after approximately 1 hour of operating room storage. The autologous blood was preserved in a condition of high quality, and retransfusion

  12. Lymphoscintigraphy and autologous stem cell implantation

    International Nuclear Information System (INIS)

    Peña, Yamile; Batista, Juan F.; Perera, Alejandro; Torres, Leonel A.; Sánchez, Elvia L.; Sánchez, Yolaine; Ducat, Luis; Prats, Anais; Hernández, Porfirio; Romero, Susana; Goicochea, Pedro; Quintela, Ana M.

    2016-01-01

    Lymphoscintigraphy is the criterion standard technique for the diagnosis of lymphedema. Advances of the application of autologous hematopoietic stem cells in ischemic disorders of lower limbs have increased the attention of researchers in this field. Aim: To determine the usefulness of lymphoscintigraphy for the assessment the efficacy of autologous stem cell implantation in patients with chronic lymphedema of the upper and lower limbs. Methods: Sixty-five patients were included. Clinical evaluation and lymphoscintigraphy were performed before and six months after stem cells implantation. The stem cells implantations were carried out by multiple superficial and deep injections in the trajectory of the lymphatic vessels and also in the inguinal region. A volume of 0.75 to 1.00 mL of cell suspension (1.0-2.2 x 109 stem cells) was administered in each injection site. Lymphoscintigraphy: Whole-body scans were acquired at 20 minutes, 1 hour, and 3 hours after administration of 185 to 259 MBq (5–7mCi) of 99m Tc-albumin nanocolloids in the interdigital space of both limbs. The anatomy and function of the lymphatic system were evaluated. Results: Functional assessment before implantation of stem cells showed that 69.2% of the patients had severe lymphatic insufficiency. The 61.5% of patients showed clinical improvement, confirmed by the results of the lymphoscintigraphy. The 46.1% of the cases evaluated showed a clear improvement. The study showed that the isotopic lymphography can evaluate the therapeutic response and its intensity. Conclusion: Lymphoscintigraphy is a useful technique for the evaluation and monitoring of autologous stem cell transplantation in patients with chronic lymphedema. (author)

  13. Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

    Science.gov (United States)

    Jeanplong, Ferenc; Falconer, Shelley J.; Oldham, Jenny M.; Thomas, Mark; Gray, Tarra S.; Hennebry, Alex; Matthews, Kenneth G.; Kemp, Frederick C.; Patel, Ketan; Berry, Carole; Nicholas, Gina; McMahon, Christopher D.

    2013-01-01

    Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (Pmyostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product. PMID:24312578

  14. Autologous orbicularis muscle for filling facial folds-an experimental and clinical study.

    Science.gov (United States)

    Schellini, Silvana Artioli; Hirai, Flavio Eduardo; Hoyama, Erika; Mattos, Maila Karina; Chaves, Fernando Rodrigo; Pellizon, Claudia Helena; Padovani, Carlos Roberto

    2009-01-01

    To present a technique for filling facial folds by using autologous orbicularis oculi muscle, based on an experimental model. two studies are presented: (1) an experimental study using 15 albino guinea-pigs from which a strip of the sural triceps muscle was removed and implanted in the subcutaneous tissue of the dorsal area. The animals were sacrificed 7, 30 and 60 days after the implantation, and the material was histologically evaluated. And (2) an interventional prospective clinical trial carried out on 20 patients referred to blepharoplasty surgery. They received autologous preseptal orbicularis muscle for filling facial folds. The results where evaluated by patients satisfaction and clinical exam. the sural tricep muscle, when implanted in the subcutaneous tissue, resulted in fibrosis. The patients whom received autologous orbicularis muscle implanted for filling facial folds showed that the procedure can be successfully carried out. autologous preseptal orbicularis muscle is a good material for filling facial folds. Cicatricial tissue will be formed on its implantation site, filling the tissue gap that forms the folds on the skin.

  15. Anticancer activity of biologically synthesized silver and gold nanoparticles on mouse myoblast cancer cells and their toxicity against embryonic zebrafish

    International Nuclear Information System (INIS)

    Ramachandran, Rajan; Krishnaraj, Chandran; Sivakumar, Allur Subramaniyan; Prasannakumar, Palaniappan; Abhay Kumar, V.K.; Shim, Kwan Seob; Song, Chul-Gyu; Yun, Soon-Il

    2017-01-01

    The aim of this study was to evaluate the anticancer activity of bioinspired silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) against mouse myoblast cancer cells (C 2 C 12 ). Both AgNPs and AuNPs were biologically synthesized using Spinacia oleracea Linn., aqueous leaves extract. UV–Vis. spectrophotometer, high resolution-transmission electron microscopy (HR-TEM), field emission-scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) studies supported the successful synthesis of AgNPs and AuNPs. Both these NPs have shown cytotoxicity against C 2 C 12 cells even at very low concentration (5 μg/mL). Acridine orange/Ethidium bromide (AO/EB) dual staining confirmed the apoptotic morphological features. The levels of caspase enzymes (caspase-3 and caspase-7) were significantly up-regulated in NPs treated myoblast cells than the plant extract. Furthermore, in zebrafish embryo toxicity study, AgNPs showed 100% mortality at 3 μg/mL concentration while AuNPs exhibited the same at much higher concentration (300 mg/mL). Taken together, these results provide a preliminary guidance for the development of biomaterials based drugs to fight against the fatal diseases for example cancer. - Highlights: • Anticancer activity was done for the first time against mouse myoblast cells. • AgNPs showed 100% growth inhibition against C 2 C 12 cells at 20 μg/mL concentration. • AO/EB dual staining and caspase assays confirmed the apoptotic features. • Nanoparticles treated embryos showed yolk sac edema and tail malformation. • AgNPs were found to be more toxic to embryonic zebrafishes than the AuNPs.

  16. Anticancer activity of biologically synthesized silver and gold nanoparticles on mouse myoblast cancer cells and their toxicity against embryonic zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Ramachandran, Rajan [Centre for Advanced Studies in Botany, School of Life Sciences, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu (India); Krishnaraj, Chandran [Department of Food Science & Technology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); M/s. Eureka Forbes Ltd, R & D Centre, Kudlu, Bangalore (India); Sivakumar, Allur Subramaniyan [Department of Animal Biotechnology, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Prasannakumar, Palaniappan [Advanced Biomedical Imaging Center, Department of Electronic Engineering, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Abhay Kumar, V.K. [M/s. Eureka Forbes Ltd, R & D Centre, Kudlu, Bangalore (India); Shim, Kwan Seob [Department of Animal Biotechnology, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Song, Chul-Gyu [Advanced Biomedical Imaging Center, Department of Electronic Engineering, Chonbuk National University, Jeonju 54896 (Korea, Republic of); Yun, Soon-Il, E-mail: siyun@jbnu.ac.kr [Department of Food Science & Technology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2017-04-01

    The aim of this study was to evaluate the anticancer activity of bioinspired silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) against mouse myoblast cancer cells (C{sub 2}C{sub 12}). Both AgNPs and AuNPs were biologically synthesized using Spinacia oleracea Linn., aqueous leaves extract. UV–Vis. spectrophotometer, high resolution-transmission electron microscopy (HR-TEM), field emission-scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) studies supported the successful synthesis of AgNPs and AuNPs. Both these NPs have shown cytotoxicity against C{sub 2}C{sub 12} cells even at very low concentration (5 μg/mL). Acridine orange/Ethidium bromide (AO/EB) dual staining confirmed the apoptotic morphological features. The levels of caspase enzymes (caspase-3 and caspase-7) were significantly up-regulated in NPs treated myoblast cells than the plant extract. Furthermore, in zebrafish embryo toxicity study, AgNPs showed 100% mortality at 3 μg/mL concentration while AuNPs exhibited the same at much higher concentration (300 mg/mL). Taken together, these results provide a preliminary guidance for the development of biomaterials based drugs to fight against the fatal diseases for example cancer. - Highlights: • Anticancer activity was done for the first time against mouse myoblast cells. • AgNPs showed 100% growth inhibition against C{sub 2}C{sub 12} cells at 20 μg/mL concentration. • AO/EB dual staining and caspase assays confirmed the apoptotic features. • Nanoparticles treated embryos showed yolk sac edema and tail malformation. • AgNPs were found to be more toxic to embryonic zebrafishes than the AuNPs.

  17. Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Siemionow, M; Cwykiel, J; Heydemann, A; Garcia-Martinez, J; Siemionow, K; Szilagyi, E

    2018-04-01

    Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin-deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 10 6 ) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

  18. Clinical Allogeneic and Autologous Islet Cell Transplantation: Update

    Directory of Open Access Journals (Sweden)

    Shinichi Matsumoto

    2011-06-01

    Full Text Available Islet cell transplantation is categorized as a β-cell replacement therapy for diabetic patients who lack the ability to secrete insulin. Allogeneic islet cell transplantation is for the treatment of type 1 diabetes, and autologous islet cell transplantation is for the prevention of surgical diabetes after a total pancreatectomy. The issues of allogeneic islet cell transplantation include poor efficacy of islet isolation, the need for multiple donor pancreata, difficulty maintaining insulin independence and undesirable side effects of immunosuppressive drugs. Those issues have been solved step by step and allogeneic islet cell transplantation is almost ready to be the standard therapy. The donor shortage will be the next issue and marginal and/or living donor islet cell transplantation might alleviate the issue. Xeno-islet cell transplantation, β-cell regeneration from human stem cells and gene induction of the naïve pancreas represent the next generation of β-cell replacement therapy. Autologous islet cell transplantation after total pancreatectomy for the treatment of chronic pancreatitis with severe abdominal pain is the standard therapy, even though only limited centers are able to perform this treatment. Remote center autologous islet cell transplantation is an attractive option for hospitals performing total pancreatectomies without the proper islet isolation facilities.

  19. An invention of thermo-responsive polymer surface, yielding cell sheet based regenerative therapies in cardiology and ophthalmology

    Directory of Open Access Journals (Sweden)

    Sawa Y

    2015-12-01

    /2009/091/000590 after standardizing the procedures for collection of autologous bone marrow cells in January 2010. Out of the 104 patients, 68 patients met the inclusion criteria for the trial follow up. Of the total, 45 patients could not be followed up in spite of the team’s best efforts to trace them. Only 23 patients (34% were available for follow up. Amongst those, 19 patients (82.6% were alive at the end of the first year and 10 out of the 19 patients (52.6% had at least a 5% increase in the Ejection Fraction post therapy and had significant improvement in the functional status. The procedure was safe in all the other patients which were similar to the results of other studies reported so far. Though the results of this clinical trial infer that stem cells are not an alternative to heart transplantation they offer a 'dynamic cellular support' for regenerating functional and viable myocardium. As a step further in identifying definitive cell based therapy approaches to treat heart failure, cell sheet therapy was advocated by Prof. Sawa’s team. In Prof. Sawa’s Institute, longitudinal research team was launched in the late 1990s to develop new treatments for advanced cardiac disease by an array of basic experimental studies of surgical and regenerative approaches. As a result, placement of autologous or allogeneic stem/progenitor cell-sheets over the cardiac surface via surgical approach was proven to be the most safe and efficacious treatment for advanced cardiac failure. To grow the skeletal myoblasts as a sheet for clinical transplantation, Prof. Sawa’s team employed the thermo-responsive polymer dish developed by Prof. Okano’s team which enabled the in vitro growth of the skeletal myoblasts as patches and their easy separation for application over the ischemic/infarcted area of the heart after thorough validation in animal experiments [4]. A clinical study was, therefore, launched in 2006, wherein autologous skeletal myoblast cell-sheets were directly

  20. The composite of bone marrow concentrate and PRP as an alternative to autologous bone grafting.

    Directory of Open Access Journals (Sweden)

    Mohssen Hakimi

    Full Text Available One possible alternative to the application of autologous bone grafts represents the use of autologous bone marrow concentrate (BMC. The purpose of our study was to evaluate the potency of autologous platelet-rich plasma (PRP in combination with BMC. In 32 mini-pigs a metaphyseal critical-size defect was surgically created at the proximal tibia. The animals were allocated to four treatment groups of eight animals each (1. BMC+CPG group, 2. BMC+CPG+PRP group, 3. autograft group, 4. CPG group. In the BMC+CPG group the defect was filled with autologous BMC in combination with calcium phosphate granules (CPG, whereas in the BMC+CPG+PRP group the defect was filled with the composite of autologous BMC, CPG and autologous PRP. In the autograft group the defect was filled with autologous cancellous graft, whereas in the CPG group the defect was filled with CPG solely. After 6 weeks radiological and histomorphometrical analysis showed significantly more new bone formation in the BMC+CPG+PRP group compared to the BMC+CPG group and the CPG group. There were no significant differences between the BMC+CPG+PRP group and the autograft group. In the PRP platelets were enriched significantly about 4.7-fold compared to native blood. In BMC the count of mononuclear cells increased significantly (3.5-fold compared to the bone marrow aspirate. This study demonstrates that the composite of BMC+CPG+PRP leads to a significantly higher bone regeneration of critical-size defects at the proximal tibia in mini-pigs than the use of BMC+CPG without PRP. Furthermore, within the limits of the present study the composite BMC+CPG+PRP represents a comparable alternative to autologous bone grafting.

  1. Preeclampsia in autologous and oocyte donation pregnancy: is there a different pathophysiology?

    Science.gov (United States)

    Lashley, Lisa E E L O; Buurma, Aletta; Swings, Godelieve M J S; Eikmans, Michael; Anholts, Jacqueline D H; Bakker, Jaap A; Claas, Frans H J

    2015-06-01

    Oocyte donation (OD) is a specific method of artificial reproductive technology that is accompanied by a higher risk of preeclampsia during pregnancy. The pathophysiological mechanism underlying preeclampsia in OD pregnancies is thought to differ from preeclampsia in autologous pregnancies. As preeclampsia in autologous pregnancies is suggested to be associated with complement activation, we studied C4d deposition, circulating complement components and placental complement regulatory proteins in preeclamptic OD pregnancies. Women with uncomplicated and preeclamptic pregnancies after OD or spontaneous conception were selected. We stained the placentas for C4d, marker for complement activation, measured complement factors C1q, C3 and C4 in maternal sera and quantified the placental mRNA expression of complement regulatory proteins CD46, CD55 and CD59. A significantly (p preeclampsia compared with uncomplicated pregnancies, both OD and autologous. The level of complement factors in serum did not differ between the groups. Children born in the autologous preeclampsia group were significantly lower in birth weight (p preeclampsia pregnancies, there is excessive activation of complement in preeclamptic OD pregnancies. However, in contrast to autologous pregnancies this is not associated with counterbalancing upregulation of complement regulatory proteins. Furthermore, C4d deposition in OD pregnancies is not related to the severity of preeclampsia, suggesting another trigger or regulatory mechanism of placental C4d deposition in preeclamptic OD pregnancies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. A novel in vitro model for studying quiescence and activation of primary isolated human myoblasts

    DEFF Research Database (Denmark)

    Sellathurai, Jeeva; Cheedipudi, Sirisha; Dhawan, Jyotsna

    2013-01-01

    term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may...... be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis...

  3. Management of vocal fold scar with autologous fat implantation: perceptual results.

    Science.gov (United States)

    Neuenschwander, M C; Sataloff, R T; Abaza, M M; Hawkshaw, M J; Reiter, D; Spiegel, J R

    2001-06-01

    Vocal fold scar disrupts the mucosal wave and interferes with glottic closure. Treatment involves a multidisciplinary approach that includes voice therapy, medical management, and sometimes surgery. We reviewed the records of the first eight patients who underwent autologous fat implantation for vocal fold scar. Information on the etiology of scar, physical findings, and prior interventions were collected. Videotapes of videostroboscopic findings and perceptual voice ratings [Grade, Roughness, Breathiness, Asthenia, Strain (GRBAS)] were randomized and analyzed independently by four blinded observers. Etiology of scar included mass excision (7), vocal fold stripping (3), congenital sulcus (2), and hemorrhage (1). Prior surgical procedures performed included thyroplasty (1), autologous fat injection (9), excision of scar (2), and lysis of adhesions (2). Strobovideolaryngoscopy: Statistically significant improvement was found in glottic closure, mucosal wave, and stiffness (P = 0.05). Perceptual ratings (GRBAS): Statistically significant improvement was found in all five parameters, including overall Grade, Roughness, Breathiness, Asthenia, and Strain (P = 0.05). Patients appear to have improved vocal fold function and quality of voice after autologous fat implantation in the vocal fold. Autologous fat implantation is an important adjunctive procedure in the management of vocal fold scar, and a useful addition to the armamentarium of the experienced phonomicrosurgeon.

  4. Autologous bone marrow mononuclear cell delivery to dilated ...

    African Journals Online (AJOL)

    Autologous bone marrow mononuclear cell delivery to dilated cardiomyopathy patients: A clinical trial. PLN Kaparthi, G Namita, LK Chelluri, VSP Rao, PK Shah, A Vasantha, SK Ratnakar, K Ravindhranath ...

  5. Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity

    Directory of Open Access Journals (Sweden)

    William Dott

    2014-01-01

    Full Text Available Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR was significantly increased whereas extracellular acidification rate (ECAR, a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2·– level compared to cells in the glucose model. An antimycin A (AA dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a

  6. α-Syntrophin is involved in the survival signaling pathway in myoblasts under menadione-induced oxidative stress.

    Science.gov (United States)

    Lim, Jeong-A; Choi, Su Jin; Moon, Jae Yun; Kim, Hye Sun

    2016-05-15

    Dystrophin-deficient muscle is known to be more vulnerable to oxidative stress, but not much is known about the signaling pathway(s) responsible for this phenomenon. α-Syntrophin, a component of the dystrophin-glycoprotein complex, can function as a scaffold protein because of its multiple protein interaction domains. In this study, we investigated the role of α-syntrophin in C2 myoblasts under menadione-induced oxidative stress. We found that the protein level of α-syntrophin was elevated when cells were exposed to menadione. To investigate the function of α-syntrophin during oxidative stress, we established α-syntrophin-overexpressing and knockdown cell lines. The α-syntrophin-overexpressing cells were resistant to the menadione-induced oxidative stress. In addition, survival signalings such as protein kinase B (Akt) phosphorylation and the Bcl-2/BAX ratio were increased in these cells. On the other hand, apoptotic signals such as cleavage of caspase-3 and poly ADP ribose polymerase (PARP) were increased in the α-syntrophin knockdown cells. Furthermore, Ca(2+)influx, which is known to increase when cells are exposed to oxidative stress, decreased in the α-syntrophin-overexpressing cells, but increased in the knockdown cells. These results suggest that α-syntrophin plays a pivotal role in the survival pathway triggered by menadione-induced oxidative stress in cultured myoblasts. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

    International Nuclear Information System (INIS)

    Chen, Zirong; Jin, Guorong; Lin, Shuibin; Lin, Xiumei; Gu, Yumei; Zhu, Yujuan; Hu, Chengbin; Zhang, Qingjiong; Wu, Lizi; Shen, Huangxuan

    2012-01-01

    Highlights: ► CDA-II inhibits myogenic differentiation in a dose-dependent manner. ► CDA-II repressed expression of muscle transcription factors and structural proteins. ► CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.

  8. Efficacy of autologous platelets in macular hole surgery

    Directory of Open Access Journals (Sweden)

    Konstantinidis A

    2013-04-01

    Full Text Available Aristeidis Konstantinidis,1,2 Mark Hero,2 Panagiotis Nanos,1 Georgios D Panos1,3 1Department of Ophthalmology, University Hospital of Alexandroupolis, Alexandroupolis, Greece; 2Opthalmology Department, University Hospital Coventry and Warwickshire, Coventry, UK; 3Department of Ophthalmology, University Hospitals of Geneva, Geneva, Switzerland Abstract: The introduction of optical coherence tomography has allowed accurate measurement of the size of macular holes. A retrospective consecutive review was performed of 21 patients undergoing macular hole repair with vitrectomy, gas tamponade, and autologous platelet injection and we assessed the effect of macular hole parameters on anatomic and functional outcomes. We looked at the demographic features, final visual outcome, and anatomical closure. Twenty-one patients were included in the study. They underwent routine vitrectomy with gas tamponade (C3F8 and injection of autologous platelets. All patients were advised to maintain a facedown posture for 2 weeks. Anatomical closure was confirmed in all cases and 20 out of 21 of patients had improved postoperative visual acuity by two or more lines. In our series, the macular hole dimensions did not have much effect on the final results. The use of autologous platelets and strict facedown posture seems to be the deciding factor in good anatomical and visual outcome irrespective of macular hole dimensions. Keywords: macular hole, platelets, vitrectomy

  9. The model of pulmonary embolism caused by autologous thrombus in rabbits

    OpenAIRE

    Yu-Jiao Ding; Yang Chen

    2017-01-01

    Objective: To establish a model of pulmonary embolism in rabbits by using autologous thrombosis of rabbit ear vein, to study the method of establishing acute pulmonary embolism by using autologous thrombus and to explore the diagnostic value of oxygen partial pressure in acute pulmonary embolism. Methods: Twenty rabbits were randomly divided into normal group (n=5), 7 h group, 24h group, 1 week after model establishment Group. The arterial blood gas analysis was performed on th...

  10. Quality of harvested autologous platelets compared with stored donor platelets for use after cardiopulmonary bypass procedures.

    Science.gov (United States)

    Crowther, M; Ford, I; Jeffrey, R R; Urbaniak, S J; Greaves, M

    2000-10-01

    Platelet dysfunction has a major contribution in bleeding after cardiopulmonary bypass (CPB) and transfusion of platelets is frequently used to secure haemostasis. Allogeneic platelets prepared for transfusion are functionally impaired. Autologous platelets harvested preoperatively require a shorter storage time before transfusion and their use also avoids the risks associated with transfusion of allogeneic blood products. For the first time, we have compared the functional quality of autologous platelets with allogeneic platelets prepared by two methods, immediately before infusion. Platelet activation was assessed by P-selectin expression and fibrinogen binding using flow cytometry. We also monitored the effects of CPB surgery and re-infusion of autologous platelets on platelet function. Autologous platelet-rich plasma (PRP) contained a significantly lower (P platelets compared with allogeneic platelet preparations, and also contained a significantly higher (P platelets. Allogeneic platelets prepared by donor apheresis were more activated and less responsive than those produced by centrifugation of whole blood. In patients' blood, the percentage of platelets expressing P-selectin or binding fibrinogen increased significantly after CPB (P platelets responsive to in vitro agonists was decreased (P platelet activation during the procedure. The percentage of activated platelets decreased (statistically not significant) after re-infusion of autologous PRP. P-selectin expression had returned to pre-CPB levels 24 h post-operatively. Autologous platelet preparations display minimal activation, but remain responsive. Conservation of platelet function may contribute to the potential clinical benefits of autologous transfusion in cardiopulmonary bypass.

  11. Effect of hyperbaric oxygen therapy combined with autologous platelet concentrate applied in rabbit fibula fraction healing

    Directory of Open Access Journals (Sweden)

    Paulo Cesar Fagundes Neves

    2013-09-01

    Full Text Available OBJECTIVES: The purpose is to study the effects of hyperbaric oxygen therapy and autologous platelet concentrates in healing the fibula bone of rabbits after induced fractures. METHODS: A total of 128 male New Zealand albino rabbits, between 6-8 months old, were subjected to a total osteotomy of the proximal portion of the right fibula. After surgery, the animals were divided into four groups (n = 32 each: control group, in which animals were subjected to osteotomy; autologous platelet concentrate group, in which animals were subjected to osteotomy and autologous platelet concentrate applied at the fracture site; hyperbaric oxygen group, in which animals were subjected to osteotomy and 9 consecutive daily hyperbaric oxygen therapy sessions; and autologous platelet concentrate and hyperbaric oxygen group, in which animals were subjected to osteotomy, autologous platelet concentrate applied at the fracture site, and 9 consecutive daily hyperbaric oxygen therapy sessions. Each group was divided into 4 subgroups according to a pre-determined euthanasia time points: 2, 4, 6, and 8 weeks postoperative. After euthanasia at a specific time point, the fibula containing the osseous callus was prepared histologically and stained with hematoxylin and eosin or picrosirius red. RESULTS: Autologous platelet concentrates and hyperbaric oxygen therapy, applied together or separately, increased the rate of bone healing compared with the control group. CONCLUSION: Hyperbaric oxygen therapy and autologous platelet concentrate combined increased the rate of bone healing in this experimental model.

  12. Latissimus Dorsi and Immediate Fat Transfer (LIFT for Complete Autologous Breast Reconstruction

    Directory of Open Access Journals (Sweden)

    James M. Economides, MD

    2018-01-01

    Conclusion:. Autologous augmentation of the LD flap with lipotransfer has been used to avoid placement of an implant. We improve the technique by performing lipotransfer during index reconstruction. Furthermore, we perform lipotransfer prior to disorigination of the LD muscle to minimize trauma to the flap and increase the efficiency of fat grafting. Our experience demonstrates that this technique is a viable autologous alternative to microsurgical breast reconstruction.

  13. Engraftment potential of dermal fibroblasts following in vivo myogenic conversion in immunocompetent dystrophic skeletal muscle

    Directory of Open Access Journals (Sweden)

    Lindsey A Muir

    2014-01-01

    Full Text Available Autologous dermal fibroblasts (dFbs are promising candidates for enhancing muscle regeneration in Duchenne muscular dystrophy (DMD due to their ease of isolation, immunological compatibility, and greater proliferative potential than DMD satellite cells. We previously showed that mouse fibroblasts, after MyoD-mediated myogenic reprogramming in vivo, engraft in skeletal muscle and supply dystrophin. Assessing the therapeutic utility of this system requires optimization of conversion and transplantation conditions and quantitation of engraftment so that these parameters can be correlated with possible functional improvements. Here, we derived dFbs from transgenic mice carrying mini-dystrophin, transduced them by lentivirus carrying tamoxifen-inducible MyoD, and characterized their myogenic and engraftment potential. After cell transplantation into the muscles of immunocompetent dystrophic mdx4cv mice, tamoxifen treatment drove myogenic conversion and fusion into myofibers that expressed high levels of mini-dystrophin. Injecting 50,000 cells/µl (1 × 106 total cells resulted in a peak of ∼600 mini-dystrophin positive myofibers in tibialis anterior muscle single cross-sections. However, extensor digitorum longus muscles with up to 30% regional engraftment showed no functional improvements; similar limitations were obtained with whole muscle mononuclear cells. Despite the current lack of physiological improvement, this study suggests a viable initial strategy for using a patient-accessible dermal cell population to enhance skeletal muscle regeneration in DMD.

  14. Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

    Science.gov (United States)

    Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

    1999-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

  15. [Autologous fat grafting in children].

    Science.gov (United States)

    Baptista, C; Bertrand, B; Philandrianos, C; Degardin, N; Casanova, D

    2016-10-01

    Lipofilling or fat grafting transfer is defined as a technique of filling soft tissue by autologous fat grafting. The basic principle of lipofilling is based on a harvest of adipose tissue, followed by a reinjection after treatment. Lipofilling main objective is a volume defect filling, but also improving cutaneous trophicity. Lipofilling specificities among children is mainly based on these indications. Complications of autologous fat grafting among children are the same as those in adults: we distinguish short-term complications (intraoperative and perioperative) and the medium and long-term complications. The harvesting of fat tissue is the main limiting factor of the technique, due to low percentage of body fat of children. Indications of lipofilling among children may be specific or similar to those in adults. There are two types of indications: cosmetic, in which the aim of lipofilling is correcting a defect density, acquired (iatrogenic, post-traumatic scar) or malformation (otomandibular dysplasia, craniosynostosis, Parry Romberg syndrom, Poland syndrom, pectus excavatum…). The aim of functional indications is correcting a velar insufficiency or lagophthalmos. In the paediatric sector, lipofilling has become an alternative to the conventional techniques, by its reliability, safety, reproducibility, and good results. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  16. Efficient non-viral reprogramming of myoblasts to stemness with a single small molecule to generate cardiac progenitor cells.

    Directory of Open Access Journals (Sweden)

    Zeeshan Pasha

    Full Text Available The current protocols for generation of induced pluripotent stem (iPS cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs using small molecules.SMs from young male Oct3/4-GFP(+ transgenic mouse were treated with DNA methyltransferase (DNMT inhibitor, RG108. Two weeks later, GFP(+ colonies of SM derived iPS cells (SiPS expressing GFP and with morphological similarity of mouse embryonic stem (ESCs were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity, expressed SSEA1, displayed ES cell specific pluripotency markers and formed teratoma in nude mice. Optimization of culture conditions for embryoid body (EBs formation yielded spontaneously contracting EBs having morphological, molecular, and ultra-structural similarities with cardiomyocytes and expressed early and late cardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicating that SiPS were similar to ESCs in miR profile. Four weeks after transplantation into the immunocompetent mice model of acute myocardial infarction (n = 12 per group, extensive myogenesis was observed in SiPS transplanted hearts as compared to DMEM controls (n = 6 per group. A significant reduction in fibrosis and improvement in global heart function in the hearts transplanted with SiPS derived cardiac progenitor cells were observed.Reprogramming of SMs by DNMT inhibitor is a simple, reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function.

  17. Mastectomy Skin Necrosis After Breast Reconstruction: A Comparative Analysis Between Autologous Reconstruction and Implant-Based Reconstruction.

    Science.gov (United States)

    Sue, Gloria R; Lee, Gordon K

    2018-05-01

    Mastectomy skin necrosis is a significant problem after breast reconstruction. We sought to perform a comparative analysis on this complication between patients undergoing autologous breast reconstruction and patients undergoing 2-stage expander implant breast reconstruction. A retrospective review was performed on consecutive patients undergoing autologous breast reconstruction or 2-stage expander implant breast reconstruction by the senior author from 2006 through 2015. Patient demographic factors including age, body mass index, history of diabetes, history of smoking, and history of radiation to the breast were collected. Our primary outcome measure was mastectomy skin necrosis. Fisher exact test was used for statistical analysis between the 2 patient cohorts. The treatment patterns of mastectomy skin necrosis were then analyzed. We identified 204 patients who underwent autologous breast reconstruction and 293 patients who underwent 2-stage expander implant breast reconstruction. Patients undergoing autologous breast reconstruction were older, heavier, more likely to have diabetes, and more likely to have had prior radiation to the breast compared with patients undergoing implant-based reconstruction. The incidence of mastectomy skin necrosis was 30.4% of patients in the autologous group compared with only 10.6% of patients in the tissue expander group (P care in the autologous group, only 3.2% were treated with local wound care in the tissue expander group (P skin necrosis is significantly more likely to occur after autologous breast reconstruction compared with 2-stage expander implant-based breast reconstruction. Patients with autologous reconstructions are more readily treated with local wound care compared with patients with tissue expanders, who tended to require operative treatment of this complication. Patients considering breast reconstruction should be counseled appropriately regarding the differences in incidence and management of mastectomy skin

  18. Reduced use of allogeneic platelets through high-yield perioperative autologous plateletpheresis and reinfusion.

    Science.gov (United States)

    Alberts, Melissa; Bandarenko, Nicholas; Gaca, Jeffrey; Lockhart, Evelyn; Milano, Carmelo; Alexander, Stanlin; Linder, Dean; Lombard, Frederick W; Welsby, Ian J

    2014-05-01

    Intraoperative autologous platelet (PLT) collection as part of a multimodal blood conservation program carries a Class IIa recommendation from the Societies of Thoracic Surgeons and Cardiovascular Anesthesiologists, but achieving a suitable PLT yield limits its application. A novel, autologous, intraoperative, high-yield plateletpheresis collection program was established and retrospectively analyzed to identify potential improvements over previously reported plateletpheresis protocols. Targeting complex cardiothoracic surgery patients without recent anti-PLT agents, thrombocytopenia, or severe anemia, the program aimed to achieve a PLT yield of at least one standard apheresis unit (3.0 × 10(11) ) within 60 to 90 minutes and using an automated plateletpheresis device (Trima, Terumo BCT). Anesthetized and invasively monitored patients underwent plateletpheresis via a large-bore, indwelling central line placed for the surgery. Collection-related data for quality control purposes and subsequent PLT transfusion requirements were analyzed and reported. Forty-two patients donated autologous PLTs between 2011 and 2012. PLT yield was 4.5 (3.9-5.0) × 10(11) , which significantly exceeds previously reported yields, and procedure duration was 53.2 (48.4-57.9) minutes. As anticipated, postcollection PLT count decreased from 268 (242-293) × 10(9) to 182 (163-201) × 10(9) /L; hypocalcemia was minimized by infusion of 1 g of CaCl2 . Autologous PLT yield was inversely correlated with allogeneic PLT use, and avoidance of allogeneic PLT transfusion was increased when the autologous yield was the equivalent of 2 or more apheresis units. High-yield, intraoperative autologous PLT collection is achievable using an automated plateletpheresis device. Initial experience shows a reduction in reliance on allogeneic PLTs for complex cardiothoracic surgery. © 2013 American Association of Blood Banks.

  19. Autologous Adipose-Derived Tissue Matrix Part I: Biologic Characteristics.

    Science.gov (United States)

    Schendel, Stephen A

    2017-10-01

    Autologous collagen is an ideal soft tissue filler and may serve as a matrix for stem cell implantation and growth. Procurement of autologous collagen has been limited, though, secondary to a sufficient source. Liposuction is a widely performed and could be a source of autologous collagen. The amount of collagen and its composition in liposuctioned fat remains unknown. The purpose of this research was to characterize an adipose-derived tissue-based product created using ultrasonic cavitation and cryo-grinding. This study evaluated the cellular and protein composition of the final product. Fat was obtained from individuals undergoing routine liposuction and was processed by a 2 step process to obtain only the connective tissue. The tissue was then evaluated by scanning electronic microscope, Western blot analysis, and flow cytometry. Liposuctioned fat was obtained from 10 individuals with an average of 298 mL per subject. After processing an average of 1 mL of collagen matrix was obtained from each 100 mL of fat. Significant viable cell markers were present in descending order for adipocytes > CD90+ > CD105+ > CD45+ > CD19+ > CD144+ > CD34+. Western blot analysis showed collagen type II, III, IV, and other proteins. Scanning electronic microscope study showed a regular pattern of cross-linked, helical collagen. Additionally, vital staing demonstrated that the cells were still viable after processing. Collagen and cells can be easily obtained from liposuctioned fat by ultrasonic separation without alteration of the overall cellular composition of the tissue. Implantation results in new collagen and cellular growth. Collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat and may provide long term results. 5. © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com

  20. Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle

    NARCIS (Netherlands)

    Vliet, A.K. van; Nègre-Arrariou, P.; Thiel, G.C.F. van; Bolhuis, P.A.; Cohen, L.H.

    1996-01-01

    Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 ± 6 nM and 4.0 ± 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 ± 38 nM). Through

  1. Long-term use and follow-up of autologous and homologous cartilage graft in rhinoplasty

    Directory of Open Access Journals (Sweden)

    Ghasemali Khorasani

    2016-05-01

    Full Text Available Background: Cartilage grafting is used in rhinoplasty and reconstructive surgeries. Autologous rib and nasal septum cartilage (auto graft is the preferred source of graft material in rhinoplasty, however, homologous cartilage (allograft has been extensively used to correct the nasal framework in nasal deformities. Autologous cartilage graft usage is restricted with complication of operation and limiting availability of tissue for extensive deformities. Alternatively, preserved costal cartilage allograft represents a readily available and easily contoured material. The current study was a formal systematic review of complications associated with autologous versus homologous cartilage grafting in rhinoplasty patients. Methods: In this cohort retrospective study, a total of 124 patients undergone primary or revision rhinoplasty using homologous or autologus grafts with postoperative follow-up ranging from 6 to 60 months were studied. The types of grafts and complications related to the grafts were evaluated. This included evaluation for warping, infection, resorption, mobility and fracture. Results: The total complications related to the cartilage grafts were 7 cases, which included 1 warped in auto graft group, three cases of graft displacement (two in allograft group and one in auto graft group and three fractures in allograft group. No infection and resorption was recorded. Complication rate (confidence interval 0.95 in autologous and homologous group were 1.25(0.4-3.88 and 2.08(0.78-5.55 in 1000 months follow up. There was no statistically significant difference between autologous and homologous group complications. Onset of complication in autologous and homologous group were 51.23(49.27-53.19 and 58.7(54.51-62.91 month respectively (P=0.81. Conclusion: The allograft cartilage has the advantage of avoiding donor-site scar. Moreover, it provides the same benefits as autologous costal cartilage with comparable complication rate. Therefore, it

  2. Chondrocyte-seeded type I/III collagen membrane for autologous chondrocyte transplantation

    DEFF Research Database (Denmark)

    Niemeyer, Philipp; Lenz, Philipp; Kreuz, Peter C

    2010-01-01

    PURPOSE: We report the 2-year clinical results and identify prognostic factors in patients treated with autologous chondrocyte transplantation by use of a collagen membrane to seed the chondrocytes (ACT-CS). METHODS: This is a prospective study of 59 patients who were treated with ACT......-CS represents a technical modification of membrane-associated autologous chondrocyte transplantation that combines easy handling and attractive application properties with reliable clinical results 24 months after surgery, especially in patients with isolated cartilage defects. Even though the failure rate...

  3. Modulation of Protein Quality Control and Proteasome to Autophagy Switch in Immortalized Myoblasts from Duchenne Muscular Dystrophy Patients

    Directory of Open Access Journals (Sweden)

    Marion Wattin

    2018-01-01

    Full Text Available The maintenance of proteome integrity is of primary importance in post-mitotic tissues such as muscle cells; thus, protein quality control mechanisms must be carefully regulated to ensure their optimal efficiency, a failure of these processes being associated with various muscular disorders. Duchenne muscular dystrophy (DMD is one of the most common and severe forms of muscular dystrophies and is caused by mutations in the dystrophin gene. Protein quality control modulations have been diversely observed in degenerating muscles of patients suffering from DMD or in animal models of the disease. In this study, we investigated whether modulations of protein quality control mechanisms already pre-exist in undifferentiated myoblasts originating from DMD patients. We report for the first time that the absence of dystrophin in human myoblasts is associated with protein aggregation stress characterized by an increase of protein aggregates. This stress is combined with BAG1 to BAG3 switch, NFκB activation and up-regulation of BAG3/HSPB8 complexes that ensure preferential routing of misfolded/aggregated proteins to autophagy rather than to deficient 26S proteasome. In this context, restoration of pre-existing alterations of protein quality control processes might represent an alternative strategy for DMD therapies.

  4. The Efficacy and Safety of Autologous Transfusion in Unilateral Total Knee Arthroplasty

    OpenAIRE

    Yoo, Moon-Jib; Park, Hee-Gon; Ryu, Jee-Won; Kim, Jeong-Sang

    2015-01-01

    Purpose Although allogeneic blood transfusion is the most common method of transfusion in total knee arthroplasty (TKA), there are reports showing significant decrease in the amount of allogeneic transfusion and incidence of side effects after combined use of autologous transfusion. The purpose of this study is to investigate the efficacy of using an autologous transfusion device in TKA. Materials and Methods Patients who underwent TKA at our institution from January 2003 to January 2014 were...

  5. Recovery of autologous sickle cells by hypotonic wash.

    Science.gov (United States)

    Wilson, Emily; Kezeor, Kelly; Crosby, Monica

    2018-01-01

    It is important to isolate autologous red blood cells (RBCs) from transfused RBCs in samples from recently transfused patients to ensure that accurate serologic results are obtained. Typically, this isolation can be performed using methods that separate patient reticulocytes from transfused, older donor RBCs. Patients with sickle cell disease (SCD), however, characteristically have RBCs with altered membrane and morphological features, causing their RBCs to take on a sickle-shape appearance different from the biconcave disc-shape appearance of "normal" RBCs. These characteristics enable the use of hypotonic saline solution to lyse normal RBCs while allowing "sickle cells" to remain intact. Because many patients with SCD undergo frequent transfusions to treat their condition, the use of hypotonic saline solution provides a rapid method to obtain autologous RBCs for serologic testing from this patient population using standard laboratory equipment and supplies.

  6. Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

    Science.gov (United States)

    Rancourt, Ann; Dufresne, Sébastien S; St-Pierre, Guillaume; Lévesque, Julie-Christine; Nakamura, Haruka; Kikuchi, Yodai; Satoh, Masahiko S; Frenette, Jérôme; Sato, Sachiko

    2018-06-12

    The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), in which mutations in the dystrophin gene disrupts the firm adhesion. In patients with DMD, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in patients with DMD, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina and increases the efficiency of the myogenesis. Galectin-3 is an oligosaccharide-binding protein and is known to be involved in cell-cell interactions and cell-matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle, although its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the synthesis of binding partners (oligosaccharides) of galectin-3, promote myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased muscle-force production. The results suggest that treatment with N-acetylglucosamine might mitigate the burden of DMD.-Rancourt, A., Dufresne, S. S., St-Pierre, G., Lévesque, J.-C., Nakamura, H., Kikuchi, Y., Satoh, M. S., Frenette, J., Sato, S. Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

  7. Effects of Ghrelin on Triglyceride Accumulation and Glucose Uptake in Primary Cultured Rat Myoblasts under Palmitic Acid-Induced High Fat Conditions

    Directory of Open Access Journals (Sweden)

    Lingling Han

    2015-01-01

    Full Text Available This study aimed to study the effects of acylated ghrelin on glucose and triglyceride metabolism in rat myoblasts under palmitic acid- (PA- induced high fat conditions. Rat myoblasts were treated with 0, 10−11, 10−9, or 10−7 M acylated ghrelin and 0.3 mM PA for 12 h. Triglyceride accumulation was determined by Oil-Red-O staining and the glycerol phosphate dehydrogenase-peroxidase enzymatic method, and glucose uptake was determined by isotope tracer. The glucose transporter 4 (GLUT4, AMP-activated protein kinase (AMPK, acetyl-CoA carboxylase (ACC, and uncoupling protein 3 (UCP3 were assessed by RT-PCR and western blot. Compared to 0.3 mM PA, ghrelin at 10−9 and 10−7 M reduced triglyceride content (5.855 ± 0.352 versus 5.030 ± 0.129 and 4.158 ± 0.254 mM, P<0.05 and prevented PA-induced reduction of glucose uptake (1.717 ± 0.264 versus 2.233 ± 0.333 and 2.333 ± 0.273 10−2 pmol/g/min, P<0.05. The relative protein expression of p-AMPKα/AMPKα, UCP3, and p-ACC under 0.3 mM PA was significantly reduced compared to controls (all P<0.05, but those in the 10−9 and 10−7 M ghrelin groups were significantly protected from 0.3 mM PA (all P<0.05. In conclusion, acylated ghrelin reduced PA-induced triglyceride accumulation and prevented the PA-induced decrease in glucose uptake in rat myoblasts. These effects may involve fatty acid oxidation.

  8. Autologous transplantation of the internal limiting membrane for refractory macular holes.

    Science.gov (United States)

    Morizane, Yuki; Shiraga, Fumio; Kimura, Shuhei; Hosokawa, Mio; Shiode, Yusuke; Kawata, Tetsuhiro; Hosogi, Mika; Shirakata, Yukari; Okanouchi, Toshio

    2014-04-01

    To determine the effectiveness of autologous transplantation of the internal limiting membrane (ILM) for refractory macular holes. Prospective, interventional case series. Ten eyes of 10 consecutive patients who underwent autologous transplantation of the ILM for the treatment of refractory macular holes were studied. The primary diseases in these patients were large idiopathic macular holes that had existed for more than 1 year (4 eyes), a traumatic macular hole (1 eye), myopic foveoschisis (2 eyes), foveoschisis resulting from pit-macular syndrome (2 eyes), and proliferative diabetic retinopathy (1 eye). Apart from the 5 eyes with idiopathic or traumatic macular holes, macular holes developed in the other 5 eyes after initial vitrectomies with ILM removal. In all eyes, regular macular hole surgery failed to achieve closure. The main outcome measures used in this study were macular hole closure and best-corrected visual acuity (BCVA). Macular holes were closed successfully in 9 eyes (90%) after autologous transplantation of the ILM. The postoperative BCVAs were significantly better than the preoperative BCVAs (P = .007, paired t test). Postoperative BCVAs improved by more than 0.2 logarithm of the minimal angle of resolution units in 8 eyes (80%) and were unchanged in 2 eyes (20%). Although this is a pilot study, the results suggest that autologous transplantation of the ILM may contribute to improved anatomic and visual outcomes in the treatment of refractory macular holes and may warrant further investigation. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. [Penile augmentation and elongation using autologous dermal-fat strip grafting].

    Science.gov (United States)

    Yang, Zhe; Li, Yang-qun; Tang, Yong; Chen, Wen; Li, Qiang; Zhou, Chuan-de; Zhao, Mu-xin; Hu, Chun-mei

    2012-05-01

    To investigate the effect of autologous dermal-fat strip grafting in penile augmentation and elongation. From May 2004 to December 2010, 24 patients underwent penile enhancement with free dermal-fat strip grafting. Through suprapubic incision, the superior suspensory ligament and part deep suspensory ligament are cutted off to lengthen the penis. The resulted dead space is filled with the autologous dermal-fat strip (6.0-9.5 cm in length, 1.2-1.5 cm in width and 0.6-0.8 cm in depth) to enhance the penis. Primary healing was achieved in 23 cases. Incisional fat liquefaction happened in one case which healed after dressing change. The penile appearance was satisfactory both at rest or erection. The penile length and circumference increased by 2.5-4.8 cm (average, 3.2 cm) and 1.8-3.0 cm (average, 2.4 cm), respectively. 18 patients were followed up for 3 months to 5 years. All the patients were satisfactory on the cosmetic and functional results. No complication happened. It is safe and effective for penile augmention and elongation with autologous dermal-fat strip grafting and disconnection of penile suspensory ligament.

  10. Proteomics of Skeletal Muscle

    DEFF Research Database (Denmark)

    Deshmukh, Atul

    2016-01-01

    , of altered protein expressions profiles and/or their posttranslational modifications (PTMs). Mass spectrometry (MS)-based proteomics offer enormous promise for investigating the molecular mechanisms underlying skeletal muscle insulin resistance and exercise-induced adaptation; however, skeletal muscle......Skeletal muscle is the largest tissue in the human body and plays an important role in locomotion and whole body metabolism. It accounts for ~80% of insulin stimulated glucose disposal. Skeletal muscle insulin resistance, a primary feature of Type 2 diabetes, is caused by a decreased ability...... of muscle to respond to circulating insulin. Physical exercise improves insulin sensitivity and whole body metabolism and remains one of the most promising interventions for the prevention of Type 2 diabetes. Insulin resistance and exercise adaptations in skeletal muscle might be a cause, or consequence...

  11. Osteoarthritis treatment using autologous conditioned serum after placebo

    NARCIS (Netherlands)

    Rutgers, Marijn; Creemers, Laura B; Auw Yang, Kiem Gie; Raijmakers, Natasja J H; Dhert, Wouter J A; Saris, Daniel B F

    BACKGROUND AND PURPOSE: Autologous conditioned serum (ACS) is a disease-modifying drug for treatment of knee osteoarthritis, and modest superiority over placebo was reported in an earlier randomized controlled trial (RCT). We hypothesized that when given the opportunity, placebo-treated patients

  12. Comparative analysis of autologous blood transfusion and allogeneic blood transfusion in surgical patients

    OpenAIRE

    Long, Miao-Yun; Liu, Zhong-Han; Zhu, Jian-Guang

    2014-01-01

    Objective: To investigate application effects of autologous blood transfusion and allogeneic blood transfusion in surgically treated patients receiving spine surgery, abdomen surgery and ectopic pregnancy surgery. Methods: 130 patients who would undergo selective operations were divided into autologous transfusion group and allogeneic transfusion group. Both groups received the same anesthesia, and there was no significant difference in transfusion volume or fluid infusion volume. Results: Th...

  13. Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yong Jin; Lee, You La; Ahn, Sohn Joo; Choi, Chang Ik; Lee, Sang Woo; Ahn, Byeong Cheol; Lee, Jae Tae [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)

    2007-07-01

    To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring.

  14. Autologous fat graft as treatment of post short stature surgical correction scars.

    Science.gov (United States)

    Maione, Luca; Memeo, Antonio; Pedretti, Leopoldo; Verdoni, Fabio; Lisa, Andrea; Bandi, Valeria; Giannasi, Silvia; Vinci, Valeriano; Mambretti, Andrea; Klinger, Marco

    2014-12-01

    Surgical limb lengthening is undertaken to correct pathological short stature. Among the possible complications related to this procedure, painful and retractile scars are a cause for both functional and cosmetic concern. Our team has already shown the efficacy of autologous fat grafting in the treatment of scars with varying aetiology, so we decided to apply this technique to scars related to surgical correction of dwarfism. A prospective study was conducted to evaluate the efficacy of autologous fat grafting in the treatment of post-surgical scars in patients with short-limb dwarfism using durometer measurements and a modified patient and observer scar assessment scale (POSAS), to which was added a parameter to evaluate movement impairment. Between January 2009 and September 2012, 36 children (28 female and 8 male) who presented retractile and painful post-surgical scars came to our unit and were treated with autologous fat grafting. Preoperative and postoperative mean durometer measurements were analysed using the analysis of variance (ANOVA) test and POSAS parameters were studied using the Wilcoxon rank sum test. There was a statistically significant reduction in all durometer measurements (p-value treatment with autologous fat grafting. Surgical procedures to camouflage scars on lower limbs are not often used as a first approach and non-surgical treatments often lead to unsatisfactory results. In contrast, our autologous fat grafting technique in the treatment of post-surgical scars has been shown to be a valuable option in patients with short-limb dwarfism. There was a reduction of skin hardness and a clinical improvement of all POSAS parameters in all patients treated. Moreover, the newly introduced POSAS parameter appears to be reliable and we recommend that it is included to give a more complete evaluation of patient perception. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Use of autologous and microsurgical breast reconstruction by U.S. plastic surgeons.

    Science.gov (United States)

    Kulkarni, Anita R; Sears, Erika Davis; Atisha, Dunya M; Alderman, Amy K

    2013-09-01

    Concern exists that plastic surgeons are performing fewer autologous and microsurgical breast reconstructions, despite superior long-term outcomes. The authors describe the proportion of U.S. plastic surgeons performing these procedures and evaluate motivating factors and perceived barriers. A random national sample of American Society of Plastic Surgeons members was surveyed (n = 325; response rate, 76 percent). Surgeon and practice characteristics were assessed, and two multiple logistic regression models were created to evaluate factors associated with (1) high-volume autologous providers and (2) microsurgical providers. Qualitative assessments of motivating factors and barriers to microsurgery were also performed. Fewer than one-fifth of plastic surgeons perform autologous procedures for more than 50 percent of their breast cancer patients, and only one-quarter perform any microsurgical breast reconstruction. Independent predictors of a high-volume autologous practice include involvement with resident education (odds ratio, 2.57; 95 percent CI, 1.26 to 5.24) and a microsurgical fellowship (odds ratio, 2.09; 95 percent CI, 1.04 to 4.27). Predictors of microsurgical breast reconstruction include involvement with resident education (odds ratio, 6.8; 95 percent CI, 3.32 to 13.91), microsurgical fellowship (odds ratio, 2.4; 95 percent CI, 1.16 to 4.95), and high breast reconstruction volume (odds ratio, 6.68; 95 percent CI, 1.76 to 25.27). The primary motivator for microsurgery is superior outcomes, and the primary deterrents are time and reimbursement. The proportion of U.S. plastic surgeons with a high-volume autologous or microsurgical breast reconstruction practice is low. Involvement with resident education appears to facilitate both, whereas time constraints and reimbursement are primary deterrents. Future efforts should focus on improving the feasibility and accessibility of all types of breast reconstruction.

  16. Skeletal surveys in multiple myeloma

    International Nuclear Information System (INIS)

    Sebes, J.I.; Niell, H.B.; Palmieri, G.M.A.; Reidy, T.J.

    1986-01-01

    Thirty-three patients with multiple myeloma were studied with serial skeletal surveys, serum immunoglobulin levels, and postabsorptive urinary hydroxyproline (Spot-HYPRO) determinations. Twenty receiving chemotherapy were also followed with skeletal surveys in order to evaluate bone response to treatment. A close association was found between skeletal findings and changes in immunoglubulin levels with positive correlation in 71% of the patients. A similar association was found between skeletal disease and Spot-HYPRO level changes in 65%. Five of 12 patients (42%) with partial or complete clinical response to chemotherapy, demonstrated improvement in the appearance of skeletal lesions. Positive correlation between the roentgenographic changes and clinical markers of myeloma as well as therapeutic response, indicates that skeletal surveys are useful and effective in monitoring patients with multiple myeloma. (orig.)

  17. Computer-assisted midface reconstruction in Treacher Collins syndrome part 1: skeletal reconstruction.

    Science.gov (United States)

    Herlin, Christian; Doucet, Jean Charles; Bigorre, Michèle; Khelifa, Hatem Cheikh; Captier, Guillaume

    2013-10-01

    Treacher Collins syndrome (TCS) is a severe and complex craniofacial malformation affecting the facial skeleton and soft tissues. The palate as well as the external and middle ear are also affected, but his prognosis is mainly related to neonatal airway management. Methods of zygomatico-orbital reconstruction are numerous and currently use primarily autologous bone, lyophilized cartilage, alloplastic implants, or even free flaps. This work developed a reliable "customized" method of zygomatico-orbital bony reconstruction using a generic reference model tailored to each patient. From a standard computed tomography (CT) acquisition, we studied qualitatively and quantitatively the skeleton of four individuals with TCS whose age was between 6 and 20 years. In parallel, we studied 40 controls at the same age to obtain a morphometric database of reference. Surgical simulation was carried out using validated software used in craniofacial surgery. The zygomatic hypoplasia was very important quantitatively and morphologically in all TCS individuals. Orbital involvement was mainly morphological, with volumes comparable to the controls of the same age. The control database was used to create three-dimensional computer models to be used in the manufacture of cutting guides for autologous cranial bone grafts or alloplastic implants perfectly adapted to each patient's morphology. Presurgical simulation was also used to fabricate custom positioning guides permitting a simple and reliable surgical procedure. The use of a virtual database allowed us to design a reliable and reproducible skeletal reconstruction method for this rare and complex syndrome. The use of presurgical simulation tools seem essential in this type of craniofacial malformation to increase the reliability of these uncommon and complex surgical procedures, and to ensure stable results over time. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights

  18. LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration

    Science.gov (United States)

    Wang, Lijun; Zhao, Yu; Bao, Xichen; Zhu, Xihua; Kwok, Yvonne Ka-yin; Sun, Kun; Chen, Xiaona; Huang, Yongheng; Jauch, Ralf; Esteban, Miguel A; Sun, Hao; Wang, Huating

    2015-01-01

    Emerging studies document the roles of long non-coding RNAs (LncRNAs) in regulating gene expression at chromatin level but relatively less is known how they regulate DNA methylation. Here we identify an lncRNA, Dum (developmental pluripotency-associated 2 (Dppa2) Upstream binding Muscle lncRNA) in skeletal myoblast cells. The expression of Dum is dynamically regulated during myogenesis in vitro and in vivo. It is also transcriptionally induced by MyoD binding upon myoblast differentiation. Functional analyses show that it promotes myoblast differentiation and damage-induced muscle regeneration. Mechanistically, Dum was found to silence its neighboring gene, Dppa2, in cis through recruiting Dnmt1, Dnmt3a and Dnmt3b. Furthermore, intrachromosomal looping between Dum locus and Dppa2 promoter is necessary for Dum/Dppa2 interaction. Collectively, we have identified a novel lncRNA that interacts with Dnmts to regulate myogenesis. PMID:25686699

  19. Whole-body MRI in comparison to skeletal scintigraphy for detection of skeletal metastases in patients with solid tumors

    International Nuclear Information System (INIS)

    Ghanem, N.; Altehoefer, C.; Winterer, J.; Schaefer, O.; Bley, T.A.; Langer, M.; Kelly, T.; Moser, E.

    2004-01-01

    The aim of this study was to compare the diagnostic efficacy of whole-body magnetic resonance imaging (WB-MRI) as a new and rapid examination technique with skeletal scintigraphy for detection of skeletal metastases from solid tumors. In 129 patients with solid malignant tumors, WB-MRI was performed for individual comparison with skeletal scintigraphy. Examinations were performed with the innovative AngioSURF trademark rolling table with integrated phased array surface coil and coronary TIRM sequences for different body regions. The results for WB-MRI and skeletal scintigraphy were concordant in 81% of the cases, whereby both procedures excluded skeletal metastases in 43%. WB-MRI and skeletal scintigraphy demonstrated skeletal metastases in 38% of the cases, whereby WB-MRI provided more comprehensive findings in 45%. In 12% of the cases, skeletal scintigraphy was superior to WB-MRI and in 19% the findings were discordant, whereby WB-MRI detected skeletal metastases in 15 cases which had not been found on skeletal scintigraphy. In nine cases, skeletal scintigraphy was positive when the WB-MRI was negative. In 60% of the cases, WB-MRI evidenced tumor-associated findings. WB-MRI represents a promising new staging technique for detection of skeletal metastases, which is more sensitive in many cases than skeletal scintigraphy in detecting and assessing the extent of skeletal metastases - and tumor-associated findings that are relevant for treatment strategy. (orig.) [de

  20. Outcomes of Autologous Fascia Pubovaginal Sling for Patients with Transvaginal Mesh Related Complications Requiring Mesh Removal.

    Science.gov (United States)

    McCoy, Olugbemisola; Vaughan, Taylor; Nickles, S Walker; Ashley, Matt; MacLachlan, Lara S; Ginsberg, David; Rovner, Eric

    2016-08-01

    We reviewed the outcomes of the autologous fascial pubovaginal sling as a salvage procedure for recurrent stress incontinence after intervention for polypropylene mesh erosion/exposure and/or bladder outlet obstruction in patients treated with prior transvaginal synthetic mesh for stress urinary incontinence. In a review of surgical databases at 2 institutions between January 2007 and June 2013 we identified 46 patients who underwent autologous fascial pubovaginal sling following removal of transvaginal synthetic mesh in simultaneous or staged fashion. This cohort of patients was evaluated for outcomes, including subjective and objective success, change in quality of life and complications between those who underwent staged vs concomitant synthetic mesh removal with autologous fascial pubovaginal sling placement. All 46 patients had received at least 1 prior mesh sling for incontinence and 8 (17%) had received prior transvaginal polypropylene mesh for pelvic organ prolapse repair. A total of 30 patients underwent concomitant mesh incision with or without partial excision and autologous sling placement while 16 underwent staged autologous sling placement. Mean followup was 16 months. Of the patients 22% required a mean of 1.8 subsequent interventions an average of 6.5 months after autologous sling placement with no difference in median quality of life at final followup. At last followup 42 of 46 patients (91%) and 35 of 46 (76%) had achieved objective and subjective success, respectively. There was no difference in subjective success between patients treated with a staged vs a concomitant approach (69% vs 80%, p = 0.48). Autologous fascial pubovaginal sling placement after synthetic mesh removal can be performed successfully in patients with stress urinary incontinence as a single or staged procedure. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  1. Autologous fibrin sealant (Vivostat®) in the neurosurgical practice: Part I: Intracranial surgical procedure

    Science.gov (United States)

    Graziano, Francesca; Certo, Francesco; Basile, Luigi; Maugeri, Rosario; Grasso, Giovanni; Meccio, Flavia; Ganau, Mario; Iacopino, Domenico G.

    2015-01-01

    Background: Hemorrhages, cerebrospinal fluid (CSF) fistula and infections are the most challenging postoperative complications in Neurosurgery. In this study, we report our preliminary results using a fully autologous fibrin sealant agent, the Vivostat® system, in achieving hemostasis and CSF leakage repair during cranio-cerebral procedures. Methods: From January 2012 to March 2014, 77 patients were studied prospectively and data were collected and analyzed. Autologous fibrin sealant, taken from patient's blood, was prepared with the Vivostat® system and applied on the resection bed or above the dura mater to achieve hemostasis and dural sealing. The surgical technique, time to bleeding control and associated complications were recorded. Results: A total of 79 neurosurgical procedures have been performed on 77 patients. In the majority of cases (98%) the same autologous fibrin glue provided rapid hemostasis and dural sealing. No patient developed allergic reactions or systemic complications in association with its application. There were no cases of cerebral hematoma, swelling, infection, or epileptic seizures after surgery whether in the immediate or in late period follow-up. Conclusions: In this preliminary study, the easy and direct application of autologous fibrin sealant agent helped in controlling cerebral bleeding and in providing prompt and efficient dural sealing with resolution of CSF leaks. Although the use of autologous fibrin glue seems to be safe, easy, and effective, further investigations are strongly recommended to quantify real advantages and potential limitations. PMID:25984391

  2. Funding analysis of bilateral autologous free-flap breast reconstructions in Australia.

    Science.gov (United States)

    Sinha, Shiba; Ruskin, Olivia; McCombe, David; Morrison, Wayne; Webb, Angela

    2015-08-01

    Bilateral breast reconstructions are being increasingly performed. Autologous free-flap reconstructions represent the gold standard for post-mastectomy breast reconstruction but are resource intensive. This study aims to investigate the difference between hospital reimbursement and true cost of bilateral autologous free-flap reconstructions. Retrospective analysis of patients who underwent bilateral autologous free-flap reconstructions at a single Australian tertiary referral centre was performed. Hospital reimbursement was determined from coding analysis. A true cost analysis was also performed. Comparisons were made considering the effect of timing, indication and complications of the procedure. Forty-six bilateral autologous free-flap procedures were performed (87 deep inferior epigastric perforators (DIEPs), four superficial inferior epigastric artery perforator flaps (SIEAs) and one muscle-sparing free transverse rectus abdominis myocutaneous flap (MS-TRAM)). The mean funding discrepancy between hospital reimbursement and actual cost was $12,137 ± $8539 (mean ± standard deviation (SD)) (n = 46). Twenty-four per cent (n = 11) of the cases had been coded inaccurately. If these cases were excluded from analysis, the mean funding discrepancy per case was $9168 ± $7453 (n = 35). Minor and major complications significantly increased the true cost and funding discrepancy (p = 0.02). Bilateral free-flap breast reconstructions performed in Australian public hospitals result in a funding discrepancy. Failure to be economically viable threatens the provision of this procedure in the public system. Plastic surgeons and hospital managers need to adopt measures in order to make these gold-standard procedures cost neutral. Copyright © 2015 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

  3. Autologous pubovaginal slings: back to the future or a lost art?

    Directory of Open Access Journals (Sweden)

    Bang SL

    2016-01-01

    Full Text Available Shieh-Ling Bang, Mohammed Belal Department of Urology, Queen Elizabeth Hospital, Birmingham, UK Abstract: Stress urinary incontinence (SUI is an under-diagnosed problem affecting up to 50% of women worldwide. SUI is a source of psychological distress to the individual and also imposes a financial burden to the individual and the health care system. The role of surgery in the treatment in SUI has evolved steadily in the last two decades. The synthetic mid-urethral sling and its different insertion methods have gained widespread popularity and are now the most frequently used surgical interventions for women with SUI in Europe. As the use of synthetic slings becomes more widespread, an increasing number of complications are being reported. With the recent concerns surrounding the use of synthetic transvaginal meshes in organ prolapse surgery, synthetic slings have been put under further scrutiny. It is imperative for health care providers to be aware of the current issues associated with synthetic slings and the alternative surgical options available. Traditional autologous pubovaginal slings (PVS have re-emerged as a viable alternative to synthetic slings in light of the issues with synthetic slings. The re-adoption of autologous PVS has however, been slow due to the technical difficulty of the surgery and perceived higher morbidity rates. In this article, we will discuss the various aspects of autologous PVS and its indications as an alternative to synthetic slings. We will also touch on the current evidence and controversies for synthetic mesh slings. Keywords: autologous pubovaginal sling, stress urinary incontinence, synthetic sling, erosions 

  4. Dietary fish oil delays hypoxic skeletal muscle fatigue and enhances caffeine-stimulated contractile recovery in the rat in vivo hindlimb.

    Science.gov (United States)

    Peoples, Gregory E; McLennan, Peter L

    2017-06-01

    Oxygen efficiency influences skeletal muscle contractile function during physiological hypoxia. Dietary fish oil, providing docosahexaenoic acid (DHA), reduces the oxygen cost of muscle contraction. This study used an autologous perfused rat hindlimb model to examine the effects of a fish oil diet on skeletal muscle fatigue during an acute hypoxic challenge. Male Wistar rats were fed a diet rich in saturated fat (SF), long-chain (LC) n-6 polyunsaturated fatty acids (n-6 PUFA), or LC n-3 PUFA DHA from fish oil (FO) (8 weeks). During anaesthetised and ventilated conditions (normoxia 21% O 2 (SaO 2 -98%) and hypoxia 14% O 2 (SaO 2 -89%)) the hindlimb was perfused at a constant flow and the gastrocnemius-plantaris-soleus muscle bundle was stimulated via sciatic nerve (2 Hz, 6-12V, 0.05 ms) to established fatigue. Caffeine (2.5, 5, 10 mM) was supplied to the contracting muscle bundle via the arterial cannula to assess force recovery. Hypoxia, independent of diet, attenuated maximal twitch tension (normoxia: 82 ± 8; hypoxia: 41 ± 2 g·g -1 tissue w.w.). However, rats fed FO sustained higher peak twitch tension compared with the SF and n-6 PUFA groups (P recovery was enhanced in the FO-fed animals (SF: 41 ± 3; n-6 PUFA: 40 ± 4; FO: 52 ± 7% recovery; P < 0.05). These results support a physiological role of DHA in skeletal muscle membranes when exposed to low-oxygen stress that is consistent with the attenuation of muscle fatigue under physiologically normoxic conditions.

  5. Bio-artificial pleura using an autologous dermal fibroblast sheet

    Science.gov (United States)

    Kanzaki, Masato; Takagi, Ryo; Washio, Kaoru; Kokubo, Mami; Yamato, Masayuki

    2017-10-01

    Air leaks (ALs) are observed after pulmonary resections, and without proper treatment, can produce severe complications. AL prevention is a critical objective for managing patients after pulmonary resection. This study applied autologous dermal fibroblast sheets (DFS) to close ALs. For sealing ALs in a 44-year-old male human patient with multiple bullae, a 5 × 15-mm section of skin was surgically excised. From this skin specimen, primary dermal fibroblasts were isolated and cultured for 4 weeks to produce DFSs that were harvested after a 10-day culture. ALs were completely sealed using surgical placement of these autologous DFSs. DFS were found to be a durable long-term AL sealant, exhibiting requisite flexibility, elasticity, durability, biocompatibility, and usability, resulting reliable AL closure. DFS should prove to be an extremely useful tissue-engineered pleura substitute.

  6. Lyophilized skeletal imaging composition

    International Nuclear Information System (INIS)

    Vanduzee, B.F.

    1983-01-01

    This invention encompasses a process for producing a dry-powder skeletal imaging kit. An aqueous solution of a diphosphonate, a stannous reductant, and, optionally, a stabilizer is prepared. The solution is adjusted to a pH within the range 4.2 to 4.8 and the pH-adjusted solution is then lyophilized. The adjustment of pH, within a particular range, during the process of manufacturing lyophilized diphosphonate containing skeletal imaging kits yields a kit which produces a technetium skeletal imaging agent with superior imaging properties. This improved performance is manifested through faster blood clearance and higher skeletal uptake of the technetium imaging agent

  7. Research progresses in treating diabetic foot with autologous stem cell transplantation

    International Nuclear Information System (INIS)

    Qin Hanlin; Gao Bin

    2010-01-01

    Because the distal arteries of lower extremities become narrowed or even occluded in diabetic foot, the clinical therapeutic results for diabetic foot have been unsatisfactory so far. Autologous stem cell transplantation that has emerged in recent years is a new, safe and effective therapy for diabetic foot, which achieves its excellent clinical success in restoring the blood supply of ischemic limb by way of therapeutic angiogenesis. Now autologous stem cell transplantation has become one of the hot points in medical research both at home and abroad, moreover, it has brought a new hope of cure to the patients with diabetic foot. (authors)

  8. Autologous dental pulp stem cells in periodontal regeneration: a case report.

    Science.gov (United States)

    Aimetti, Mario; Ferrarotti, Francesco; Cricenti, Luca; Mariani, Giulia Maria; Romano, Federica

    2014-01-01

    Histologic findings in animal models suggest that the application of dental pulp stem cells (DPSCs) may promote periodontal regeneration in infrabony defects. This case report describes the clinical and radiographic regenerative potential of autologous DPSCs in the treatment of human noncontained intraosseous defects. A chronic periodontitis patient with one vital third molar requiring extraction was surgically treated. The third molar was extracted and used as an autologous DPSCs source to regenerate the infrabony defect on the mandibular right second premolar. At the 1-year examination, the defect was completely filled with bonelike tissue as confirmed through the reentry procedure.

  9. [Construction of a capsular tissue-engineered ureteral stent seeded with autologous urothelial cells].

    Science.gov (United States)

    Tan, Haisong; Fu, Weijun; Li, Jianqiang; Wang, Zhongxin; Li, Gang; Ma, Xin; Dong, Jun; Gao, Jiangping; Wang, Xiaoxiong; Zhang, Xu

    2013-01-01

    To investigate the feasibility of constructing a capsular poly L-lactic acid (PLLA) ureteral stent seeded with autologous urothelial cells using tissue engineering methods. The capsular ureteral stent was constructed by subcutaneously embedding PLLA ureteral stent in the back of beagles for 3 weeks to induce the formation of connective tissue on the surfaces. After decellularization of the stent, the expanded autologous urothelial cells were seeded on the stent. The surface structure and cell adhesion of the stent were observed using HE staining, scanning electron microscope (SEM) and immunocytochemical staining. MTT assay was used to evaluate urothelial cell proliferation on the capsular PLLA ureteral stent and on circumferential small intestinal submucosa graft. HE staining and VIII factor immunohistochemistry revealed numerous capillaries in the connective tissue encapsulating the stent without obvious local inflammatory response. The results of SEM and immunocytochemical staining showed that the capsule contained rich collagenic fibers forming three-dimensional structures, and the seeded autologous urothelial cells could adhere and well aligned on the surface. MTT assay showed normal growth of the cells on the stent as compared with the cells grown on circumferential small intestinal submucosa graft. The capsular PLLA ureteral stent allows adhesion and proliferation of autologous urothelial cells and shows a potential in applications of constructing tissue-engineered ureter.

  10. Autologous bone marrow transplantation following chemotherapy and irradiation in dogs with spontaneous lymphomas

    International Nuclear Information System (INIS)

    Bowles, C.A.; Bull, M.; McCormick, K.; Kadin, M.; Lucas, D.

    1980-01-01

    Thirty dogs with spontaneous lymphomas were administered two to six cycles of chemotherapy and were randomized into 3 groups to receive 800 rads of total body irradiation and autologous bone marrow transplantation. Of 10 dogs irradiated after chemotherapy-induced remission and infused with remission marrow (group 1), 8 (80%) had successful grafts and experienced remissions lasting 62 to 1024 days. Of 9 dogs irradiated during remission and infused with remission marrow mixed with autologous tumor cells (group 2), 6 (66%) had remission lasting 15 to 45 days. Eleven dogs with progressive tumor growth (relapse) following chemotherapy were irradiated and infused with remission marrow (group 3). Tumor remission lasting 39 to 350 days was observed in 5 dogs (45%) in this group, and 6 dogs died in less than 30 days. Dogs in groups 1 to 3 had median survival times of 216, 60, and 45 days, respectively. The prolonged survival times for dogs in group 1 compared to dogs in groups 2 and 3 suggest that protocols involving irradiation and autologous marrow grafting in this model would be most effective when these protocols are applied to animals having a minimum tumor burden at the time of irradiation and when the grafting is done with tumor-free autologous marrow

  11. Discovery of a mammalian splice variant of myostatin that stimulates myogenesis.

    Directory of Open Access Journals (Sweden)

    Ferenc Jeanplong

    Full Text Available Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05, which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a

  12. Skeletal imaging composition

    International Nuclear Information System (INIS)

    Vanduzee, B.F.; Degenhardt, C.R.

    1983-01-01

    This invention is based on the discovery that the adjustment of pH, within a particular range, during the process of manufacturing lyophilized diphosphonate-containing skeletal imaging kits yields a kit which produces a technetium skeletal imaging agent with superior imaging properties. This increased performance is manifested through faster blood clearance and higher skeletal uptake of the technetium imaging agent. The process for producing a dry-powder imaging kit comprises the steps of: preparing a solution of a diphosphonate carrier, stannous reductant, and a stabilizer in water; adjusting the pH to between 5.5 and 6.5; and lyophilizing the solution

  13. Wnt/β-catenin signaling changes C2C12 myoblast proliferation and differentiation by inducing Id3 expression

    International Nuclear Information System (INIS)

    Zhang, Long; Shi, Songting; Zhang, Juan; Zhou, Fangfang; Dijke, Peter ten

    2012-01-01

    Highlights: ► Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. ► Wnt3a induces Id3 expression via canonical Wnt/β-catenin pathway. ► Wnt3a-induced Id3 expression does not depend on BMP signaling activation. ► Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a β-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/β-catenin induced gene in myoblast cell fate determination.

  14. Resorbable screws for fixation of autologous bone grafts

    NARCIS (Netherlands)

    Raghoebar, GM; Liem, RSB; Bos, RRM; van der Wal, JE; Vissink, A

    The aim of this study was to evaluate the suitability of resorbable screws made of poly (D,L-lactide) acid (PDLLA) for fixation of autologous bone grafts related to graft regeneration and osseointegration of dental implants. In eight edentulous patients suffering from insufficient retention of their

  15. Experience with predonated autologous blood transfusion in open ...

    African Journals Online (AJOL)

    Objectives: To find out the practicability, the acceptability, the effectiveness and the safety level of pre-donated, autologous blood transfusion (ABT) in patients who underwent open prostatectomy. Study design: Prospective. Patients and methods: It was a prospective study carried out in Nigeria over a 5-year period.

  16. Vaccination with apoptosis colorectal cancer cell pulsed autologous ...

    African Journals Online (AJOL)

    To investigate vaccination with apoptosis colorectal cancer (CRC) cell pulsed autologous dendritic cells (DCs) in advanced CRC, 14 patients with advanced colorectal cancer (CRC) were enrolled and treated with DCs vaccine to assess toxicity, tolerability, immune and clinical responses to the vaccine. No severe toxicity ...

  17. Maintenance of DNA repair capacity in differentiating rat muscle cells in vitro

    International Nuclear Information System (INIS)

    Koval, T.M.; Kaufman, S.J.

    1981-01-01

    Unscheduled DNA synthesis was measured at several times during the differentiation of cultured rat skeletal muscle cells in response to exposures to 254 nm UV light. There was no change in the amount of repair DNA synthesis as the cells fuse and differentiate from postmitotic prefusion myoblasts to multinucleated contracting myotubes. (author)

  18. Effects of curcumin and ursolic acid on the mitochondrial coupling efficiency and hydrogen peroxide emission of intact skeletal myoblasts.

    Science.gov (United States)

    Tueller, Daniel J; Harley, Jackson S; Hancock, Chad R

    2017-10-21

    Curcumin may improve blood glucose management, but the mechanism is not fully established. We demonstrated that curcumin (40 μM) reduced the mitochondrial coupling efficiency (percentage of oxygen consumption coupled to ATP synthesis) of intact skeletal muscle cells. A 30-minute pretreatment with curcumin reduced mitochondrial coupling efficiency by 17.0 ± 0.4% relative to vehicle (p Curcumin pretreatment also decreased the rate of hydrogen peroxide emission by 43 ± 13% compared to vehicle (p curcumin revealed a 40 ± 4% increase in the rate of oxygen consumption upon curcumin administration (p curcumin-pretreated cells after permeabilization of cell membranes (p > 0.7). The interaction between curcumin and ursolic acid, another natural compound that may improve blood glucose management, was also examined. Pretreatment with ursolic acid (0.12 μM) increased the mitochondrial coupling efficiency of intact cells by 4.1 ± 1.1% relative to vehicle (p curcumin when the two compounds were used in combination. The observed changes to mitochondrial coupling efficiency and hydrogen peroxide emission were consistent with the established effects of curcumin on blood glucose control. Our findings also show that changes to mitochondrial coupling efficiency after curcumin pretreatment may go undetected unless cells are assessed in the intact condition. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Autologous Dendritic Cells Pulsed with Allogeneic Tumor Cell Lysate in Mesothelioma: From Mouse to Human.

    Science.gov (United States)

    Aerts, Joachim G J V; de Goeje, Pauline L; Cornelissen, Robin; Kaijen-Lambers, Margaretha E H; Bezemer, Koen; van der Leest, Cor H; Mahaweni, Niken M; Kunert, André; Eskens, Ferry A L M; Waasdorp, Cynthia; Braakman, Eric; van der Holt, Bronno; Vulto, Arnold G; Hendriks, Rudi W; Hegmans, Joost P J J; Hoogsteden, Henk C

    2018-02-15

    Purpose: Mesothelioma has been regarded as a nonimmunogenic tumor, which is also shown by the low response rates to treatments targeting the PD-1/PD-L1 axis. Previously, we demonstrated that autologous tumor lysate-pulsed dendritic cell (DC) immunotherapy increased T-cell response toward malignant mesothelioma. However, the use of autologous tumor material hampers implementation in large clinical trials, which might be overcome by using allogeneic tumor cell lines as tumor antigen source. The purpose of this study was to investigate whether allogeneic lysate-pulsed DC immunotherapy is effective in mice and safe in humans. Experimental Design: First, in two murine mesothelioma models, mice were treated with autologous DCs pulsed with either autologous or allogeneic tumor lysate or injected with PBS (negative control). Survival and tumor-directed T-cell responses of these mice were monitored. Results were taken forward in a first-in-human clinical trial, in which 9 patients were treated with 10, 25, or 50 million DCs per vaccination. DC vaccination consisted of autologous monocyte-derived DCs pulsed with tumor lysate from five mesothelioma cell lines. Results: In mice, allogeneic lysate-pulsed DC immunotherapy induced tumor-specific T cells and led to an increased survival, to a similar extent as DC immunotherapy with autologous tumor lysate. In the first-in-human clinical trial, no dose-limiting toxicities were established and radiographic responses were observed. Median PFS was 8.8 months [95% confidence interval (CI), 4.1-20.3] and median OS not reached (median follow-up = 22.8 months). Conclusions: DC immunotherapy with allogeneic tumor lysate is effective in mice and safe and feasible in humans. Clin Cancer Res; 24(4); 766-76. ©2017 AACR . ©2017 American Association for Cancer Research.

  20. Autologous fat graft in irradiated orbit postenucleation for retinoblastoma.

    Science.gov (United States)

    Klinger, Francesco; Maione, Luca; Vinci, Valeriano; Lisa, Andrea; Barbera, Federico; Balia, Laura; Caviggioli, Fabio; Di Maria, Alessandra

    2018-01-05

    Autologous fat grafting has been extensively and successfully adopted in a number of pathologic conditions in regenerative surgery especially on irradiated fields in order to improve pain symptoms and tissue trophism promoting scar release. In the present study, we report our experience with autologous fat grafting for the treatment of postirradiation fibrosis and pain on three consecutive patients undergoing orbital enucleation for locally advanced retinoblastoma (RB) and subsequent radiotherapy. We selected three consecutive patients who underwent orbital enucleation for locally advanced RB and subsequent local radiotherapy showing severe reduction in orbital volume and eyelid length and retraction due to fibrosis, spontaneous local pain exacerbated after digital pressure with no possibility to place an ocular implant. They underwent autologous fat grafting in the orbital cavity and results were evaluated by clinical examination at 5 and 14 days, and 1, 3, 6 months, and 1 year after surgery. A significant release of scar retraction, reduction of fibrosis and orbital rim contraction together with an important improvement of pain symptoms was observed in all patients. The local changes observed enabled an ease placement of an ocular prosthetic implant (implant). No local or systemic complication occurred. Fat grafting is a promising treatment for patients showing radiotherapy related complication in the orbital area and it should be adopted by all oculoplastic surgeon in order to improve pain syndrome creating the ideal local conditions for the placement of an ocular prosthetic implant.

  1. Surgical Patients\\' Knowledge and Acceptance of Autologous Blood ...

    African Journals Online (AJOL)

    Background: Homologous blood transfusion carries a well-documented array of risks especially in an HIV endemic environment like Nigeria. It is therefore imperative to consider other forms of restoring blood volume in surgical patients. Autologous blood transfusion (ABT) is one of the ways the problem of HIV transmission ...

  2. Tissue Engineered Skeletal Myofibers can Directly "Sense" Gravitational Force Changes

    Science.gov (United States)

    Vandenburgh, Herman H.; Shansky, J.; DelTatto, M.; Lee, Peter; Meir, J.

    1999-01-01

    Long-term manned space flight requires a better understanding of skeletal muscle atrophy resulting from microgravity. Atrophy most likely results from changes at both the systemic level (e.g. decreased circulating growth hormone, increased circulating glucocorticoids) and locally (e.g. decreased myofiber resting tension). Differentiated skeletal myofibers in tissue culture have provided a model system over the last decade for gaining a better understanding of the interactions of exogenous growth factors, endogenous growth factors, and muscle fiber tension in regulating protein turnover rates and muscle cell growth. Tissue engineering these cells into three dimensional bioartificial muscle (BAM) constructs has allowed us to extend their use to Space flight studies for the potential future development of countermeasures. Embryonic avian muscle cells were isolated and BAMs tissue engineered as described previously. The myoblasts proliferate and fuse into aligned postmitotic myofibers after ten to fourteen days in vitro. A cylindrical muscle-like structure containing several thousand myofibers is formed which is approximately 30 mm in length, 2-3 mm in diameter, and attached at each end. For the Space Shuttle experiments, the BAMs were transferred to 55 mL bioreactor cartridges (6 BAMs/cartridge). At Kennedy Space Center, the cartridges were mounted in two Space Tissue Loss (STL) Modules (three to four cartridges per Module) and either maintained as ground controls or loaded in a Mid-Deck locker of the Space Shuttle. The BAM cartridges were continuously perfused during the experiment at 1.5 mL/ min with tissue culture medium. Eighteen BAMs were flown for nine days on Mission STS66 while eighteen BAMs served as ground controls. The complete experiment was repeated on Mission STS77 with twenty four BAMs in each group. BAMs could be maintained in a healthy state for at least 30 days in the perfusion bioreactor cartridges. The BAM muscle fibers directly detected both the

  3. A Nationwide Analysis of Cost Variation for Autologous Free Flap Breast Reconstruction.

    Science.gov (United States)

    Billig, Jessica I; Lu, Yiwen; Momoh, Adeyiza O; Chung, Kevin C

    2017-11-01

    Cost variation among hospitals has been demonstrated for surgical procedures. Uncovering these differences has helped guide measures taken to reduce health care spending. To date, the fiscal consequence of hospital variation for autologous free flap breast reconstruction is unknown. To investigate factors that influence cost variation for autologous free flap breast reconstruction. A secondary cross-sectional analysis was performed using the Healthcare Cost and Utilization Project National Inpatient Sample database from 2008 to 2010. The dates of analysis were September 2016 to February 2017. The setting was a stratified sample of all US community hospitals. Participants were female patients who were diagnosed as having breast cancer or were at high risk for breast cancer and underwent autologous free flap breast reconstruction. Variables of interest included demographic data, hospital characteristics, length of stay, complications (surgical and systemic), and inpatient cost. The study used univariate and generalized linear mixed models to examine associations between patient and hospital characteristics and cost. A total of 3302 patients were included in the study, with a median age of 50 years (interquartile range, 44-57 years). The mean cost for autologous free flap breast reconstruction was $22 677 (interquartile range, $14 907-$33 391). Flap reconstructions performed at high-volume hospitals were significantly more costly than those performed at low-volume hospitals ($24 360 vs $18 918, P Logistic regression demonstrated that hospital volume correlated with increased cost (Exp[β], 1.06; 95% CI, 1.02-1.11; P = .003). Fewer surgical complications (16.4% [169 of 1029] vs 23.7% [278 of 1174], P cost variation among patients undergoing autologous free flap breast reconstruction. Experience, as measured by a hospital's volume, provides quality health care with fewer complications but is more costly. Longer length of stay contributed to regional

  4. Comparative Study of Skeletal Stability between Postoperative Skeletal Intermaxillary Fixation and No Skeletal Fixation after Bilateral Sagittal Split Ramus Osteotomy

    DEFF Research Database (Denmark)

    Hartlev, Jens; Godtfredsen, Erik; Andersen, Niels Trolle

    2014-01-01

    OBJECTIVES: The purpose of the present study was to evaluate skeletal stability after mandibular advancement with bilateral sagittal split osteotomy. MATERIAL AND METHODS: Twenty-six patients underwent single-jaw bilateral sagittal split osteotomy (BSSO) to correct skeletal Class II malocclusion....

  5. Specific Factors Influence the Success of Autologous and Allogeneic Hematopoietic Stem Cell Transplantation

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    Thissiane L. Gonçalves

    2009-01-01

    Full Text Available Successful hematopoietic stem cell transplantation (HSCT, both autologous and allogeneic, requires a rapid and durable engraftment, with neutrophil (>500/µL and platelet (>20,000/µL reconstitution. Factors influencing engraftment after autologous or allogeneic HSCT were investigated in 65 patients: 25 autologous peripheral stem cell transplantation (PBSCT and 40 allogeneic bone marrow transplantation (BMT patients. The major factor affecting engraftment was the graft source for HSCT. Neutrophil and platelet recovery were more rapid in autologous PBSCT than in allogeneic BMT [neutrophil occurring in median on day 10.00 (09.00/11.00 and 19.00 (16.00/23.00 and platelet on day 11.00 (10.00/13.00 and 21.00 (18.00/25.00, respectively; p < 0.0001]. The type of disease also affected engraftment, where multiple myeloma (MM and lymphoma showed faster engraftment when compared with leukemia, syndrome myelodysplastic (SMD and aplastic anemia (AA and MM presented the best overall survival (OS in a period of 12 months. Other factors included the drug used in the conditioning regimen (CR, where CBV, melphalan (M-200 and FluCy showed faster engraftment and M-200 presented the best OS, in a period of 12 months and age, where 50–59 years demonstrated faster engraftment. Sex did not influence neutrophil and platelet recovery.

  6. Complications Following Autologous Latissimus Flap Breast Reconstruction

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    Mufid Burgić

    2010-02-01

    Full Text Available Use of an autologous latissimus flap in breast reconstruction accounts for a supple and natural look of reconstructed breast. Most common postoperative complication, seroma, became more of a rule then an exception when it comes to postoperative evaluation of the patients who underwent this reconstructive procedure. A retrospective study analysing and evaluating different complication rates in 20 patients who underwent breast reconstruction by autologous latissimus flap, was conducted. All patients included in the study were operated at the Department of plastic surgery of Hôpital Civil in Strasbourg, France, between 1996 and 2008. The complication rates were noted as follows: seroma in 19 of our 20 patients (95%, late hypertrophic scarring in 3 patients (15%, postoperative surgical site hematoma in 3 patients (15%, and 2 patients (10% presented postoperative chronic back pain. Different options used in seroma treatment and prevention (subcutaneous-fascia anchor sutures of donor site, application of corticosteroids by injection into donor site postoperatively, passive drainage can reduce seroma formation and thus overall complication rates, leading to much faster patient’s recovery time and return to normal daily activities.

  7. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle

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    Helena Escobar

    2016-01-01

    Full Text Available Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6. Cg-Dysfprmd Prkdcscid/J (Scid/BLA/J mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy.

  8. A novel approach for in vitro meat production.

    Science.gov (United States)

    Pandurangan, Muthuraman; Kim, Doo Hwan

    2015-07-01

    The present review describes the possibility of in vitro meat production with the help of advanced co-culturing methods. In vitro meat production method could be a possible alternative for the conventional meat production. Originally, the research on in vitro meat production was initiated by the National Aeronautics and Space Administration (NASA) for space voyages. The required key qualities for accepting in vitro meat for consumption would be good efficiency ratio, increased protein synthesis rate in skeletal muscles, and mimicking the conventional meat qualities. In vitro culturing of meat is possible with the use of skeletal muscle tissue engineering, stem cell, cell co-culture, and tissue culture methods. Co-culture of myoblast and fibroblast is believed as one of the major techniques for in vitro meat production. In our lab, we have co-cultured myoblast and fibroblast. We believe that a billion pounds of in vitro meat could be produced from one animal for consumption. However, we require a great deal of research on in vitro meat production.

  9. An atlas of normal skeletal scintigraphy

    International Nuclear Information System (INIS)

    Flanagan, J.J.; Maisey, M.N.

    1985-01-01

    This atlas was compiled to provide the neophyte as well as the experienced radiologist and the nuclear medicine physician with a reference on normal skeletal scintigraphy as an aid in distinguishing normal variations in skeletal uptake from abnormal findings. Each skeletal scintigraph is labeled, and utilizing an identical scale, a relevant skeletal photograph and radiograph are placed adjacent to the scintigraph

  10. The exercised skeletal muscle: a review

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    Marina Marini

    2010-09-01

    Full Text Available The skeletal muscle is the second more plastic tissue of the body - second to the nervous tissue only. In fact, both physical activity and inactivity contribute to modify the skeletal muscle, by continuous signaling through nerve impulses, mechanical stimuli and humoral clues. In turn, the skeletal muscle sends signals to the body, thus contributing to its homeostasis. We'll review here the contribute of physical exercise to the shaping of skeletal muscle, to the adaptation of its mass and function to the different needs imposed by different physical activities and to the attainment of the health benefits associated with active skeletal muscles. Focus will primarily be on the molecular pathways and on gene regulation that result in skeletal muscle adaptation to exercise.

  11. Evidence for the involvement of the CXCL12 system in the adaptation of skeletal muscles to physical exercise.

    Science.gov (United States)

    Puchert, Malte; Adams, Volker; Linke, Axel; Engele, Jürgen

    2016-09-01

    The chemokine CXCL12 and its primary receptor, CXCR4, not only promote developmental myogenesis, but also muscle regeneration. CXCL12 chemoattracts CXCR4-positive satellite cells/blood-borne progenitors to the injured muscle, promotes myoblast fusion, partially with existing myofibers, and induces angiogenesis in regenerating muscles. Interestingly, the mechanisms underlying muscle regeneration are in part identical to those involved in muscular adaptation to intensive physical exercise. These similarities now prompted us to determine whether physical exercise would impact the CXCL12 system in skeletal muscle. We found that CXCL12 and CXCR4 are upregulated in the gastrocnemius muscle of rats that underwent a four-week period of constrained daily running exercise on a treadmill. Double-staining experiments confirmed that CXCL12 and CXCR4 are predominantly expressed in MyHC-positive muscle fibers. Moreover, these training-dependent increases in CXCL12 and CXCR4 expression also occurred in rats with surgical coronary artery occlusion, implying that the muscular CXCL12 system is still active in skeletal myopathy resulting from chronic heart failure. Expression of the second CXCL12 receptor, CXCR7, which presumably acts as a scavenger receptor in muscle, was not affected by training. Attempts to dissect the molecular events underlying the training-dependent effects of CXCL12 revealed that the CXCL12-CXCR4 axis activates anabolic mTOR-p70S6K signaling and prevents upregulation of the catabolic ubiquitin ligase MurF-1 in C2C12 myotubes, eventually increasing myotube diameters. Together, these findings point to a pivotal role of the CXCL12-CXCR4 axis in exercise-induced muscle maintenance and/or growth. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. in Skeletal Muscle

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    Espen E. Spangenburg

    2011-01-01

    Full Text Available Triglyceride storage is altered across various chronic health conditions necessitating various techniques to visualize and quantify lipid droplets (LDs. Here, we describe the utilization of the BODIPY (493/503 dye in skeletal muscle as a means to analyze LDs. We found that the dye was a convenient and simple approach to visualize LDs in both sectioned skeletal muscle and cultured adult single fibers. Furthermore, the dye was effective in both fixed and nonfixed cells, and the staining seemed unaffected by permeabilization. We believe that the use of the BODIPY (493/503 dye is an acceptable alternative and, under certain conditions, a simpler method for visualizing LDs stored within skeletal muscle.

  13. Autologous mesenchymal stem cells: clinical applications in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Mazzini, Letizia; Mareschi, Katia; Ferrero, Ivana; Vassallo, Elena; Oliveri, Giuseppe; Boccaletti, Riccardo; Testa, Lucia; Livigni, Sergio; Fagioli, Franca

    2006-07-01

    Our study was aimed to evaluate the feasibility and safety of intraspinal cord implantation of autologous mesenchymal stem cells (MSCs) in a few well-monitored amyotrophic lateral sclerosis (ALS) patients. Seven patients affected by definite ALS were enrolled in the study and two patients were treated for compassionate use and monitored for at least 3 years. Bone marrow was collected from the posterior iliac crest according to the standard procedure and MSCs were expanded ex vivo according to Pittenger's protocol. The cells were suspended in 2 ml autologous cerebrospinal fluid and transplanted into the spinal cord by a micrometric pump injector. The in vitro expanded MSCs did not show any bacterial o fungal contamination, hemopoietic cell contamination, chromosomic alterations and early cellular senescence. No patient manifested major adverse events such as respiratory failure or death. Minor adverse events were intercostal pain irradiation and leg sensory dysesthesia, both reversible after a mean period of 6 weeks. No modification of the spinal cord volume or other signs of abnormal cell proliferation were observed. A significant slowing down of the linear decline of the forced vital capacity was evident in four patients 36 months after MSCs transplantation. Our results demonstrate that direct injection of autologous expanded MSCs into the spinal cord of ALS patients is safe, with no significant acute or late toxicity, and well tolerated. The clinical results seem to be encouraging.

  14. Effect of oxygen tension on bioenergetics and proteostasis in young and old myoblast precursor cells.

    Science.gov (United States)

    Konigsberg, M; Pérez, V I; Ríos, C; Liu, Y; Lee, S; Shi, Y; Van Remmen, H

    2013-01-01

    In the majority of studies using primary cultures of myoblasts, the cells are maintained at ambient oxygen tension (21% O2), despite the fact that physiological O2 at the tissue level in vivo is much lower (~1-5% O2). We hypothesized that the cellular response in presence of high oxygen concentration might be particularly important in studies comparing energetic function or oxidative stress in cells isolated from young versus old animals. To test this, we asked whether oxygen tension plays a role in mitochondrial bioenergetics (oxygen consumption, glycolysis and fatty acid oxidation) or oxidative damage to proteins (protein disulfides, carbonyls and aggregates) in myoblast precursor cells (MPCs) isolated from young (3-4 m) and old (29-30 m) C57BL/6 mice. MPCs were grown under physiological (3%) or ambient (21%) O2 for two weeks prior to exposure to an acute oxidative insult (H2O2). Our results show significantly higher basal mitochondrial respiration in young versus old MPCs, an increase in basal respiration in young MPCs maintained at 3% O2 compared to cells maintained at 21% O2, and a shift toward glycolytic metabolism in old MPCs grown at 21% O2. H2O2 treatment significantly reduced respiration in old MPCs grown at 3% O2 but did not further repress respiration at 21% O2 in old MPCs. Oxidative damage to protein was higher in cells maintained at 21% O2 and increased in response to H2O2 in old MPCs. These data underscore the importance of understanding the effect of ambient oxygen tension in cell culture studies, in particular studies measuring oxidative damage and mitochondrial function.

  15. Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice.

    Science.gov (United States)

    Al Jaam, Bilal; Heu, Katy; Pennarubia, Florian; Segelle, Alexandre; Magnol, Laetitia; Germot, Agnès; Legardinier, Sébastien; Blanquet, Véronique; Maftah, Abderrahman

    2016-09-01

    Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1(cax/cax) (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1(cax/cax) mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1(cax/cax) SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7(+)/MYOD(-) progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice. © 2016 The Authors.

  16. Caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress.

    Science.gov (United States)

    Zhang, Qiang; Liu, Jianing; Chen, Shulan; Liu, Jing; Liu, Lijuan; Liu, Guirong; Wang, Fang; Jiang, Wenxin; Zhang, Caixia; Wang, Shuangyu; Yuan, Xiao

    2016-04-01

    It is well recognized that mandibular growth, which is caused by a variety of functional appliances, is considered to be the result of both neuromuscular and skeletal adaptations. Accumulating evidence has demonstrated that apoptosis plays an important role in the adaptation of skeletal muscle function. However, the underlying mechanism of apoptosis that is induced by stretch continues to be incompletely understood. Endoplasmic reticulum stress (ERS), a newly defined signaling pathway, initiates apoptosis. This study seeks to determine if caspase-12 is involved in stretch-induced apoptosis mediated endoplasmic reticulum stress in myoblast and its underlying mechanism. Apoptosis was assessed by Hochest staining, DAPI staining and annexin V binding and PI staining. ER chaperones, such as GRP78, CHOP and caspase-12, were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Furthermore, caspase-12 inhibitor was used to value the mechanism of the caspase-12 pathway. Apoptosis of myoblast, which is subjected to cyclic stretch, was observed in a time-dependent manner. We found that GRP78 mRNA and protein were significantly increased and CHOP and caspase-12 were activated in myoblast that was exposed to cyclic stretch. Caspase-12 inhibition reduced stretch-induced apoptosis, and caspase-12 activated caspase-3 to induce apoptosis. We concluded that caspase-12 played an important role in stretch-induced apoptosis that is associated by endoplasmic reticulum stress by activating caspase-3.

  17. Protective effect of Rhus coriaria fruit extracts against hydrogen peroxide-induced oxidative stress in muscle progenitors and zebrafish embryos

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    Fadia Najjar

    2017-12-01

    Full Text Available Background and Purpose Oxidative stress is involved in normal and pathological functioning of skeletal muscle. Protection of myoblasts from oxidative stress may improve muscle contraction and delay aging. Here we studied the effect of R. coriaria sumac fruit extract on human myoblasts and zebrafish embryos in conditions of hydrogen peroxide-induced oxidative stress. Study Design and Methods Crude ethanolic 70% extract (CE and its fractions was obtained from sumac fruits. The composition of sumac ethyl acetate EtOAc fraction was studied by 1H NMR. The viability of human myoblasts treated with CE and the EtOAc fraction was determined by trypan blue exclusion test. Oxidative stress, cell cycle and adhesion were analyzed by flow cytometry and microscopy. Gene expression was analyzed by qPCR. Results The EtOAc fraction (IC50 2.57 µg/mL had the highest antioxidant activity and exhibited the best protective effect against hydrogen peroxide-induced oxidative stress. It also restored cell adhesion. This effect was mediated by superoxide dismutase 2 and catalase. Pre-treatment of zebrafish embryos with low concentrations of the EtOAc fraction protected them from hydrogen peroxide-induced death in vivo. 1H NMR analysis revealed the presence of gallic acid in this fraction. Conclusion Rhus coriaria extracts inhibited or slowed down the progress of skeletal muscle atrophy by decreasing oxidative stress via superoxide dismutase 2 and catalase-dependent mechanisms.

  18. Autologous Bone Marrow Stem Cell Infusion (AMBI therapy for Chronic Liver Diseases

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    Rajkumar JS

    2007-01-01

    Full Text Available Liver Cirrhosis is the end stage of chronic liver disease which may happen due to alcoholism, viral infections due to Hepatitis B, Hepatitis C viruses and is difficult to treat. Liver transplantation is the only available definitive treatment which is marred by lack of donors, post operative complications such as rejection and high cost. Autologous bone marrow stem cells have shown a lot of promise in earlier reported animal studies and clinical trials. We have in this study administered in 22 patients with chronic liver disease, autologous bone marrow stem cell whose results are presented herewith.

  19. Pulmonary heart valve replacement using stabilized acellular xenogeneic scaffolds; effects of seeding with autologous stem cells

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    Harpa Marius Mihai

    2015-12-01

    Full Text Available Background: We hypothesized that an ideal heart valve replacement would be acellular valve root scaffolds seeded with autologous stem cells. To test this hypothesis, we prepared porcine acellular pulmonary valves, seeded them with autologous adipose derived stem cells (ADSCs and implanted them in sheep and compared them to acellular valves.

  20. Congenital anomalies and normal skeletal variants

    International Nuclear Information System (INIS)

    Guebert, G.M.; Yochum, T.R.; Rowe, L.J.

    1987-01-01

    Congenital anomalies and normal skeletal variants are a common occurrence in clinical practice. In this chapter a large number of skeletal anomalies of the spine and pelvis are reviewed. Some of the more common skeletal anomalies of the extremities are also presented. The second section of this chapter deals with normal skeletal variants. Some of these variants may simulate certain disease processes. In some instances there are no clear-cut distinctions between skeletal variants and anomalies; therefore, there may be some overlap of material. The congenital anomalies are presented initially with accompanying text, photos, and references, beginning with the skull and proceeding caudally through the spine to then include the pelvis and extremities. The normal skeletal variants section is presented in an anatomical atlas format without text or references

  1. Unicameral bone cysts: a comparison of injection of steroid and grafting with autologous bone marrow.

    Science.gov (United States)

    Cho, H S; Oh, J H; Kim, H-S; Kang, H G; Lee, S H

    2007-02-01

    Open surgery is rarely justified for the initial treatment of a unicameral bone cyst, but there is some debate concerning the relative effectiveness of closed methods. This study compared the results of steroid injection with those of autologous bone marrow grafting for the treatment of unicameral bone cysts. Between 1990 and 2001, 30 patients were treated by steroid injection and 28 by grafting with autologous bone marrow. The overall success rates were 86.7% and 92.0%, respectively (p>0.05). The success rate after the initial procedure was 23.3% in the steroid group and 52.0% in those receiving autologous bone marrow (p0.05). The mean number of procedures required was 2.19 (1 to 5) and 1.57 (1 to 3) (p0.05), and the rate of recurrence after the initial procedure was 41.7% and 13.3% in the steroid and in the autologous bone marrow groups, respectively (p<0.05). Although the overall rates of success of both methods were similar, the steroid group had higher recurrence after a single procedure and required more injections to achieve healing.

  2. Scintigraphic detection of carotid atherosclerosis with indium-111-labeled autologous platelets

    International Nuclear Information System (INIS)

    Davis, H.H.; Siegel, B.A.; Sherman, L.A.; Heaton, W.A.; Naidich, T.P.; Joist, J.R.; Welch, M.J.

    1980-01-01

    Using autologous platelets labeled with indium-111-oxine, we studied the localization of platelets on arterial lesions by radionuclide scintigraphy in 34 patients with suspected cerebrovascular disease. The imaging results were compared with the findings of contrast angiography in 23 patients, 16 of whom were receiving antiplatelet and/or anticoagulant drugs during the platelet imaging study. Angiography demonstrated atherosclerotic lesions at 33 sites in the extracranial arteries of 16 of these patients. There was accumulation of 111 In-platelets at 20 of these sites (61%) and at three other sites without definite angiographic abnormalities. Lesions with stenoses 111 In-labeled autologous platelets may be useful for evaluating the pathophysiologic characteristics of atherosclerotic lesions in patients with cerebrovascular disease

  3. Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects

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    Daniela GUZMÁN-URIBE

    Full Text Available Abstract Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%, meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes. Objective The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Material and Methods Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group. Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. Results It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Conclusion Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

  4. Skeletal Stability after Large Mandibular Advancement (> 10 mm) with Bilateral Sagittal Split Osteotomy and Skeletal Elastic Intermaxillary Fixation

    DEFF Research Database (Denmark)

    Schwartz, Kristoffer; Rodrigo, Maria; Jensen, Thomas

    2016-01-01

    OBJECTIVES: The aim of the present study was to assess the skeletal stability after large mandibular advancement (> 10 mm) with bilateral sagittal split osteotomy and skeletal elastic intermaxillary fixation and to correlate the skeletal stability with the vertical facial type. MATERIAL AND METHODS......: A total of 33 consecutive patients underwent bimaxillary surgery to correct skeletal Class II malocclusion with a mandibular advancement (> 10 mm) measured at B-point and postoperative skeletal elastic intermaxillary fixation for 16 weeks. Skeletal stability was evaluated using lateral cephalometric...... radiographs obtained preoperative (T1), 8 weeks postoperatively (T2), and 18 month postoperatively (T3). B-point and pogonion (Pog) was used to measure the skeletal relapse and the mandibular plane angle (MP-angle) was used to determine the vertical facial type. RESULTS: The mean advancement from T1 to T2...

  5. Evidences of autologous fat grafting for the treatment of keloids and hypertrophic scars

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    VINÍCIUS ZOLEZI DA SILVA

    Full Text Available SUMMARY Introduction Since the 1980s, the use of autologous fat grafting has been growing in plastic surgery. Recently, this procedure has come to be used as a treatment for keloids and hypertrophic scars mainly due to the lack of satisfactory results with other techniques. So far, however, it lacks more consistent scientific evidence to recommend its use. The aim of this study was to review the current state of autologous fat grafting for the treatment of keloids and hypertrophic scars, their benefits and scientific evidences in the literature. Method A review in the Pubmed database was performed using the keywords “fat grafting and scar”, “fat grafting and keloid scar” and “fat grafting and hypertrophic scar.” Inclusion criteria were articles written in English and published in the last 10 years, resulting in 15 studies. Results These articles indicate that autologous fat grafting carried out at sites with pathological scars leads to a reduction of the fibrosis and pain, an increased range of movement in areas of scar contraction, an increase in their flexibility, resulting in a better quality of scars. Conclusion So far, evidences suggest that autologous fat grafting for the treatment of keloids and hypertrophic scars is associated with a better quality of scars, leading to esthetic and functional benefits. However, this review has limitations and these findings should be treated with reservations, since they mostly came from studies with low levels of evidence.

  6. Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts

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    Sachchida Nand Pandey

    2015-05-01

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is believed to be caused by aberrant expression of double homeobox 4 (DUX4 due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01 and primary (4.7 fold, p < 0.01 FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells.

  7. Regeneration of blood-forming organs after autologous leukocyte transfusion in lethally irradiated dogs. II. Distribution and cellularity of the marrow in irradiated and transfused animals

    International Nuclear Information System (INIS)

    Calvo, W.; Fliedner, T.M.; Herbst, E.; Huegl, E.; Bruch, C.

    1976-01-01

    Dogs were given transfusions of cryopreserved autologous mononuclear blood leukocytes after 1200 roentgens (R) (midline dose) whole-body x-irradiation. Bone marrow repopulation was studied by means of histomorphological methods at days 9 and 10 after transfusion of an average of 3 x 10 9 , 7 x 10 9 , 13 x 10 9 , and 31 x 10 9 cells. The return of marrow cellularity to normal values was related to the number of cells transfused. With low cell doses (3 x 10 9 and 7 x 10 9 ), the marrow regeneration at 10 days was focal. There were groups of cells (colonies) showing either erythropoiesis, myelopoiesis, or megakaryocytopoiesis in the osteal niches of the trabecular bones. Frequently such niches were seen showing complete cellular recovery next to niches with complete aplasia. With higher cell doses, all niches showed hemopoietic regeneration, and the cellularity approached normal values. No hemopoietic regeneration was observed in those skeletal parts that do not show hemopoiesis, even under normal circumstances

  8. Skeletal manifestations of juvenile hypothyroidism and the impact of treatment on skeletal system.

    Science.gov (United States)

    Gutch, Manish; Philip, Rajeev; Philip, Renjit; Toms, Ajit; Saran, Sanjay; Gupta, K K

    2013-10-01

    Thyroid hormone mediates growth and development of the skeleton through its direct effects and through its permissive effects on growth hormone. The effect of hypothyroidism on bone is well described in congenital hypothyroidism, but the impact of thyroid hormone deficiency on a growing skeleton, as it happens with juvenile hypothyroidism, is less defined. In addition, the extent to which the skeletal defects of juvenile hypothyroidism revert on the replacement of thyroid hormone is not known. A study was undertaken in 29 juvenile autoimmune hypothyroid patients to study the skeletal manifestations of juvenile hypothyroidism and the impact of treatment of hypothyroidism on the skeletal system of juvenile patients. Hypothyroidism has a profound impact on the skeletal system and delayed bone age, dwarfism, and thickened bands at the metaphyseal ends being the most common findings. Post treatment, skeletal findings like delayed bone age and dwarfism improved significantly, but there were no significant changes in enlargement of sella, presence of wormian bones, epihyseal dysgenesis, vertebral changes and thickened band at the metaphyseal ends. With the treatment of hypothyroidism, there is an exuberant advancement of bone age, the catch up of bone age being approximately double of the chronological age advancement.

  9. Feasibility of pre-operative autologous blood donation in Indian patients with elective orthopaedic surgery.

    Science.gov (United States)

    Saluja, Karan; Marwaha, Neelam; Thakral, Beenu; Goni, Vijay; Sharma, R R; Puri, G D

    2006-11-01

    Pre-operative autologous blood donation (PABD) in elective orthopaedic surgeries is a well known procedure in the West. We initiated this programme at a tertiary care hospital in north India to study its feasibility in Indian patients. In a prospective case-control study, 144 patients undergoing primary total hip or knee replacement, inter-vertebral discectomy, mal-union and non-union reconstruction were educated and motivated to pre-donate. Patients fulfilling the inclusion criteria and making autologous donation formed the PABD group (n=22). Patients eligible for PABD, but unwilling to participate; age, sex, pre-operative haemoglobin and operative procedure matched acted as controls (n=27). Unit(s) collected was processed like an allogeneic unit. Unit(s) found reactive for infectious markers or not utilized was discarded. Mean blood losses, transfusion trigger, allogeneic exposure and wastage between the two groups were compared. Of the 144 patients motivated, 40 per cent of the eligible subjects pre-deposited. The main motivational factor was fear of getting infection from someone's blood. Cardiac events and anaemia prevented 61.8 per cent patients to participate. Of the 50 units ordered, autologous units with a mean of 1.4 units/patient contributed 62 per cent. For total hip and total knee replacement (THR and TKR), autologous units met 76.2 and 80 per cent respectively of the total blood requirement. A significant decrease in the allogeneic exposure was observed between PABD and control group (18.2 vs 66.7%); 32.3 per cent of the autologous units were discarded. Comprehensive PABD programme may be an effective method for reducing the need for allogeneic transfusion in patients undergoing joint replacement surgeries in our country, where transfusion transmitted infections due to high percentage of replacement donations and lack of sensitive assays for testing are still a cause for concern.

  10. Impact of Postoperative Antibiotic Prophylaxis Duration on Surgical Site Infections in Autologous Breast Reconstruction.

    Science.gov (United States)

    Drury, Kerry E; Lanier, Steven T; Khavanin, Nima; Hume, Keith M; Gutowski, Karol A; Thornton, Brian P; Hansen, Nora M; Murphy, Robert X; Fine, Neil A; Kim, John Y S

    2016-02-01

    Although some surgeons prescribe prolonged postoperative antibiotics after autologous breast reconstruction, evidence is lacking to support this practice. We used the Tracking Operations and Outcomes for Plastic Surgeons database to evaluate the association between postoperative antibiotic duration and the rate of surgical site infection (SSI) in autologous breast reconstruction. The intervention of interest for this study was postoperative duration of antibiotic prophylaxis: either discontinued 24 hours after surgery or continued beyond 24 hours. The primary outcome variable of interest for this study was the presence of SSI within 30 days of autologous breast reconstruction. Cohort characteristics and 30-day outcomes were compared using χ² and Fischer exact tests for categorical variables and Student t tests for continuous variables. Multivariate logistic regression was used to control for confounders. A total of 1036 patients met inclusion criteria for our study. Six hundred fifty-nine patients (63.6%) received antibiotics for 24 hours postoperatively, and 377 patients (36.4%) received antibiotics for greater than 24 hours. The rate of SSI did not differ significantly between patients given antibiotics for only 24 hours and those continued on antibiotics beyond the 24-hour postoperative time period (5.01% vs 2.92%, P = 0.109). Furthermore, antibiotic duration was not predictive of SSI in multivariate regression modeling. We did not find a statistically significant difference in the rate of SSI in patients who received 24 hours of postoperative antibiotics compared to those that received antibiotics for greater than 24 hours. These findings held for both purely autologous reconstruction as well as latissimus dorsi reconstruction in conjunction with an implant. Thus, our study does not support continuation of postoperative antibiotics beyond 24 hours after autologous breast reconstruction.

  11. Thimerosal-induced apoptosis in mouse C2C12 myoblast cells occurs through suppression of the PI3K/Akt/survivin pathway.

    Directory of Open Access Journals (Sweden)

    Wen-Xue Li

    Full Text Available BACKGROUND: Thimerosal, a mercury-containing preservative, is one of the most widely used preservatives and found in a variety of biological products. Concerns over its possible toxicity have reemerged recently due to its use in vaccines. Thimerosal has also been reported to be markedly cytotoxic to neural tissue. However, little is known regarding thimerosal-induced toxicity in muscle tissue. Therefore, we investigated the cytotoxic effect of thimerosal and its possible mechanisms on mouse C2C12 myoblast cells. METHODOLOGY/PRINCIPAL FINDINGS: The study showed that C2C12 myoblast cells underwent inhibition of proliferation and apoptosis after exposure to thimerosal (125-500 nM for 24, 48 and 72 h. Thimerosal caused S phase arrest and induced apoptosis as assessed by flow cytometric analysis, Hoechst staining and immunoblotting. The data revealed that thimerosal could trigger the leakage of cytochrome c from mitochondria, followed by cleavage of caspase-9 and caspase-3, and that an inhibitor of caspase could suppress thimerosal-induced apoptosis. Thimerosal inhibited the phosphorylation of Akt(ser473 and survivin expression. Wortmannin, a PI3K inhibitor, inhibited Akt activity and decreased survivin expression, resulting in increased thimerosal-induced apoptosis in C2C12 cells, while the activation of PI3K/Akt pathway by mIGF-I (50 ng/ml increased the expression of survivin and attenuated apoptosis. Furthermore, the inhibition of survivin expression by siRNA enhanced thimerosal-induced cell apoptosis, while overexpression of survivin prevented thimerosal-induced apoptosis. Taken together, the data show that the PI3K/Akt/survivin pathway plays an important role in the thimerosal-induced apoptosis in C2C12 cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that in C2C12 myoblast cells, thimerosal induces S phase arrest and finally causes apoptosis via inhibition of PI3K/Akt/survivin signaling followed by activation of the mitochondrial apoptotic

  12. Expression of Gla proteins during fish skeletal development

    OpenAIRE

    Gavaia, Paulo J.

    2006-01-01

    Senegal sole skeletal development; Skeletal malformations; Skeletal malformation in mediterranean species; Senegal sole skeletal deformities; Zebra fish as model system: skeletal development; Identification of bone cells / skeletal development; Spatial - temporal pattern of bgp expression; Single cell resolution: localization of bgp mRNA; Single cell resolution: Immunolocalization of Bgp; Single cell resolution: localization of mgp mRNA; Single cell resolution: Immunolocalization of Mgp; An i...

  13. Autologous Chondrocyte Implantation in Osteoarthritic Surroundings

    DEFF Research Database (Denmark)

    Ossendorff, Robert; Grad, Sibylle; Stoddart, Martin J

    2018-01-01

    BACKGROUND: Autologous chondrocyte implantation (ACI) fails in up to 20% of cases. Advanced intra-articular degeneration paired with an inflammatory environment may be closely related to implantation failure. Certain cytokines have been identified to play a major role during early osteoarthritis....... PURPOSE: To investigate the effects of tumor necrosis factor α (TNFα) and its potential inhibition by adalimumab on cartilage regeneration in an in vitro model of ACI. STUDY DESIGN: Controlled laboratory study. METHODS: Bovine articular chondrocytes were cultivated and transferred at passage 3 to fibrin...

  14. Hyaline cartilage degenerates after autologous osteochondral transplantation.

    Science.gov (United States)

    Tibesku, C O; Szuwart, T; Kleffner, T O; Schlegel, P M; Jahn, U R; Van Aken, H; Fuchs, S

    2004-11-01

    Autologous osteochondral grafting is a well-established clinical procedure to treat focal cartilage defects in patients, although basic research on this topic remains sparse. The aim of the current study was to evaluate (1) histological changes of transplanted hyaline cartilage of osteochondral grafts and (2) the tissue that connects the transplanted cartilage with the adjacent cartilage in a sheep model. Both knee joints of four sheep were opened surgically and osteochondral grafts were harvested and simultaneously transplanted to the contralateral femoral condyle. The animals were sacrificed after three months and the received knee joints were evaluated histologically. Histological evaluation showed a complete ingrowth of the osseous part of the osteochondral grafts. A healing or ingrowth at the level of the cartilage could not be observed. Histological evaluation of the transplanted grafts according to Mankin revealed significantly more and more severe signs of degeneration than the adjacent cartilage, such as cloning of chondrocytes and irregularities of the articular surface. We found no connecting tissue between the transplanted and the adjacent cartilage and histological signs of degeneration of the transplanted hyaline cartilage. In the light of these findings, long-term results of autologous osteochondral grafts in human beings have to be followed critically.

  15. Persistent seropositivity for yellow fever in a previously vaccinated autologous hematopoietic stem cell transplantation recipient.

    Science.gov (United States)

    Hayakawa, Kayoko; Takasaki, Tomohiko; Tsunemine, Hiroko; Kanagawa, Shuzo; Kutsuna, Satoshi; Takeshita, Nozomi; Mawatari, Momoko; Fujiya, Yoshihiro; Yamamoto, Kei; Ohmagari, Norio; Kato, Yasuyuki

    2015-08-01

    The duration of a protective level of yellow fever antibodies after autologous hematopoietic stem cell transplantation in a previously vaccinated person is unclear. The case of a patient who had previously been vaccinated for yellow fever and who remained seropositive for 22 months after autologous peripheral blood stem cell transplantation for malignant lymphoma is described herein. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Autologous Blood and Platelet-Rich Plasma Injections for Treatment of Lateral Epicondylitis.

    Science.gov (United States)

    Calandruccio, James H; Steiner, Murphy M

    2017-07-01

    Lateral epicondylitis (tennis elbow) is a frequent cause of elbow pain; most patients (80%-90%) are successfully treated with standard nonoperative methods (rest, nonsteroidal anti-inflammatory drugs, bracing, and physical therapy). Autologous blood injections and platelet-rich plasma injections are the two most frequently used orthobiologic techniques in the treatment of lateral epicondylitis. Studies of the effectiveness of autologous blood injections and platelet-rich plasma report varying outcomes, some citing significant clinical relief and others reporting no beneficial effect. More research is needed to determine how to best use orthobiologics in the treatment of lateral epicondylitis. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Skeletal muscle performance and ageing.

    Science.gov (United States)

    Tieland, Michael; Trouwborst, Inez; Clark, Brian C

    2018-02-01

    The world population is ageing rapidly. As society ages, the incidence of physical limitations is dramatically increasing, which reduces the quality of life and increases healthcare expenditures. In western society, ~30% of the population over 55 years is confronted with moderate or severe physical limitations. These physical limitations increase the risk of falls, institutionalization, co-morbidity, and premature death. An important cause of physical limitations is the age-related loss of skeletal muscle mass, also referred to as sarcopenia. Emerging evidence, however, clearly shows that the decline in skeletal muscle mass is not the sole contributor to the decline in physical performance. For instance, the loss of muscle strength is also a strong contributor to reduced physical performance in the elderly. In addition, there is ample data to suggest that motor coordination, excitation-contraction coupling, skeletal integrity, and other factors related to the nervous, muscular, and skeletal systems are critically important for physical performance in the elderly. To better understand the loss of skeletal muscle performance with ageing, we aim to provide a broad overview on the underlying mechanisms associated with elderly skeletal muscle performance. We start with a system level discussion and continue with a discussion on the influence of lifestyle, biological, and psychosocial factors on elderly skeletal muscle performance. Developing a broad understanding of the many factors affecting elderly skeletal muscle performance has major implications for scientists, clinicians, and health professionals who are developing therapeutic interventions aiming to enhance muscle function and/or prevent mobility and physical limitations and, as such, support healthy ageing. © 2017 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.

  18. Comparison between sensitivity of autologous skin serum test and autologous plasma skin test in patients with Chronic Idiopathic Urticaria for detection of antibody against IgE or IgE receptor (FcεRIα).

    Science.gov (United States)

    Sajedi, Vahid; Movahedi, Masoud; Aghamohammadi, Asghar; Aghamohamadi, Asghar; Gharagozlou, Mohammad; Ghareguzlou, Mohammad; Shafiei, Alireza; Soheili, Habib; Sanajian, Nahal

    2011-06-01

    Intradermal injection of autologous serum and plasma elicit a cutaneous reactivity in almost 45-60% of patients with Chronic Idiopathic Urticaria (CIU). This reactivity is associated with the presence of auto antibodies against IgE or IgE receptors. This study was carried out to compare the cutaneous reactivity of autologous serum and plasma skin tests in a series of patients with CIU for diagnosis of auto antibodies against IgE or IgE receptor. Fifty eight patients with CIU were injected intradermally with autologous serum and plasma (anticoagulated by citrate). Histamine was used as positive control and normal saline as negative control. The study group was checked by routine laboratory tests (CBC, U/A etc), allergens with skin prick tests, and serum IgE level, and auto antibodies against thyroid as well. Duration of urticaria was another factor which was assessed.There was no significant difference between positive ASST and positive APST patients for the above mentioned tests. 77.6% of the patients were Positive for APST and 65.5% were ASST positive. Duration of urticaria was longer in patients with positive ASST and APST than ASST and APST negative patients, although the difference was not statistically significant.Autologus serum skin test (ASST) and autologous plasma skin test (APST) could be used for estimation of duration and severity of urticaria and planning for the treatment.

  19. Noncultured Autologous Adipose-Derived Stem Cells Therapy for Chronic Radiation Injury

    Science.gov (United States)

    Akita, Sadanori; Akino, Kozo; Hirano, Akiyoshi; Ohtsuru, Akira; Yamashita, Shunichi

    2010-01-01

    Increasing concern on chronic radiation injuries should be treated properly for life-saving improvement of wound management and quality of life. Recently, regenerative surgical modalities should be attempted with the use of noncultured autologous adipose-derived stem cells (ADSCs) with temporal artificial dermis impregnated and sprayed with local angiogenic factor such as basic fibroblast growth factor, and secondary reconstruction can be a candidate for demarcation and saving the donor morbidity. Autologous adipose-derived stem cells, together with angiogenic and mitogenic factor of basic fibroblast growth factor and an artificial dermis, were applied over the excised irradiated skin defect and tested for Patients who were uneventfully healed with minimal donor-site morbidity, which lasts more than 1.5 years. PMID:21151652

  20. Noncultured Autologous Adipose-Derived Stem Cells Therapy for Chronic Radiation Injury

    Directory of Open Access Journals (Sweden)

    Sadanori Akita

    2010-01-01

    Full Text Available Increasing concern on chronic radiation injuries should be treated properly for life-saving improvement of wound management and quality of life. Recently, regenerative surgical modalities should be attempted with the use of noncultured autologous adipose-derived stem cells (ADSCs with temporal artificial dermis impregnated and sprayed with local angiogenic factor such as basic fibroblast growth factor, and secondary reconstruction can be a candidate for demarcation and saving the donor morbidity. Autologous adipose-derived stem cells, together with angiogenic and mitogenic factor of basic fibroblast growth factor and an artificial dermis, were applied over the excised irradiated skin defect and tested for Patients who were uneventfully healed with minimal donor-site morbidity, which lasts more than 1.5 years.

  1. A role for Insulin-like growth factor 2 in specification of the fast skeletal muscle fibre

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    Ting Tao

    2007-06-01

    Full Text Available Abstract Background Fibre type specification is a poorly understood process beginning in embryogenesis in which skeletal muscle myotubes switch myosin-type to establish fast, slow and mixed fibre muscle groups with distinct function. Growth factors are required to establish slow fibres; it is unknown how fast twitch fibres are specified. Igf-2 is an embryonically expressed growth factor with established in vitro roles in skeletal muscle. Its localisation and role in embryonic muscle differentiation had not been established. Results Between E11.5 and E15.5 fast Myosin (FMyHC localises to secondary myotubes evenly distributed throughout the embryonic musculature and gradually increasing in number so that by E15.5 around half contain FMyHC. The Igf-2 pattern closely correlates with FMyHC from E13.5 and peaks at E15.5 when over 90% of FMyHC+ myotubes also contain Igf-2. Igf-2 lags FMyHC and it is absent from muscle myotubes until E13.5. Igf-2 strongly down-regulates by E17.5. A striking feature of the FMyHC pattern is its increased heterogeneity and attenuation in many fibres from E15.5 to day one after birth (P1. Transgenic mice (MIG which express Igf-2 in all of their myotubes, have increased FMyHC staining, a higher proportion of FMyHC+ myotubes and loose their FMyHC staining heterogeneity. In Igf-2 deficient mice (MatDi FMyHC+ myotubes are reduced to 60% of WT by E15.5. In vitro, MIG induces a 50% excess of FMyHC+ and a 30% reduction of SMHyC+ myotubes in C2 cells which can be reversed by Igf-2-targeted ShRNA resulting in 50% reduction of FMyHC. Total number of myotubes was not affected. Conclusion In WT embryos the appearance of Igf-2 in embryonic myotubes lags FMyHC, but by E15.5 around 45% of secondary myotubes contain both proteins. Forced expression of Igf-2 into all myotubes causes an excess, and absence of Igf-2 suppresses, the FMyHC+ myotube component in both embryonic muscle and differentiated myoblasts. Igf-2 is thus required, not for

  2. Oncological outcomes of patients with Ewing's sarcoma: is there a difference between skeletal and extra-skeletal Ewing's sarcoma?

    Science.gov (United States)

    Pradhan, A; Grimer, R J; Spooner, D; Peake, D; Carter, S R; Tillman, R M; Abudu, A; Jeys, L

    2011-04-01

    The aim of this study was to identify whether there was any difference in patient, tumour, treatment or outcome characteristics between patients with skeletal or extra-skeletal Ewing's sarcoma. We identified 300 patients with new primary Ewing's sarcoma diagnosed between 1980 and 2005 from the centres' local database. There were 253 (84%) with skeletal and 47 (16%) with extra-skeletal Ewing's sarcomas. Although patients with skeletal Ewing's were younger (mean age 16.8 years) than those with extra-skeletal Ewing's sarcoma (mean age 27.5 years), there was little difference between the groups in terms of tumour stage or treatment. Nearly all the patients were treated with chemotherapy and most had surgery. There was no difference in the overall survival of patients with skeletal (64%) and extra-skeletal Ewing's sarcoma (61%) (p = 0.85), and this was also the case when both groups were split by whether they had metastases or not. This large series has shown that the oncological outcomes of Ewing's sarcoma are related to tumour characteristics and patient age, and not determined by whether they arise in bone or soft tissue.

  3. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming.

    Science.gov (United States)

    Mitani, Yasuyuki; Vagnozzi, Ronald J; Millay, Douglas P

    2017-01-01

    Knowledge regarding cellular fusion and nuclear reprogramming may aid in cell therapy strategies for skeletal muscle diseases. An issue with cell therapy approaches to restore dystrophin expression in muscular dystrophy is obtaining a sufficient quantity of cells that normally fuse with muscle. Here we conferred fusogenic activity without transdifferentiation to multiple non-muscle cell types and tested dystrophin restoration in mouse models of muscular dystrophy. We previously demonstrated that myomaker, a skeletal muscle-specific transmembrane protein necessary for myoblast fusion, is sufficient to fuse 10T 1/2 fibroblasts to myoblasts in vitro. Whether myomaker-mediated heterologous fusion is functional in vivo and whether the newly introduced nonmuscle nuclei undergoes nuclear reprogramming has not been investigated. We showed that mesenchymal stromal cells, cortical bone stem cells, and tail-tip fibroblasts fuse to skeletal muscle when they express myomaker. These cells restored dystrophin expression in a fraction of dystrophin-deficient myotubes after fusion in vitro. However, dystrophin restoration was not detected in vivo although nuclear reprogramming of the muscle-specific myosin light chain promoter did occur. Despite the lack of detectable dystrophin reprogramming by immunostaining, this study indicated that myomaker could be used in nonmuscle cells to induce fusion with muscle in vivo, thereby providing a platform to deliver therapeutic material.-Mitani, Y., Vagnozzi, R. J., Millay, D. P. In vivo myomaker-mediated heterologous fusion and nuclear reprogramming. © FASEB.

  4. Autologous conjunctiva transplantation with stem cells on edge of cornea for recurrent pterygium

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    Yun Wang

    2013-10-01

    Full Text Available AIM: To observe the clinical effectiveness and practicality the autologous conjunctiva transplantation with stem cells on edge of cornea for recurrent pterygium.METHODS: Of the 53 recurrent pterygium patients(57 eyes, after all pathological tissues were removed, underwent the autologous conjunctiva transplantation with stem cells on edge of cornea which were locked above conjunctival transplantation of the operated eye.RESULTS: Postopretive follow-up was 1-12 months for all 57 eyes, of which 3 eyes(5%relapsed. The corneoscleral autolysis was occurred in one eye and surgery treatment was conducted. Corneal wounds were healing and transplantations survived well for the remaining 53 patients without obvious surgical marks. Cure rate was 93%.CONCLUSION: Autologous conjunctiva transplantation with stem cells on edge of cornea for recurrent pterygium can meet the aesthetic requirements of the some patients, with the advantages of obtaining material easily, faster wound healing, lower postoperative recurrence rate, meeting the aesthetic needs of some patients and improving postoperative results. Thus, it is an ideal surgery and is worthy of applying on primary hospital.

  5. EFFICACY OF LOCAL INFILTRATION OF AUTOLOGOUS BLOOD VERSUS LOCAL CORTICOSTEROID INJECTION- THE TREATMENT OF CHRONIC TENNIS ELBOW

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    Rajeev Kumar Roy

    2017-05-01

    Full Text Available BACKGROUND Chronic painful tendon disorders are common in both sport persons and common individuals. 1,2 Lateral epicondylitis (tennis elbow is relatively more common among active individuals in the general population. 3 Typical signs and symptoms include pain and tenderness over the lateral epicondyle, exacerbated by resisted wrist extension and passive wrist flexion and impaired grip strength. The aim of the study is to find whether autologous blood provides comparable functional outcome over local steroids and hence whether it can replace steroids in treatment of tennis elbow. MATERIALS AND METHODS Patients with nontraumatic elbow pain attending the Orthopaedics Outpatient Department of Nalanda Medical College Hospital from January 2016 to August 2016. The participating subjects were randomly grouped into two groups (steroid (Group A and autologous blood (Group B. Pain in the subject’s affected elbow was measured using Visual Analogue Score (VAS. RESULTS Initially, both the groups had comparable initial VAS scores. At 1 month follow up, steroid group showed a significantly greater improvement in mean VAS scores when compared to autologous blood group. However, at 6 months follow up, steroid group showed no statistically significant difference in mean VAS scores when compared to autologous blood group. CONCLUSION From the current study, we concluded that both local corticosteroid and autologous blood were equally efficacious in the treatment of chronic lateral epicondylitis of elbow.

  6. Human T-Cell Clones from Autoimmune Thyroid Glands: Specific Recognition of Autologous Thyroid Cells

    Science.gov (United States)

    Londei, Marco; Bottazzo, G. Franco; Feldmann, Marc

    1985-04-01

    The thyroid glands of patients with autoimmune diseases such as Graves' disease and certain forms of goiter contain infiltrating activated T lymphocytes and, unlike cells of normal glands, the epithelial follicular cells strongly express histocompatability antigens of the HLA-DR type. In a study of such autoimmune disorders, the infiltrating T cells from the thyroid glands of two patients with Graves' disease were cloned in mitogen-free interleukin-2 (T-cell growth factor). The clones were expanded and their specificity was tested. Three types of clones were found. One group, of T4 phenotype, specifically recognized autologous thyroid cells. Another, also of T4 phenotype, recognized autologous thyroid or blood cells and thus responded positively in the autologous mixed lymphocyte reaction. Other clones derived from cells that were activated in vivo were of no known specificity. These clones provide a model of a human autoimmune disease and their analysis should clarify mechanisms of pathogenesis and provide clues to abrogating these undesirable immune responses.

  7. Skeletal manifestations of juvenile hypothyroidism and the impact of treatment on skeletal system

    Directory of Open Access Journals (Sweden)

    Manish Gutch

    2013-01-01

    Full Text Available Thyroid hormone mediates growth and development of the skeleton through its direct effects and through its permissive effects on growth hormone. The effect of hypothyroidism on bone is well described in congenital hypothyroidism, but the impact of thyroid hormone deficiency on a growing skeleton, as it happens with juvenile hypothyroidism, is less defined. In addition, the extent to which the skeletal defects of juvenile hypothyroidism revert on the replacement of thyroid hormone is not known. A study was undertaken in 29 juvenile autoimmune hypothyroid patients to study the skeletal manifestations of juvenile hypothyroidism and the impact of treatment of hypothyroidism on the skeletal system of juvenile patients. Hypothyroidism has a profound impact on the skeletal system and delayed bone age, dwarfism, and thickened bands at the metaphyseal ends being the most common findings. Post treatment, skeletal findings like delayed bone age and dwarfism improved significantly, but there were no significant changes in enlargement of sella, presence of wormian bones, epihyseal dysgenesis, vertebral changes and thickened band at the metaphyseal ends. With the treatment of hypothyroidism, there is an exuberant advancement of bone age, the catch up of bone age being approximately double of the chronological age advancement.

  8. Bortezomib consolidation after autologous stem cell transplantation in multiple myeloma

    DEFF Research Database (Denmark)

    Mellqvist, Ulf-Henrik; Gimsing, Peter; Hjertner, Oyvind

    2013-01-01

    The Nordic Myeloma Study Group conducted an open randomized trial to compare bortezomib as consolidation therapy given after high-dose therapy and autologous stem cell transplantation (ASCT) with no consolidation in bortezomib-naive patients with newly diagnosed multiple myeloma. Overall, 370...

  9. Skeletal muscle and fetal alcohol spectrum disorder.

    Science.gov (United States)

    Myrie, Semone B; Pinder, Mark A

    2018-04-01

    Skeletal muscle is critical for mobility and many metabolic functions integral to survival and long-term health. Alcohol can affect skeletal muscle physiology and metabolism, which will have immediate and long-term consequences on health. While skeletal muscle abnormalities, including morphological, biochemical, and functional impairments, are well-documented in adults that excessively consume alcohol, there is a scarcity of information about the skeletal muscle in the offspring prenatally exposed to alcohol ("prenatal alcohol exposure"; PAE). This minireview examines the available studies addressing skeletal muscle abnormalities due to PAE. Growth restriction, fetal alcohol myopathy, and abnormalities in the neuromuscular system, which contribute to deficits in locomotion, are some direct, immediate consequences of PAE on skeletal muscle morphology and function. Long-term health consequences of PAE-related skeletal abnormalities include impaired glucose metabolism in the skeletal muscle, resulting in glucose intolerance and insulin resistance, leading to an increased risk of type 2 diabetes. In general, there is limited information on the morphological, biochemical, and functional features of skeletal abnormalities in PAE offspring. There is a need to understand how PAE affects muscle growth and function at the cellular level during early development to improve the immediate and long-term health of offspring suffering from PAE.

  10. Autologous stem-cell transplantation in Hodgkin’s lymphoma: analysis of a therapeutic option

    Directory of Open Access Journals (Sweden)

    Adriano de Moraes Arantes

    2011-06-01

    Full Text Available Objective: To report the clinical progress of patients with Hodgkin’slymphoma treated with autologous transplantation after failure orrelapse of first-line treatment with chemotherapy and/or radiationtherapy. Methods: The results of a retrospective analysis of 31patients submitted to autologous transplantation as second-linetreatment, between April 2000 and December 2008, were analyzed.Fourteen men and seventeen women, with a median age of 27 years,were submitted to autologous transplantation for relapsed (n = 21or refractory (n = 10 Hodgkin’s lymphoma. Results: Mortalityrelated to treatment in the first 100 days after transplant was 3.2%.With a mean follow-up period of 18 months (range: 1 to 88 months,the probability of global survival and progression-free survival in18 months was 84 and 80%, respectively. The probability of globalsurvival and progression-free survival at 18 months for patients withchemosensitive relapses (n = 21 was 95 and 90%, respectively,versus 60 and 45% for patients with relapses resistant to chemotherapy(n = 10 (p = 0.001 for global survival; p = 0.003 for progressionfreesurvival. In the multivariate analysis, absence of disease or pretransplant disease < 5 cm were favorable factors for global survival (p= 0.02; RR: 0.072; 95%CI: 0.01-0.85 and progression-free survival (p= 0.01; RR: 0.040; 95%CI: 0.007-0.78. Conclusion: Autologous transplantation of stem-cells is a therapeutic option for Hodgkin’s lymphoma patients after the first relapse. Promising results were observed in patients with a low tumor burden at transplant.

  11. Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

    Directory of Open Access Journals (Sweden)

    Julio Alonso-Padilla

    2015-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease.Genetically engineered parasitic strains are used for high throughput screening (HTS of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6 and was validated against a series of known anti-trypanosomatid drugs.We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

  12. Review of autologous blood transfusion at the Kenyatta National ...

    African Journals Online (AJOL)

    Objective: This study was performed over a three- month period to establish the pattern of autologous blood transfusion with specific focus on age, sex, type of surgery, duration of hospital stay and religious beliefs. Design: Hospital based prospective study. Setting: The study was conducted at the Kenyatta National Hospital ...

  13. Augmentation of the Nasal Dorsum Using the Multistrip Autologous Cartilage Technique.

    Science.gov (United States)

    Liu, Liqiang; Bu, Zhaoyun; Fan, Jincai; Tian, Jia; Gan, Cheng; Yang, Zengjie; Jiao, Hu

    2017-12-01

    Nasal augmentation is a popular modern technique requested by many Asian people. There are two kinds of autologous cartilage used to augment the nose at present: carved as a monobloc or diced into pieces. Each approach has its pros and cons. The authors performed their surgical technique on a group of 28 patients. Twenty of these patients had undergone rhinoplasties performed before referral to our hospital; eight of these patients had undergone a primary rhinoplasty. Bilateral conchal, nasal septum, or rib cartilage was harvested; deep temporal fascia or abdominal muscle fascia to be prepared for packing stripped cartilage was also removed at this time. The cartilage was placed on a plastic cutting board and cut into strips with a transverse section of 1 × 1 mm. Then, these strips were packed and covered by fascia to form the grafts. The median follow-up was 23 months (range, 12 to 48 months). Twenty-two patients were satisfied with their augmented noses. Through examinations, biopsies, and magnetic resonance imaging scans, less resorption was observed with the multistrip autologous cartilage technique. Junctional stepoffs, excessive prominence, and slanting grafts occurred in three patients, two of whom had revisions. Using multistrip autologous cartilage grafts is an easier method to perform and could be another alternative technique for augmentative and reconstructive rhinoplasties.

  14. Functional analysis of SH3 domain containing ring finger 2 during the myogenic differentiation of quail myoblast cells

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    Si Won Kim

    2017-08-01

    Full Text Available Objective Owing to the public availability of complete genome sequences, including avian species, massive bioinformatics analyses may be conducted for computational gene prediction and the identification of gene regulatory networks through various informatics tools. However, to evaluate the biofunctional activity of a predicted target gene, in vivo and in vitro functional genomic analyses should be a prerequisite. Methods Due to a lack of quail genomic sequence information, we first identified the partial genomic structure and sequences of the quail SH3 domain containing ring finger 2 (SH3RF2 gene. Subsequently, SH3RF2 was knocked out using clustered regularly interspaced short palindromic repeat/Cas9 technology and single cell-derived SH3RF2 mutant sublines were established to study the biofunctional activity of SH3RF2 in quail myoblast (QM7 cells during muscle differentiation. Results Through a T7 endonuclease I assay and genotyping analysis, we established an SH3RF2 knockout (KO QM7#4 subline with 61 and 155 nucleotide deletion mutations in SH3RF2. After the induction of myotube differentiation, the expression profiles were analyzed and compared between regular QM7 and SH3RF2 KO QM7#4 cells by global RNA sequencing and bioinformatics analysis. Conclusion We did not detect any statistically significant role of SH3RF2 during myotube differentiation in QM7 myoblast cells. However, additional experiments are necessary to examine the biofunctional activity of SH3RF2 in cell proliferation and muscle growth.

  15. Protein incorporation by isolated amphibian oocytes. VI. Comparison of autologous and xenogeneic vitellogenins

    Energy Technology Data Exchange (ETDEWEB)

    Wallace, R A; Deufel, R A; Misulovin, Z

    1980-01-01

    1. Labeled vitellogenins were isolated from the sera of several amphibians, a turtle, and a pigeon, and were incubated in vitro with oocytes from Xenopus laevis and Rana pipiens. 2. Oocytes from X. laevis sequestered vitellogenin from salamanders, turtle, and pigeon at rates comparable to that for autologous vitellogenin, while anuran vitellogenins were sequestered at somewhat lower rates. 3. Oocytes from R. pipiens sequestered X. laevis vitellogenin at a rate comparable to autologous vitellogenin, while salamander, turtle, and pigeon vitellogenins were sequestered at faster rates. 4. All vitellogenins examined appear to be recognized and incorporated specifically by X. laevis and R. pipiens oocytes.

  16. [Consensus of the Deutsche Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC) on Autologous Fat Grafting].

    Science.gov (United States)

    Giunta, R E; Horch, R E; Prantl, L; Baur, E M; Herold, C; Kamolz, L; Lehnhardt, M; Noah, E M; Rennekampff, O; Richter, D; Schaefer, D J; Ueberreiter, K

    2016-12-01

    On occasion of the Munich Plastic Symposium in Munich the board of the Deutsche Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC) together with a group of experts who were also involved in the preparation of the recently published S2K guideline "Autologous Fat Grafting", prepared a consensus statement from a plastic-surgical point of view so to evaluate current spects and taking into account the current legal framework: 1. Autologous Fat Grafting is a long established treatment in plastic surgery and does not differ from other tissue grafts. 2. Mechanical processing of autologous fat does not provide any substantial change tot he tissue. 3. If other treatment methods to enrich progenitor cells of autolous fat i. e. by an enzymatic process have evidence that autologous adipose tissue or cells were substantially changed, classification as a drug could come in question under current german law (application of AMG/ATMP). © Georg Thieme Verlag KG Stuttgart · New York.

  17. Immediate breast reconstruction using autologous skin graft associated with breast implant.

    Science.gov (United States)

    Dutra, A K; Andrade, W P; Carvalho, S M T; Makdissi, F B A; Yoshimatsu, E K; Domingues, M C; Maciel, M S

    2012-02-01

    Immediate breast reconstruction with skin graft is still little mentioned in the literature. Follow-up studies regarding the technique aspects are particularly scarce. The objective was to detail immediate breast reconstruction using autologous skin graft. Patients (n = 49) who underwent mastectomies and autologous immediate breast reconstruction with skin graft associated with a breast implant at A. C. Camargo Hospital (São Paulo, Brazil) between January 2007 and July 2010 were included. Information on clinical data, technique details and clinical outcome were prospectively collected. Following mastectomy, the autologous full-thickness skin graft was obtained through an inframammary fold incision along the contralateral breast in most patients. The skin graft was placed on the surface of the pectoralis major muscle after adjustments to conform to the mastectomy defect. A minimum of 10-month follow-up period was established. Patients' age ranged from 35 to 55 years and all received a silicone gel textured surface implant to obtain the necessary breast mound. The mean surgical time was 45 min, and the mean amount of skin resection was 4.5 cm in the largest diameter. Follow-up ranged from 10 to 35 months (median 23). All patients had silicone-gel textured surface implants to perform the breast mound reconstruction. No complications were observed in 87.8% of reconstructions. Forty-six patients (94%) had no complaints about the donor-site aesthetics. The result was a breast mound with a central ellipse of healed skin graft. Three (6%) poor results were observed. Thirty-six patients (67%) reported the results as good or very good. Our results lead us to conclude that autologous skin graft provided a reliable option in immediate breast reconstruction to skin-sparing mastectomy defects. The technique accomplished a single-stage implant breast reconstruction when there is inadequate skin coverage. Copyright © 2011 British Association of Plastic, Reconstructive and

  18. Improving oocyte quality by transfer of autologous mitochondria from fully grown oocytes

    DEFF Research Database (Denmark)

    Kristensen, Stine Gry; Pors, Susanne Elisabeth; Andersen, Claus Yding

    2017-01-01

    options using autologous mitochondria to potentially augment pregnancy potential in ART. Autologous transfer of mitochondria from the patient's own germline cells has attracted much attention as a possible new treatment to revitalize deficient oocytes. IVF births have been reported after transfer...... of oogonial precursor cell-derived mitochondria; however, the source and quality of the mitochondria are still unclear. In contrast, fully grown oocytes are loaded with mitochondria which have passed the genetic bottleneck and are likely to be of high quality. An increased supply of such oocytes could...... with high quality mitochondria can be obtained from natural or stimulated ovaries and potentially be used to improve both quality and quantity of oocytes available for fertility treatment....

  19. Lysis of fresh human solid tumors by autologous lymphocytes activated in vitro with lectins

    International Nuclear Information System (INIS)

    Mazumder, A.; Grimm, E.A.; Zhang, H.Z.; Rosenberg, S.A.

    1982-01-01

    Human peripheral blood lymphocytes (PBL), obtained from patients with a variety of cancers, were incubated in vitro with phytohemagglutinin, concanavalin A, and crude or lectin-free T-cell growth factors. The lectin-activated PBL of nine patients were capable of lysing fresh autologous tumor during a 4-hr 51Cr release assay. Multiple metastases from the same patient were equivalently lysed by these activated autologous PBL. No lysis of fresh PBL or lectin-induced lymphoblast cell targets was seen, although tumor, PBL, and lymphoblast cells were shown to be equally lysable using allosensitized cells. The activated cells could be expanded without loss of cytotoxicity in crude or lectin-free T-cell growth factors. The generation of cells lytic to fresh autologous tumor was dependent on the presence of adherent cells, although the lytic cell itself was not adherent. Proliferation was not involved in the induction of lytic cells since equal lysis was induced in irradiated and nonirradiated lymphocytes. Lectin was not required in the lytic assay, and the addition of alpha-methyl-D-mannoside to concanavalin A-activated lymphoid cells did not increase the lysis of fresh tumor cells. Activation by lectin for 3 days appears to be an efficient and convenient method for generating human cells lytic to fresh autologous tumor. These lytic cells may be of value for studies of the cell-mediated lysis of human tumor and possibly for tumor immunotherapy as well

  20. THE PORTION OF AUTOLOGOUS BLOOD IN ORTHOPEDIC DEPARTMENT OF GENERAL HOSPITAL NOVA GORICA IN FIVE YEARS PERIOD (1996–2000

    Directory of Open Access Journals (Sweden)

    Janka Černe-Zavadlav

    2002-04-01

    Full Text Available Background. The presentation of autologous blood donation with analysis of used blood and the percentage of autologous blood on Orthopedic Department in the years 1996–2000.Methods. From card-index of autologous blood donors we analysed 363 preoperative autologous blood donations.We followed the number of doses in one patient and type of operating procedure.We analysed the usage of blood from transfusion issue protocols and the usage of postoperative autotransfusion from patient protocols.Results. 91% of all preoperative blood donations in our hospital in five years period were from Orthopedic Department. There were 280 operating procedures (hip and knee arthroplasty that needed blood transfusion. 196 of these (70% were included in preoperative blood donation programme. We collected 330 doses: 1 dose in 61 cases, 2 doses in 100, 3 doses in 34 and 4 doses in 1 case or 1.68 doses per patient. The relationship between used autologous and allogenic blood were 46 : 54 (doses or 38 : 62 (mL. Autologous blood represented 38% of all used blood on Orthopedic Department, only 11% of autologous blood were discarded.Conclusions. The program of preoperative blood donation is well organized on The Orthopedic Department of our Hospital. To our experience we make the most of profit of autotransfusion (to avoid risks of allogenic blood, optimal patient colaboration, low percentage of discarded blood with two predonated doses in combination with postoperative autotransfusion. Regard to The Law of Blood supply (may 2000 we are going to introduce this protocol of preoperative blood donation for all programed operating procedures in our Hospital, which need blood transfusion.

  1. Human skeletal muscle releases leptin in vivo

    DEFF Research Database (Denmark)

    Wolsk, Emil; Grøndahl, Thomas Sahl; Pedersen, Bente Klarlund

    2012-01-01

    Leptin is considered an adipokine, however, cultured myocytes have also been found to release leptin. Therefore, as proof-of-concept we investigated if human skeletal muscle synthesized leptin by measuring leptin in skeletal muscle biopsies. Following this, we quantified human skeletal muscle...... was unaltered. During saline infusion the adipose tissue release averaged 0.8 ± 0.3 ng min(-1) 100g tissue(-1) whereas skeletal muscle release was 0.5 ± 0.1 ng min(-1) 100g tissue(-1). In young healthy humans, skeletal muscle contribution to whole body leptin production could be substantial given the greater...

  2. Treatment of articular cartilage lesions of the knee by microfracture or autologous chondrocyte implantation: a systematic review.

    Science.gov (United States)

    Oussedik, Sam; Tsitskaris, Konstantinos; Parker, David

    2015-04-01

    We performed a systematic review of the treatment of articular cartilage lesions of the knee by microfracture or autologous chondrocyte implantation to determine the differences in patient outcomes after these procedures. We searched PubMed/Medline, Embase, and The Cochrane Library databases in the period from January 10 through January 20, 2013, and included 34 articles in our qualitative analysis. All studies showed improvement in outcome scores in comparison with baseline values, regardless of the treatment modality. The heterogeneity of the results presented in the studies precluded a meta-analysis. Microfracture appears to be effective in smaller lesions and is usually associated with a greater proportion of fibrocartilage production, which may have an effect on durability and eventual failure. Autologous chondrocyte implantation is an effective treatment that may result in a greater proportion of hyaline-like tissue at the repair site, which may in turn have a beneficial effect on durability and failure; it appears to be effective in larger lesions. Autologous chondrocyte implantation with periosteum has been shown to be associated with symptomatic cartilage hypertrophy more frequently than autologous chondrocyte implantation with collagen membrane. Matrix-associated autologous chondrocyte implantation is technically less challenging than the other techniques available, and in lesions greater than 4 cm(2), it has been shown to be more effective than microfracture. Level IV, systematic review of Level I-IV studies. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

  3. AMPK in skeletal muscle function and metabolism

    DEFF Research Database (Denmark)

    Kjøbsted, Rasmus; Hingst, Janne Rasmuss; Fentz, Joachim

    2018-01-01

    Skeletal muscle possesses a remarkable ability to adapt to various physiologic conditions. AMPK is a sensor of intracellular energy status that maintains energy stores by fine-tuning anabolic and catabolic pathways. AMPK's role as an energy sensor is particularly critical in tissues displaying...... highly changeable energy turnover. Due to the drastic changes in energy demand that occur between the resting and exercising state, skeletal muscle is one such tissue. Here, we review the complex regulation of AMPK in skeletal muscle and its consequences on metabolism (e.g., substrate uptake, oxidation......, and storage as well as mitochondrial function of skeletal muscle fibers). We focus on the role of AMPK in skeletal muscle during exercise and in exercise recovery. We also address adaptations to exercise training, including skeletal muscle plasticity, highlighting novel concepts and future perspectives...

  4. Evolution of Autologous Chondrocyte Repair and Comparison to Other Cartilage Repair Techniques

    Directory of Open Access Journals (Sweden)

    Ashvin K. Dewan

    2014-01-01

    Full Text Available Articular cartilage defects have been addressed using microfracture, abrasion chondroplasty, or osteochondral grafting, but these strategies do not generate tissue that adequately recapitulates native cartilage. During the past 25 years, promising new strategies using assorted scaffolds and cell sources to induce chondrocyte expansion have emerged. We reviewed the evolution of autologous chondrocyte implantation and compared it to other cartilage repair techniques. Methods. We searched PubMed from 1949 to 2014 for the keywords “autologous chondrocyte implantation” (ACI and “cartilage repair” in clinical trials, meta-analyses, and review articles. We analyzed these articles, their bibliographies, our experience, and cartilage regeneration textbooks. Results. Microfracture, abrasion chondroplasty, osteochondral grafting, ACI, and autologous matrix-induced chondrogenesis are distinguishable by cell source (including chondrocytes and stem cells and associated scaffolds (natural or synthetic, hydrogels or membranes. ACI seems to be as good as, if not better than, microfracture for repairing large chondral defects in a young patient’s knee as evaluated by multiple clinical indices and the quality of regenerated tissue. Conclusion. Although there is not enough evidence to determine the best repair technique, ACI is the most established cell-based treatment for full-thickness chondral defects in young patients.

  5. Autologous hematopoietic stem cell transplantation in classical Hodgkin's lymphoma

    Directory of Open Access Journals (Sweden)

    Afonso José Pereira Cortez

    2011-02-01

    Full Text Available BACKGROUND: Hodgkin's lymphoma has high rates of cure, but in 15% to 20% of general patients and between 35% and 40% of those in advanced stages, the disease will progress or will relapse after initial treatment. For this group, hematopoietic stem cell transplantation is considered one option of salvage therapy. OBJECTIVES: To evaluate a group of 106 patients with Hodgkin's lymphoma, who suffered relapse or who were refractory to treatment, submitted to autologous hematopoietic stem cell transplantation in a single transplant center. METHODS: A retrospective study was performed with data collected from patient charts. The analysis involved 106 classical Hodgkin's lymphoma patients who were consecutively submitted to high-dose chemotherapy followed by autologous transplants in a single institution from April 1993 to December 2006. RESULTS: The overall survival rates of this population at five and ten years were 86% and 70%, respectively. The disease-free survival was approximately 60% at five years. Four patients died of procedure-related causes but relapse of classical Hodgkin's lymphoma after transplant was the most frequent cause of death. Univariate analysis shows that sensitivity to pre-transplant treatment and hemoglobin < 10 g/dL at diagnosis had an impact on patient survival. Unlike other studies, B-type symptoms did not seem to affect overall survival. Lactic dehydrogenase and serum albumin concentrations analyzed at diagnosis did not influence patient survival either. CONCLUSION: Autologous hematopoietic stem cell transplantation is an effective treatment strategy for early and late relapse in classical Hodgkin's lymphoma for cases that were responsive to pre-transplant chemotherapy. Refractory to treatment is a sign of worse prognosis. Additionally, a hemoglobin concentration below 10 g/dL at diagnosis of Hodgkin's lymphoma has a negative impact on the survival of patients after transplant. As far as we know this relationship has not

  6. Autologous Blood Pleurodesis In Patients With Persistent Air Leaks

    Directory of Open Access Journals (Sweden)

    Agkajanzadeh M

    2003-10-01

    Full Text Available Persistent air leaks occur after Spontaneous pneumothorax both primary and secondary, and after lungs trauma and lung surgeries are sever problems encountered chest surgeons with. Persistent air leak causes longer patients hospitalization."nMaterials and Methods: We used autologous blood pleurodesis in patients with persistent air leak for 30patients with more than 8 days air leaks, during a three years period 1377-1380 (1999-2002."nResults: The patients had 19 years up to 70 years old. Eight patients had thoracotomy and lobectomy and /or segmentectomies 6 with primary pneumothorax, 10 with secondary pneumothorax, and four with penetrated or blunt thoracic traumas. Blood was obtained from femoral or brachial veins and 70-150 mis. Injected in chest tubes. Chest bottle was first lied 80cm higher than body levels. After 24 hours repositioned in normal levels, and patients were supervised. Via chest tube we injected blood 70-100ml.for young patients, and 100-150 ml for older patients into intra pleural space. There were no clamped chest tubes. There were no pain, respiratory distress, fever, or cough in pleurodesized patients. The only patient's complaint was local pain in femoral vein or brachial vein because blood sampling and blood obtaining, although there was no local visible complication as hematoma or bleeding. After 48 hours in 24 patients air leak ceased. In six patients because persistent air leak autologous blood pleurodesis repeated, two patients after 48hours"nair leak ceased, remaining four patients underwent for thoracotomies, success rate"nwas 86.6%."nConclusion: According above success rate we suggest autologous blood pleurodesis in patients with persistent air leak is a reliable, effective, and no complicated procedure for persistent air leaks.

  7. Autologous proliferative therapies in recalcitrant lateral epicondylitis.

    Science.gov (United States)

    Tetschke, Elisa; Rudolf, Margit; Lohmann, Christoph H; Stärke, Christian

    2015-09-01

    This study investigates the clinical effects of autologous conditioned plasma (ACP) injections and low-level laser application as therapy options for chronic lateral epicondylitis. A total of 52 patients with chronic lateral epicondylitis were evaluated in this study; 26 of these patients received three ACP injections and the control group, with 26 patients, received 12 laser applications, with standardized physical therapy for all patients afterward. Control examinations took place before treatment, after 2 and 6 mos, and in the 1 yr final follow-up. The control examination included the visual analog scale for pain and Disabilities of the Arm, Shoulder and Hand outcome measure scores. The analysis at final follow-up after 1 yr showed that both treatment options resulted in successful therapy outcome for the patients. In total, 63.5 % were successfully treated. Successful treatment was defined as more than 30% improvement in the visual analog score and more than 10.2 points in the Disabilities of the Arm, Shoulder and Hand score. Both groups showed a significant improvement in time response. This study demonstrates the beneficial effects of autologous proliferative therapies in the treatment of lateral epicondylitis. The data show that laser application and ACP therapy lead to a clinical improvement in epicondylopathia. Especially the new treatment with ACP can be highlighted as an alternative and as an easy-to-apply therapy option for clinical practice.

  8. Cost-effectiveness analysis of implants versus autologous perforator flaps using the BREAST-Q.

    Science.gov (United States)

    Matros, Evan; Albornoz, Claudia R; Razdan, Shantanu N; Mehrara, Babak J; Macadam, Sheina A; Ro, Teresa; McCarthy, Colleen M; Disa, Joseph J; Cordeiro, Peter G; Pusic, Andrea L

    2015-04-01

    Reimbursement has been recognized as a physician barrier to autologous reconstruction. Autologous reconstructions are more expensive than prosthetic reconstructions, but provide greater health-related quality of life. The authors' hypothesis is that autologous tissue reconstructions are cost-effective compared with prosthetic techniques when considering health-related quality of life and patient satisfaction. A cost-effectiveness analysis from the payer perspective, including patient input, was performed for unilateral and bilateral reconstructions with deep inferior epigastric perforator (DIEP) flaps and implants. The effectiveness measure was derived using the BREAST-Q and interpreted as the cost for obtaining 1 year of perfect breast health-related quality-adjusted life-year. Costs were obtained from the 2010 Nationwide Inpatient Sample. The incremental cost-effectiveness ratio was generated. A sensitivity analysis for age and stage at diagnosis was performed. BREAST-Q scores from 309 patients with implants and 217 DIEP flap reconstructions were included. The additional cost for obtaining 1 year of perfect breast-related health for a unilateral DIEP flap compared with implant reconstruction was $11,941. For bilateral DIEP flaps compared with implant reconstructions, the cost for an additional breast health-related quality-adjusted life-year was $28,017. The sensitivity analysis demonstrated that the cost for an additional breast health-related quality-adjusted life-year for DIEP flaps compared with implants was less for younger patients and earlier stage breast cancer. DIEP flaps are cost-effective compared with implants, especially for unilateral reconstructions. Cost-effectiveness of autologous techniques is maximized in women with longer life expectancy. Patient-reported outcomes findings can be incorporated into cost-effectiveness analyses to demonstrate the relative value of reconstructive procedures.

  9. Enrichment of autologous fat grafts with ex-vivo expanded adipose tissue-derived stem cells for graft survival

    DEFF Research Database (Denmark)

    Kølle, Stig-Frederik Trojahn; Fischer-Nielsen, Anne; Mathiasen, Anders Bruun

    2013-01-01

    Autologous fat grafting is increasingly used in reconstructive surgery. However, resorption rates ranging from 25% to 80% have been reported. Therefore, methods to increase graft viability are needed. Here, we report the results of a triple-blind, placebo-controlled trial to compare the survival ...... of fat grafts enriched with autologous adipose-derived stem cells (ASCs) versus non-enriched fat grafts....

  10. Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity.

    Science.gov (United States)

    Chaillou, Thomas; Lanner, Johanna T

    2016-12-01

    Reduced oxygen (O 2 ) levels (hypoxia) are present during embryogenesis and exposure to altitude and in pathologic conditions. During embryogenesis, myogenic progenitor cells reside in a hypoxic microenvironment, which may regulate their activity. Satellite cells are myogenic progenitor cells localized in a local environment, suggesting that the O 2 level could affect their activity during muscle regeneration. In this review, we present the idea that O 2 levels regulate myogenesis and muscle regeneration, we elucidate the molecular mechanisms underlying myogenesis and muscle regeneration in hypoxia and depict therapeutic strategies using changes in O 2 levels to promote muscle regeneration. Severe hypoxia (≤1% O 2 ) appears detrimental for myogenic differentiation in vitro, whereas a 3-6% O 2 level could promote myogenesis. Hypoxia impairs the regenerative capacity of injured muscles. Although it remains to be explored, hypoxia may contribute to the muscle damage observed in patients with pathologies associated with hypoxia (chronic obstructive pulmonary disease, and peripheral arterial disease). Hypoxia affects satellite cell activity and myogenesis through mechanisms dependent and independent of hypoxia-inducible factor-1α. Finally, hyperbaric oxygen therapy and transplantation of hypoxia-conditioned myoblasts are beneficial procedures to enhance muscle regeneration in animals. These therapies may be clinically relevant to treatment of patients with severe muscle damage.-Chaillou, T. Lanner, J. T. Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity. © FASEB.

  11. Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation

    Directory of Open Access Journals (Sweden)

    Elena Barbieri

    2011-01-01

    Full Text Available This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1 , NRF1 , and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD, a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1 involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

  12. Skeletal Muscle Na+ Channel Disorders

    Directory of Open Access Journals (Sweden)

    Dina eSimkin

    2011-10-01

    Full Text Available Five inherited human disorders affecting skeletal muscle contraction have been traced to mutations in the gene encoding the voltage-gated sodium channel Nav1.4. The main symptoms of these disorders are myotonia or periodic paralysis caused by changes in skeletal muscle fiber excitability. Symptoms of these disorders vary from mild or latent disease to incapacitating or even death in severe cases. As new human sodium channel mutations corresponding to disease states become discovered, the importance of understanding the role of the sodium channel in skeletal muscle function and disease state grows.

  13. Autologous Fat Transfer for Thumb Carpometacarpal Joint Osteoarthritis: A Prospective Study.

    Science.gov (United States)

    Herold, Christian; Rennekampff, Hans-Oliver; Groddeck, Robert; Allert, Sixtus

    2017-08-01

    Most operations for carpometacarpal joint osteoarthritis of the thumb irreversibly alter or destroy the anatomy. There is a high demand for minimally invasive alternatives. The authors report the results of autologous fat transfer for treatment of thumb carpometacarpal joint osteoarthritis. In a prospective study, 50 patients with thumb carpometacarpal joint osteoarthritis were observed for 1 year after autologous fat transfer. Manual liposuction and centrifugation were performed. Pain rating according to visual analogue pain scale; objective force of pinch grip and fist closure; and Disabilities of the Arm, Shoulder, and Hand questionnaire scores before and after treatment were analyzed. The average pain in stage 2 patients preoperatively was 7.7 ± 1.3; it was 1.8 ± 1.9 after 6 months and 2.4 ± 3.1 after 12 months. Patients with stage 2 osteoarthritis demonstrated a superior benefit from this treatment compared with patients with either stage 3 or stage 4 thumb carpometacarpal joint osteoarthritis. There were similar improvements for the parameters strength and Disabilities of the Arm, Shoulder, and Hand questionnaire score. No serious adverse events were observed. Autologous fat transplantation is an appealing alternative, especially in early-stage basal joint osteoarthritis of the thumb. The low invasiveness of the procedure and early recovery of patients compared with classical procedures such as trapeziectomy, and the superior long-term results compared with classical injection therapy, make this approach feasible as a first-line therapy in early-stage basal joint osteoarthritis of the thumb. Therapeutic, IV.

  14. Effect of BCNU combined with total body irradiation or cyclophosphamide on survival of dogs after autologous marrow grafts

    International Nuclear Information System (INIS)

    Paterson, A.H.G.; English, D.

    1979-01-01

    Dogs were treated with either: (1) 750 rad total body irradiation; (2) BCNU 2 or 4 mg/kg IV 48 hours prior to 750 rad total body irradiation; or (3) BCNU 4 mg/kg IV plus cyclophosphamide 30 mg/kg IV. Results showed that of 11 dogs who received 750 rad total body irradiation and did not receive cryopreserved autologous bone marrow cells, none survived, compared to an 88% survival (31 of 35 dogs) after 750 rad total body irradiation if the dogs received stored autologous bone marrow cells. However, when the dogs were treated with BCNU 2 or 4 mg/kg prior to 750 rad total body irradiation the survival rate, despite infusion of autologous bone marrow cells, dropped to 25% (3 of 12 dogs) for BCNU 2 mg/kg, and 17% (2 of 12 dogs) for BCNU 4 mg/kg. This effect did not seem to be due to direct serum inhibition of hemopoietic cell proliferation since serum obtained at various intervals after BCNU administrations failed to inhibit CFU growth in vitro. The dogs died from hemorrhage and infection; at autopsy there was hemorrhagic pneumonitis and intestinal ulcerations with petechial hemorrhages, suggesting that the combination of BCNU and total body irradiation may have synergistic toxicity on the canine gastro-intestinal tract. When BCNU was combined with cyclophosphamide, reversal of marrow toxicity occurred in 54% (6 of 11 dogs) with stored autologous bone marrow cells compared to no survival (0 of 8 dogs) with stored autologous bone marrow cells. Thus while autologous bone marrow grafts are useful for reversal of marrow toxicity due to many therapeutic protocols, such grafts alone may not provide protection against toxicity due to the combination of high dosage BCNU and total body irradiation

  15. Intra-arterial Autologous Bone Marrow Cell Transplantation in a Patient with Upper-extremity Critical Limb Ischemia

    International Nuclear Information System (INIS)

    Madaric, Juraj; Klepanec, Andrej; Mistrik, Martin; Altaner, Cestmir; Vulev, Ivan

    2013-01-01

    Induction of therapeutic angiogenesis by autologous bone marrow mononuclear cell transplantation has been identified as a potential new option in patients with advanced lower-limb ischemia. There is little evidence of the benefit of intra-arterial cell application in upper-limb critical ischemia. We describe a patient with upper-extremity critical limb ischemia with digital gangrene resulting from hypothenar hammer syndrome successfully treated by intra-arterial autologous bone marrow mononuclear cell transplantation.

  16. High dose therapy with autologous stem cell support in malignant disorders

    International Nuclear Information System (INIS)

    Holte, H.; Kvaloey, S.O.; Engan, T.

    1996-01-01

    New biomedical knowledge may improve the diagnostic procedures and treatment provided by the Health Services, but at additional cost. In a social democratic health care system, the hospital budgets have no room for expensive, new procedures or treatments, unless these are funded through extra allocation from the central authorities. High dose therapy with autologous stem cell support in malignant disorders is an example of a new and promising, but rather expensive treatment, but its role in cancer therapy has yet to be established. The indications for testing high dose therapy with autologous stem cell support in various malignancies are discussed, with emphasis on the principles for deciding which categories of disease should have priority. The authors suggest some malignant disorder for which high dose therapy with stem cell support should be explored versus conventional treatment in randomized prospective trials. 8 refs., 1 tab

  17. Use of autologous platelet - Rich plasma in the treatment of intrabony defects

    Directory of Open Access Journals (Sweden)

    Sharath K Shetty

    2009-01-01

    Treatment of intrabony defects by autologous PRP gel alone caused significant soft tissue clinical improvement as well as hard tissue defect fill as evidenced by SSD view in spiral computed tomography.

  18. Redox Control of Skeletal Muscle Regeneration.

    Science.gov (United States)

    Le Moal, Emmeran; Pialoux, Vincent; Juban, Gaëtan; Groussard, Carole; Zouhal, Hassane; Chazaud, Bénédicte; Mounier, Rémi

    2017-08-10

    Skeletal muscle shows high plasticity in response to external demand. Moreover, adult skeletal muscle is capable of complete regeneration after injury, due to the properties of muscle stem cells (MuSCs), the satellite cells, which follow a tightly regulated myogenic program to generate both new myofibers and new MuSCs for further needs. Although reactive oxygen species (ROS) and reactive nitrogen species (RNS) have long been associated with skeletal muscle physiology, their implication in the cell and molecular processes at work during muscle regeneration is more recent. This review focuses on redox regulation during skeletal muscle regeneration. An overview of the basics of ROS/RNS and antioxidant chemistry and biology occurring in skeletal muscle is first provided. Then, the comprehensive knowledge on redox regulation of MuSCs and their surrounding cell partners (macrophages, endothelial cells) during skeletal muscle regeneration is presented in normal muscle and in specific physiological (exercise-induced muscle damage, aging) and pathological (muscular dystrophies) contexts. Recent advances in the comprehension of these processes has led to the development of therapeutic assays using antioxidant supplementation, which result in inconsistent efficiency, underlying the need for new tools that are aimed at precisely deciphering and targeting ROS networks. This review should provide an overall insight of the redox regulation of skeletal muscle regeneration while highlighting the limits of the use of nonspecific antioxidants to improve muscle function. Antioxid. Redox Signal. 27, 276-310.

  19. The Benefit of Microskin in Combination With Autologous Keratinocyte Suspension to Treat Full Skin Loss In Vivo.

    Science.gov (United States)

    Yuru, Shang; Dawei, Li; Chuanan, Shen; Kai, Yin; Li, Ma; Longzhu, Li; Dongxu, Zhao; Wenfeng, Cheng

    Patients with extensive deep burns often lack enough autologous skin to cover the wounds. This study explores a new method using microskin in combination with autologous keratinocytes in the treatment of extensive deep burn. Wounds in the combination group were treated with automicroskin at an area expansion ratio of 20:1 (wound area to automicroskin area) and autologous keratinocyte suspension, which were compared with the following treatments: no autotransplant, only allografts (control group); autologous keratinocyte suspension only (keratinocyte only group); automicroskin at an area expansion ratio of 20:1 (20:1 group); and automicroskin at an area expansion ratio of 10:1 (10:1 group, positive control). The authors used epithelialization rate (epithelialized area on day 21 divided by original wound area), hematoxylin and eosin staining, laminin, and type IV collagen immunohistochemistry to assess wound healing. The epithelialization rate of combination group (74.2% ± 8.0%) was similar to that of 10: 1 group (84.3% ± 11.9%, P = .085) and significantly (P < .05) higher than that of 20:1 group (59.2% ± 10.8%), keratinocyte only group (53.8% ± 11.5%), and control group (22.7% ± 5.5%). The hematoxylin and eosin staining and immunohistochemistry showed the epithelialization in the combination group was better than that in the keratinocyte only group and control group. Microskin in combination with autologous keratinocyte suspension can promote the reepithelialization of full-thickness wounds and reduce the requirements for automircoskin, and it is a useful option in the treatment of extensive deep burns.

  20. Determinants of the Use of Autologous Blood in Elective General ...

    African Journals Online (AJOL)

    Objective: The study reports the 7 year experience of the authors with autologous blood transfusion in elective general surgery using the predeposit method. Material and Method: Patients aged 18 years and older, presenting for elective surgery and for whom blood donation was required were encouraged to predonate one ...

  1. Is there evidence that barrier membranes prevent bone resorption in autologous bone grafts during the healing period? A systematic review

    NARCIS (Netherlands)

    Gielkens, Pepijn F. M.; Bos, Ruud R. M.; Raghoebar, Gerry M.; Stegenga, Boudewijn

    2007-01-01

    Introduction: Autologous bone is considered the "reference standard" for bone-grafting procedures. A barrier membrane covering an autologous bone graft (guided bone regeneration [GBR]) is expected to prevent graft resorption. Good clinical results have been reported for GBR, although potential

  2. A prospective randomized trial comparing homologous and autologous fibrin sealants for the control of alveolar air leak.

    Science.gov (United States)

    Kılıç, Burcu; Erşen, Ezel; Demirkaya, Ahmet; Kara, H Volkan; Alizade, Nurlan; İşcan, Mehlika; Kaynak, Kamil; Turna, Akif

    2017-09-01

    Postoperative air leak is a common complication seen after pulmonary resection. It is a significant reason of morbidity and also leads to greater hospital cost owing to prolonged length of stay. The purpose of this study is to compare homologous sealant with autologous one to prevent air leak following pulmonary resection. A total of 57 patients aged between 20 and 79 (mean age: 54.36) who underwent pulmonary resection other than pneumonectomy (lobar or sublobar resections) were analyzed. There were 47 males (83%) and 10 females (17%). Patients who intraoperatively had air leaks were randomized to receive homologous (Tisseel; n=28) or autologous (Vivostat; n=29) fibrin sealant. Differences among groups in terms of air leak, prolonged air leak, hospital stay, amount of air leak were analyzed. Indications for surgery were primary lung cancer in 42 patients (71.9%), secondary malignancy in 5 patients (8.8%), and benign disease in 10 patients (17.5%). Lobectomy was performed in 40 patients (70.2%), whereas 17 patients (29.8%) had wedge resection. Thirteen (46.4%) patients developed complications in patients receiving homologous sealant while 11 (38.0%) patients had complication in autologous sealant group (P=0.711). Median duration of air leak was 3 days in two groups. Time to intercostal drain removal was 3.39 and 3.38 days in homologous and autologous sealant group respectively (P=0.978). Mean hospital stay was 5.5 days in patients receiving homologous sealant whereas it was 5.0 days in patients who had autologous agent (P=0.140). There were no significant differences between groups in terms of measured maximum air leak (P=0.823) and mean air leak (P=0.186). There was no significant difference in the incidence of complications between two groups (P=0.711). Autologous and heterologous fibrin sealants are safe and acts similarly in terms of air leak and hospital stay in patients who had resectional surgery.

  3. Autologous osteochondral mosaicplasty or TruFit plugs for cartilage repair.

    Science.gov (United States)

    Hindle, Paul; Hendry, Jane L; Keating, John F; Biant, Leela C

    2014-06-01

    Autologous osteochondral mosaicplasty and TruFit Bone graft substitute plugs are methods used to repair symptomatic articular cartilage defects in the adult knee. There have been no comparative studies of the two techniques. This retrospective study assessed functional outcome of patients using the EQ-5D, Knee Injury and Osteoarthritis Outcome Score (KOOS) and Modified Cincinnati scores at follow-up of 1-5 years. There were 66 patients in the study (35 TruFit and 31 Mosaicplasty): 44 males and 22 females with a mean age of 37.3 years (SD 12.6). The mean BMI was 26.8. Thirty-six articular cartilage lesions were due to trauma, twenty-six due to osteochondritis dissecans and three due to non-specific degenerative change or unknown. There was no difference between the two groups age (n.s.), sex (n.s.), BMI (n.s.), defect location (n.s.) or aetiology (n.s.). The median follow-up was 22 months for the TruFit cohort and 30 months for the mosaicplasty group. There was no significant difference in the requirement for re-operation (n.s). Patients undergoing autologous mosaicplasty had a higher rate of returning to sport (p = 0.006), lower EQ-5D pain scores (p = 0.048) and higher KOOS activities of daily living (p = 0.029) scores. Sub-group analysis showed no difference related to the number of cases the surgeon performed. Patients requiring re-operation had lower outcome scores regardless of their initial procedure. This study demonstrated significantly better outcomes using two validated outcome scores (KOOS, EQ-5D), and an ability to return to sport in those undergoing autologous mosaicplasty compared to those receiving TruFit plugs. IV.

  4. Cerebellar medulloblastoma presenting with skeletal metastasis

    Directory of Open Access Journals (Sweden)

    Barai Sukanta

    2004-04-01

    Full Text Available Medulloblastomas are highly malignant brain tumours, but only rarely produce skeletal metastases. No case of medulloblastoma has been documented to have produced skeletal metastases prior to craniotomy or shunt surgery. A 21-year-old male presented with pain in the hip and lower back with difficulty in walking of 3 months′ duration. Signs of cerebellar dysfunction were present hence a diagnosis of cerebellar neoplasm or skeletal tuberculosis with cerebellar abscess formation was considered. MRI of brain revealed a lesion in the cerebellum suggestive of medulloblastoma. Bone scan revealed multiple sites of skeletal metastases excluding the lumbar vertebrae. MRI of lumbar spine and hip revealed metastases to all lumbar vertebrae and both hips. Computed tomography-guided biopsy was obtained from the L3 vertebra, which revealed metastatic deposits from medulloblastoma. Cerebrospinal fluid cytology showed the presence of medulloblastoma cells. A final diagnosis of cerebellar medulloblastoma with skeletal metastases was made. He underwent craniotomy and histopathology confirmed medulloblastoma.

  5. Successful autologous hematopoietic stem cell transplantation for a patient with rapidly progressive localized scleroderma.

    Science.gov (United States)

    Nair, Velu; Sharma, Ajay; Sharma, Sanjeevan; Das, Satyaranjan; Bhakuni, Darshan S; Narayanan, Krishnan; Nair, Vivek; Shankar, Subramanian

    2015-03-01

    Autologous hematopoietic stem cell transplant (HSCT) for rapidly progressive disease has not been reported in localized scleroderma. Our patient, a 16-year-old girl had an aggressive variant of localized scleroderma, mixed subtype (linear-generalized) with Parry Romberg syndrome, with no internal organ involvement, that was unresponsive to immunosuppressive therapy and was causing rapid disfigurement. She was administered autologous HSCT in June 2011 and has maintained drug-free remission with excellent functional status at almost 3.5 years of follow-up. © 2015 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  6. Manufacturing models permitting roll out/scale out of clinically led autologous cell therapies: regulatory and scientific challenges for comparability.

    Science.gov (United States)

    Hourd, Paul; Ginty, Patrick; Chandra, Amit; Williams, David J

    2014-08-01

    Manufacturing of more-than-minimally manipulated autologous cell therapies presents a number of unique challenges driven by complex supply logistics and the need to scale out production to multiple manufacturing sites or near the patient within hospital settings. The existing regulatory structure in Europe and the United States imposes a requirement to establish and maintain comparability between sites. Under a single market authorization, this is likely to become an unsurmountable burden beyond two or three sites. Unless alternative manufacturing approaches can be found to bridge the regulatory challenge of comparability, realizing a sustainable and investable business model for affordable autologous cell therapy supply is likely to be extremely demanding. Without a proactive approach by the regulators to close this "translational gap," these products may not progress down the development pipeline, threatening patient accessibility to an increasing number of clinician-led autologous cellular therapies that are already demonstrating patient benefits. We propose three prospective manufacturing models for the scale out/roll out of more-than-minimally manipulated clinically led autologous cell therapy products and test their prospects for addressing the challenge of product comparability with a selected expert reference panel of US and UK thought leaders. This paper presents the perspectives and insights of the panel and identifies where operational, technological and scientific improvements should be prioritized. The main purpose of this report is to solicit feedback and seek input from key stakeholders active in the field of autologous cell therapy in establishing a consensus-based manufacturing approach that may permit the roll out of clinically led autologous cell therapies. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. GRAF1 deficiency blunts sarcolemmal injury repair and exacerbates cardiac and skeletal muscle pathology in dystrophin-deficient mice.

    Science.gov (United States)

    Lenhart, Kaitlin C; O'Neill, Thomas J; Cheng, Zhaokang; Dee, Rachel; Demonbreun, Alexis R; Li, Jianbin; Xiao, Xiao; McNally, Elizabeth M; Mack, Christopher P; Taylor, Joan M

    2015-01-01

    The plasma membranes of striated muscle cells are particularly susceptible to rupture as they endure significant mechanical stress and strain during muscle contraction, and studies have shown that defects in membrane repair can contribute to the progression of muscular dystrophy. The synaptotagmin-related protein, dysferlin, has been implicated in mediating rapid membrane repair through its ability to direct intracellular vesicles to sites of membrane injury. However, further work is required to identify the precise molecular mechanisms that govern dysferlin targeting and membrane repair. We previously showed that the bin-amphiphysin-Rvs (BAR)-pleckstrin homology (PH) domain containing Rho-GAP GTPase regulator associated with focal adhesion kinase-1 (GRAF1) was dynamically recruited to the tips of fusing myoblasts wherein it promoted membrane merging by facilitating ferlin-dependent capturing of intracellular vesicles. Because acute membrane repair responses involve similar vesicle trafficking complexes/events and because our prior studies in GRAF1-deficient tadpoles revealed a putative role for GRAF1 in maintaining muscle membrane integrity, we postulated that GRAF1 might also play an important role in facilitating dysferlin-dependent plasma membrane repair. We used an in vitro laser-injury model to test whether GRAF1 was necessary for efficient muscle membrane repair. We also generated dystrophin/GRAF1 doubledeficient mice by breeding mdx mice with GRAF1 hypomorphic mice. Evans blue dye uptake and extensive morphometric analyses were used to assess sarcolemmal integrity and related pathologies in cardiac and skeletal muscles isolated from these mice. Herein, we show that GRAF1 is dynamically recruited to damaged skeletal and cardiac muscle plasma membranes and that GRAF1-depleted muscle cells have reduced membrane healing abilities. Moreover, we show that dystrophin depletion exacerbated muscle damage in GRAF1-deficient mice and that mice with dystrophin/GRAF1

  8. Sudden unilateral visual loss after autologous fat injection into the nasolabial fold

    Directory of Open Access Journals (Sweden)

    Sang Hyouk Park

    2008-10-01

    Full Text Available Sang Hyouk Park, Hae Jung Sun, Kyung Seek ChoiDepartment of ophthalmology, Soonchunhyang University College of Medicine, Seoul, KoreaAbstract: A 27-year-old female presented with sudden visual loss of her right eye after receiving an autologous fat injection into the right nasolabial fold. Fundus examination of the right eye showed multiple whitish patchy lesions with macular edema. Fluorescein angiogram showed deterioration of choroidal circulation with patchy choroidal filling and arm-to-retina circulation time and retinal arteriovenous passage time were delayed to 30 seconds and 20 seconds, respectively. There was no response in flash visual evoked potential (VEP. High dose steroid therapy (methylprednisolone 1 g/day/i.v. was done and about 2 weeks later, the disc edema subsided and retinal arteriovenous passage time of fluorescein angiogram was normalized but there was no improvement in visual acuity. Absence of a cherry red spot, deterioration of choroidal circulation with patchy choroidal fillings seen in fluorescein angiogram, and no response in flash VEP suggests multiple choroidal infarction due to perfusion defect of the short posterior ciliary artery. The autologous fat injected is thought to have entered the dorsal nasal artery and the retrograde migration of the emboli to the ophthalmic artery might have caused the multiple occlusions of the short posterior ciliary artery.Keywords: autologous fat injection, ciliary artery occlusion, ischemic optic neuropathy

  9. Spot light on skeletal muscles: optogenetic stimulation to understand and restore skeletal muscle function.

    Science.gov (United States)

    van Bremen, Tobias; Send, Thorsten; Sasse, Philipp; Bruegmann, Tobias

    2017-08-01

    Damage of peripheral nerves results in paralysis of skeletal muscle. Currently, the only treatment option to restore proper function is electrical stimulation of the innervating nerve or of the skeletal muscles directly. However this approach has low spatial and temporal precision leading to co-activation of antagonistic muscles and lacks cell-type selectivity resulting in pain or discomfort by stimulation of sensible nerves. In contrast to electrical stimulation, optogenetic methods enable spatially confined and cell-type selective stimulation of cells expressing the light sensitive channel Channelrhodopsin-2 with precise temporal control over the membrane potential. Herein we summarize the current knowledge about the use of this technology to control skeletal muscle function with the focus on the direct, non-neuronal stimulation of muscle fibers. The high temporal flexibility of using light pulses allows new stimulation patterns to investigate skeletal muscle physiology. Furthermore, the high spatial precision of focused illumination was shown to be beneficial for selective stimulation of distinct nearby muscle groups. Finally, the cell-type specific expression of the light-sensitive effector proteins in muscle fibers will allow pain-free stimulation and open new options for clinical treatments. Therefore, we believe that direct optogenetic stimulation of skeletal muscles is a very potent method for basic scientists that also harbors several distinct advantages over electrical stimulation to be considered for clinical use in the future.

  10. Specifically activated memory T cell subsets from cancer patients recognize and reject xenotransplanted autologous tumors

    Science.gov (United States)

    Beckhove, Philipp; Feuerer, Markus; Dolenc, Mathias; Schuetz, Florian; Choi, Carmen; Sommerfeldt, Nora; Schwendemann, Jochen; Ehlert, Katrin; Altevogt, Peter; Bastert, Gunther; Schirrmacher, Volker; Umansky, Viktor

    2004-01-01

    Bone marrow of breast cancer patients was found to contain CD8+ T cells specific for peptides derived from breast cancer–associated proteins MUC1 and Her-2/neu. Most of these cells had a central or effector memory phenotype (CD45RA–CD62L+ or CD45RA–CD62L–, respectively). To test their in vivo function, we separated bone marrow–derived CD45RA+ naive or CD45RA–CD45RO+ memory T cells, stimulated them with autologous dendritic cells pulsed with tumor lysate, and transferred them into NOD/SCID mice bearing autologous breast tumors and normal skin transplants. CD45RA– memory but not CD45RA+ naive T cells infiltrated autologous tumor but not skin tissues after the transfer. These tumor-infiltrating cells had a central or effector memory phenotype and produced perforin. Many of them expressed the P-selectin glycoprotein ligand 1 and were found around P-selectin+ tumor endothelium. Tumor infiltration included cluster formation in tumor tissue by memory T cells with cotransferred dendritic cells. It was associated with the induction of tumor cell apoptosis and significant tumor reduction. We thus demonstrate selective homing of memory T cells to human tumors and suggest that tumor rejection is based on the recognition of tumor-associated antigens on tumor cells and dendritic cells by autologous specifically activated central and effector memory T cells. PMID:15232613

  11. Traumatic skeletal changes

    International Nuclear Information System (INIS)

    Troeger, J.; Schofer, O.

    1985-01-01

    Skeleton scintiscanning is indicated in the following cases: (1) Suspected bone injury after clinical examination, the radiograph of the skeletal region in question contributing findings that either do not confirm suspision, or make not clear whether the changes observed are traumatic. (2) Polytrauma. (3) When the accident scenario reported by the persons taking care of the child does not sufficiently explain the skeletal changes observed, or when these persons expressly deny the possibility of a trauma being the cause of findings observed. (4) Suspected or proven battered-child syndrome. (orig./MG) [de

  12. Method of cell transplantation promoting the organization of intraarterial thrombus.

    Science.gov (United States)

    Hirano, Koji; Shimono, Takatsugu; Imanaka-Yoshida, Kyoko; Miyamoto, Keiichi; Fujinaga, Kazuya; Kajimoto, Masaki; Miyake, Yoichiro; Nishikawa, Masakatsu; Yoshida, Toshimichi; Uchida, Atsumasa; Shimpo, Hideto; Yada, Isao; Hirata, Hitoshi

    2005-08-30

    Endovascular aortic repairs have been developed as less invasive treatments for aortic aneurysms. Some aneurismal cavities, however, remain without organization, causing a re-expansion of the aneurysms. We studied cell transplantation into the aneurismal sac to promote the organization of thrombus for the complete healing of aneurysms. Skin fibroblasts and skeletal myoblasts were isolated from rats for cell transplantation. An intraarterial thrombus model was made by ligation of the carotid artery. Culture medium (medium group, n=11), collagen gel (gel group, n=11), fibroblasts with collagen gel (F group, n=15), myoblasts with collagen gel (M group, n=12), or mixture of fibroblasts and myoblasts with collagen gel (F+M group, n=14) were injected into the thrombus. After 28 days, histologically, the arterial lumens of the F and M groups were partly filled with fibrous tissues, whereas in the F+M group organization was almost completed and luminal sizes diminished. Immunohistochemical staining demonstrated that alpha-smooth muscle actin-positive cells were more abundantly contained in the organized area of the F+M group than in the other groups. We also analyzed cellular function in vitro with immunofluorescence; coculture of fibroblasts and myoblasts showed that the fraction of alpha-smooth muscle actin-positive fibroblasts increased. This phenomenon accounts for the rapid organization of thrombus in the F+M group in vivo. Cell transplantation accelerated thrombus organization. Especially, myoblasts enhanced differentiation of fibroblasts into myofibroblasts, contributing to rapid thrombus organization. Cell transplantation into unorganized spaces seems applicable to endovascular treatment of aneurysms.

  13. Gastrocnemius tendon strain in a dog treated with autologous mesenchymal stem cells and a custom orthosis.

    Science.gov (United States)

    Case, J Brad; Palmer, Ross; Valdes-Martinez, Alex; Egger, Erick L; Haussler, Kevin K

    2013-05-01

    To report clinical findings and outcome in a dog with gastrocnemius tendon strain treated with autologous mesenchymal stem cells and a custom orthosis. Clinical report. A 4-year-old spayed female Border Collie. Bone-marrow derived, autologous mesenchymal stem cells were transplanted into the tendon core lesion. A custom, progressive, dynamic orthosis was fit to the tarsus. Serial orthopedic examinations and ultrasonography as well as long-term force-plate gait analysis were utilized for follow up. Lameness subjectively resolved and peak vertical force increased from 43% to 92% of the contralateral pelvic limb. Serial ultrasonographic examinations revealed improved but incomplete restoration of normal linear fiber pattern of the gastrocnemius tendon. Findings suggest that autologous mesenchymal stem cell transplantation with custom, progressive, dynamic orthosis may be a viable, minimally invasive technique for treatment of calcaneal tendon injuries in dogs. © Copyright 2013 by The American College of Veterinary Surgeons.

  14. Indications and organisational methods for autologous blood transfusion procedures in Italy: results of a national survey.

    Science.gov (United States)

    Catalano, Liviana; Campolongo, Alessandra; Caponera, Maurizio; Berzuini, Alessandra; Bontadini, Andrea; Furlò, Giuseppe; Pasqualetti, Patrizio; Liumbruno, Giancarlo M

    2014-10-01

    Pre-operative donation of autologous blood is a practice that is now being abandoned. Alternative methods of transfusing autologous blood, other than predeposited blood, do however play a role in limiting the need for transfusion of allogeneic blood. This survey of autologous blood transfusion practices, promoted by the Italian Society of Transfusion Medicine and Immunohaematology more than 2 years after the publication of national recommendations on the subject, was intended to acquire information on the indications for predeposit in Italy and on some organisational aspects of the alternative techniques of autotransfusion. A structured questionnaire consisting of 22 questions on the indications and organisational methods of autologous blood transfusion was made available on a web platform from 15 January to 15 March, 2013. The 232 Transfusion Services in Italy were invited by e-mail to complete the online survey. Of the 232 transfusion structures contacted, 160 (69%) responded to the survey, with the response rate decreasing from the North towards the South and the Islands. The use of predeposit has decreased considerably in Italy and about 50% of the units collected are discarded because of lack of use. Alternative techniques (acute isovolaemic haemodilution and peri-operative blood salvage) are used at different frequencies across the country. The data collected in this survey can be considered representative of national practice; they show that the already very limited indications for predeposit autologous blood transfusion must be adhered to even more scrupulously, also to avoid the notable waste of resources due to unused units.Users of alternative autotransfusion techniques must be involved in order to gain a full picture of the degree of use of such techniques; multidisciplinary agreement on the indications for their use is essential in order for these indications to have an effective role in "patient blood management" programmes.

  15. Skeletal sarcoidosis; Skelettsarkoidose

    Energy Technology Data Exchange (ETDEWEB)

    Freyschmidt, J. [Klinikum Bremen-Mitte, Beratungsstelle und Referenzzentrum fuer Osteoradiologie, Bremen (Germany); Freyschmidt, P. [Dermatologische Gemeinschaftspraxis, Schwalmstadt (Germany)

    2016-10-15

    Presentation of the etiology, pathology, clinical course, radiology and differential diagnostics of skeletal sarcoidosis. Noncaseating epithelioid cell granulomas can trigger solitary, multiple or disseminated osteolysis, reactive osteosclerosis and/or granulomatous synovitis. The incidence of sarcoidosis is 10-12 per 100,000 inhabitants per year. Skeletal involvement is approximately 14 %. Skeletal involvement occurs almost exclusively in the stage of lymph node and pulmonary manifestation. Most cases of skeletal involvement are clinically asymptomatic. In the case of synovial involvement, unspecific joint complaints (arthralgia) or less commonly arthritis can occur. Typical skin alterations can be diagnostically significant. Punch out lesions osteolysis, coarse destruction and osteosclerosis can occur, which are best visualized with projection radiography and/or computed tomography. Pure bone marrow foci without interaction with the bone can only be detected with magnetic resonance imaging (MRI) and more recently with positron emission tomography (PET), mostly as incidental findings. There is a predeliction for the hand and trunk skeleton. Skeletal tuberculosis, metastases, multiple myeloma, Langerhans cell histiocytosis and sarcoid-like reactions in solid tumors must be differentiated. The key factors for correct diagnosis are thorax radiography, thorax CT and dermatological manifestations. (orig.) [German] Darstellung von Aetiologie, Pathologie, Klinik, Radiologie und Differenzialdiagnose der Skelettsarkoidose. Nichtverkaesende Epitheloidzellgranulome koennen solitaere, multiple oder disseminierte Osteolysen, reaktive Osteosklerosen und/oder eine granulomatoese Synovialitis ausloesen. Inzidenz der Sarkoidose: 10-12/100.000 Einwohner/Jahr. Skelettbeteiligung ca. 14 %. Skelettbeteiligungen kommen fast ausschliesslich im Stadium einer Lymphknoten- und pulmonalen Manifestation vor. Die meisten Skelettbeteiligungen verlaufen klinisch stumm. Bei synovialer

  16. Infusion of Autologous Retrodifferentiated Stem Cells into Patients with Beta-Thalassemia

    Directory of Open Access Journals (Sweden)

    Ilham Saleh Abuljadayel

    2006-01-01

    Full Text Available Beta-thalassemia is a genetic, red blood cell disorder affecting the beta-globin chain of the adult hemoglobin gene. This results in excess accumulation of unpaired alpha-chain gene products leading to reduced red blood cell life span and the development of severe anemia. Current treatment of this disease involves regular blood transfusion and adjunct chelation therapy to lower blood transfusion–induced iron overload. Fetal hemoglobin switching agents have been proposed to treat genetic blood disorders, such as sickle cell anemia and beta-thalassemia, in an effort to compensate for the dysfunctional form of the beta-globin chain in adult hemoglobin. The rationale behind this approach is to pair the excess normal alpha-globin chain with the alternative fetal gamma-chain to promote red blood cell survival and ameliorate the anemia. Reprogramming of differentiation in intact, mature, adult white blood cells in response to inclusion of monoclonal antibody CR3/43 has been described. This form of retrograde development has been termed “retrodifferentiation”, with the ability to re-express a variety of stem cell markers in a heterogeneous population of white blood cells. This form of reprogramming, or reontogeny, to a more pluripotent stem cell state ought to recapitulate early hematopoiesis and facilitate expression of a fetal and/or adult program of hemoglobin synthesis or regeneration on infusion and subsequent redifferentiation. Herein, the outcome of infusion of autologous retrodifferentiated stem cells (RSC into 21 patients with beta-thalassemia is described. Over 6 months, Infusion of 3-h autologous RSC subjected to hematopoietic-conducive conditions into patients with beta-thalassemia reduced mean blood transfusion requirement, increased mean fetal hemoglobin synthesis, and significantly lowered mean serum ferritin. This was always accompanied by an increase in mean corpuscular volume (MCV, mean corpuscular hemoglobin (MCH, and mean

  17. Cartilage repair: Generations of autologous chondrocyte transplantation

    International Nuclear Information System (INIS)

    Marlovits, Stefan; Zeller, Philip; Singer, Philipp; Resinger, Christoph; Vecsei, Vilmos

    2006-01-01

    Articular cartilage in adults has a limited capacity for self-repair after a substantial injury. Surgical therapeutic efforts to treat cartilage defects have focused on delivering new cells capable of chondrogenesis into the lesions. Autologous chondrocyte transplantation (ACT) is an advanced cell-based orthobiologic technology used for the treatment of chondral defects of the knee that has been in clinical use since 1987 and has been performed on 12,000 patients internationally. With ACT, good to excellent clinical results are seen in isolated post-traumatic lesions of the knee joint in the younger patient, with the formation of hyaline or hyaline-like repair tissue. In the classic ACT technique, chondrocytes are isolated from small slices of cartilage harvested arthroscopically from a minor weight-bearing area of the injured knee. The extracellular matrix is removed by enzymatic digestion, and the cells are then expanded in monolayer culture. Once a sufficient number of cells has been obtained, the chondrocytes are implanted into the cartilage defect, using a periosteal patch over the defect as a method of cell containment. The major complications are periosteal hypertrophy, delamination of the transplant, arthrofibrosis and transplant failure. Further improvements in tissue engineering have contributed to the next generation of ACT techniques, where cells are combined with resorbable biomaterials, as in matrix-associated autologous chondrocyte transplantation (MACT). These biomaterials secure the cells in the defect area and enhance their proliferation and differentiation

  18. Cartilage repair: Generations of autologous chondrocyte transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Marlovits, Stefan [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)]. E-mail: stefan.marlovits@meduniwien.ac.at; Zeller, Philip [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Singer, Philipp [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Resinger, Christoph [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Vecsei, Vilmos [Department of Traumatology, Center for Joint and Cartilage, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)

    2006-01-15

    Articular cartilage in adults has a limited capacity for self-repair after a substantial injury. Surgical therapeutic efforts to treat cartilage defects have focused on delivering new cells capable of chondrogenesis into the lesions. Autologous chondrocyte transplantation (ACT) is an advanced cell-based orthobiologic technology used for the treatment of chondral defects of the knee that has been in clinical use since 1987 and has been performed on 12,000 patients internationally. With ACT, good to excellent clinical results are seen in isolated post-traumatic lesions of the knee joint in the younger patient, with the formation of hyaline or hyaline-like repair tissue. In the classic ACT technique, chondrocytes are isolated from small slices of cartilage harvested arthroscopically from a minor weight-bearing area of the injured knee. The extracellular matrix is removed by enzymatic digestion, and the cells are then expanded in monolayer culture. Once a sufficient number of cells has been obtained, the chondrocytes are implanted into the cartilage defect, using a periosteal patch over the defect as a method of cell containment. The major complications are periosteal hypertrophy, delamination of the transplant, arthrofibrosis and transplant failure. Further improvements in tissue engineering have contributed to the next generation of ACT techniques, where cells are combined with resorbable biomaterials, as in matrix-associated autologous chondrocyte transplantation (MACT). These biomaterials secure the cells in the defect area and enhance their proliferation and differentiation.

  19. Intrathecal application of autologous bone marrow cell preparations in parkinsonian syndromes

    DEFF Research Database (Denmark)

    Storch, Alexander; Csoti, Ilona; Eggert, Karla

    2012-01-01

    A growing number of patients is treated with intrathecal application of autologous bone marrow cells (aBMCs), but clinical data are completely lacking in movement disorders. We provide first clinical data on efficacy and safety of this highly experimental treatment approach in parkinsonian...

  20. Pelvic radiograph in skeletal dysplasias: An approach

    Directory of Open Access Journals (Sweden)

    Manisha Jana

    2017-01-01

    Full Text Available The bony pelvis is constituted by the ilium, ischium, pubis, and sacrum. The pelvic radiograph is an important component of the skeletal survey performed in suspected skeletal dysplasia. Most of the common skeletal dysplasias have either minor or major radiological abnormalities; hence, knowledge of the normal radiological appearance of bony pelvis is vital for recognizing the early signs of various skeletal dysplasias. This article discusses many common and some uncommon radiological findings on pelvic radiographs along with the specific dysplasia in which they are seen; common differential diagnostic considerations are also discussed.