Dissemination of Walker 256 carcinoma cells to rat skeletal muscle

International Nuclear Information System (INIS)

Ueoka, H.; Hayashi, K.; Namba, T.; Grob, D.

1986-01-01

After injection of 10 6 Walker 256 carcinoma cells labelled with 125 I-5-iodo-2'-deoxyuridine into the tail vein, peak concentration in skeletal muscle was 46 cells/g at 60 minutes, which was lower than 169202, 1665, 555, 198 and 133 cells/g, respectively, at 30 or 60 minutes in lung, liver, spleen, kidney and heart. Because skeletal muscle constitutes 37.4% of body weight, the total number of tumor cells was 2323 cells, which was much greater than in spleen, kidney and heart with 238, 271, and 85 cells, respectively, and only less than in lung and liver, at 222857 and 11700 cells, respectively. The total number in skeletal muscle became greater than in liver at 4 hours and than in lung at 24 hours. Ten minutes after injection of 7.5 x 10 6 Walker 256 carcinoma cells into the abdominal aorta of rats, a mean of 31 colony-forming cells were recovered from the gastrocnemius, while 106 cells were recovered from the lung after injection into the tail vein. These results indicate that a large number of viable tumor cells can be arrested in skeletal muscle through circulation. The rare remote metastasis of malignancies into skeletal muscle despite constantly circulating tumor cells does not appear to be due to poor dissemination of tumor cells into muscle but due to unhospitable environment of skeletal muscle

Stem Cells for Skeletal Muscle Tissue Engineering.

Science.gov (United States)

Pantelic, Molly N; Larkin, Lisa M

2018-04-19

Volumetric muscle loss (VML) is a debilitating condition wherein muscle loss overwhelms the body's normal physiological repair mechanism. VML is particularly common among military service members who have sustained war injuries. Because of the high social and medical cost associated with VML and suboptimal current surgical treatments, there is great interest in developing better VML therapies. Skeletal muscle tissue engineering (SMTE) is a promising alternative to traditional VML surgical treatments that use autogenic tissue grafts, and rather uses isolated stem cells with myogenic potential to generate de novo skeletal muscle tissues to treat VML. Satellite cells are the native precursors to skeletal muscle tissue, and are thus the most commonly studied starting source for SMTE. However, satellite cells are difficult to isolate and purify, and it is presently unknown whether they would be a practical source in clinical SMTE applications. Alternative myogenic stem cells, including adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, perivascular stem cells, umbilical cord mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells, each have myogenic potential and have been identified as possible starting sources for SMTE, although they have yet to be studied in detail for this purpose. These alternative stem cell varieties offer unique advantages and disadvantages that are worth exploring further to advance the SMTE field toward highly functional, safe, and practical VML treatments. The following review summarizes the current state of satellite cell-based SMTE, details the properties and practical advantages of alternative myogenic stem cells, and offers guidance to tissue engineers on how alternative myogenic stem cells can be incorporated into SMTE research.

Skeletal Muscle Cell Induction from Pluripotent Stem Cells

Directory of Open Access Journals (Sweden)

Yusaku Kodaka

2017-01-01

Full Text Available Embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD. Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.

Satellite Cells and the Muscle Stem Cell Niche

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Yin, Hang; Price, Feodor

2013-01-01

Adult skeletal muscle in mammals is a stable tissue under normal circumstances but has remarkable ability to repair after injury. Skeletal muscle regeneration is a highly orchestrated process involving the activation of various cellular and molecular responses. As skeletal muscle stem cells, satellite cells play an indispensible role in this process. The self-renewing proliferation of satellite cells not only maintains the stem cell population but also provides numerous myogenic cells, which proliferate, differentiate, fuse, and lead to new myofiber formation and reconstitution of a functional contractile apparatus. The complex behavior of satellite cells during skeletal muscle regeneration is tightly regulated through the dynamic interplay between intrinsic factors within satellite cells and extrinsic factors constituting the muscle stem cell niche/microenvironment. For the last half century, the advance of molecular biology, cell biology, and genetics has greatly improved our understanding of skeletal muscle biology. Here, we review some recent advances, with focuses on functions of satellite cells and their niche during the process of skeletal muscle regeneration. PMID:23303905

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.

Homogenizing bacterial cell factories: Analysis and engineering of phenotypic heterogeneity.

Science.gov (United States)

Binder, Dennis; Drepper, Thomas; Jaeger, Karl-Erich; Delvigne, Frank; Wiechert, Wolfgang; Kohlheyer, Dietrich; Grünberger, Alexander

2017-07-01

In natural habitats, microbes form multispecies communities that commonly face rapidly changing and highly competitive environments. Thus, phenotypic heterogeneity has evolved as an innate and important survival strategy to gain an overall fitness advantage over cohabiting competitors. However, in defined artificial environments such as monocultures in small- to large-scale bioreactors, cell-to-cell variations are presumed to cause reduced production yields as well as process instability. Hence, engineering microbial production toward phenotypic homogeneity is a highly promising approach for synthetic biology and bioprocess optimization. In this review, we discuss recent studies that have unraveled the cell-to-cell heterogeneity observed during bacterial gene expression and metabolite production as well as the molecular mechanisms involved. In addition, current single-cell technologies are briefly reviewed with respect to their applicability in exploring cell-to-cell variations. We highlight emerging strategies and tools to reduce phenotypic heterogeneity in biotechnological expression setups. Here, strain or inducer modifications are combined with cell physiology manipulations to achieve the ultimate goal of equalizing bacterial populations. In this way, the majority of cells can be forced into high productivity, thus reducing less productive subpopulations that tend to consume valuable resources during production. Modifications in uptake systems, inducer molecules or nutrients represent valuable tools for diminishing heterogeneity. Finally, we address the challenge of transferring homogeneously responding cells into large-scale bioprocesses. Environmental heterogeneity originating from extrinsic factors such as stirring speed and pH, oxygen, temperature or nutrient distribution can significantly influence cellular physiology. We conclude that engineering microbial populations toward phenotypic homogeneity is an increasingly important task to take biotechnological

Glucocorticoid actions on L6 muscle cells in culture

International Nuclear Information System (INIS)

Max, S.R.; Konagaya, M.; Konagaya, Y.

1986-01-01

Glucocorticoids exert striking catabolic effects on skeletal muscle. The mechanism of these effects remains poorly understood. They employed L6 muscle cells in culture to ascertain whether intracellular glucocorticoid receptors are involved. Studies in vitro permit exploration of glucocorticoid effects in the absence of other hormonal influences. L6 myoblasts were induced to form differentiated myotubes by growth in 1% serum. L6 myotubes were found to possess a high-affinity, limited capacity intracellular glucocorticoid receptor (apparent K/sub D/ = 5 x 10 -10 M; B/sub max/ = 711 pmols/g protein) with ligand specificity similar to that of glucocorticoid receptors from classical glucocorticoid target tissues. Further, [ 3 H] triamcinolone acetonide specific binding to L6 cell homogenates was blocked by a glucocorticoid antagonist, RU38486 (11β-(4-dimethyl-aminophenyl)-17β-hydroxy-17α-(prop-l-ynyl)-estra-4,9-dien-3-one). Dexamethasone (10 -5 M) caused a 10-fold increase in the activity of gluatmine synthetase in L6 myotubes; this increase was prevented by RU38486. Similarly, dexamethasone (10 -5 M) caused a 20% decrease in [ 12 C] leucine incorporation into protein. This effect also was blocked by RU38486. Thus, induction of glutamine synthetase and diminution of protein synthesis by dexamethasone require intracellular glucocorticoid receptors. L6 cells should prove particularly valuable for further studies of glucocorticoid actions on skeletal muscle

Skeletal muscle to pancreatic β-cell cross-talk

DEFF Research Database (Denmark)

Christensen, Camilla S; P. Christensen, Dan; Lundh, Morten

2015-01-01

CONTEXT: Mechanisms explaining exercise-induced β-cell health are unknown. OBJECTIVE: To define the role of muscle contraction and acute exercise-derived soluble humoral mediators on β-cell health. DESIGN: In vitro models. SETTING: University. PARTICIPANTS: Healthy subjects. INTERVENTION...... insulin secretion in the absence of IL-1β+IFN-γ. CONCLUSIONS: Unidentified circulating humoral mediators released during exercise prevent proinflammatory cytokine-induced β-cell apoptosis. Muscle-derived mediators released during exercise suppress β-cell insulin secretion. Furthermore, muscle-derived IL-6...

Muscle satellite cell heterogeneity and self-renewal

Science.gov (United States)

Motohashi, Norio; Asakura, Atsushi

2014-01-01

Adult skeletal muscle possesses extraordinary regeneration capacities. After muscle injury or exercise, large numbers of newly formed muscle fibers are generated within a week as a result of expansion and differentiation of a self-renewing pool of muscle stem cells termed muscle satellite cells. Normally, satellite cells are mitotically quiescent and reside beneath the basal lamina of muscle fibers. Upon regeneration, satellite cells are activated, and give rise to daughter myogenic precursor cells. After several rounds of proliferation, these myogenic precursor cells contribute to the formation of new muscle fibers. During cell division, a minor population of myogenic precursor cells returns to quiescent satellite cells as a self-renewal process. Currently, accumulating evidence has revealed the essential roles of satellite cells in muscle regeneration and the regulatory mechanisms, while it still remains to be elucidated how satellite cell self-renewal is molecularly regulated and how satellite cells are important in aging and diseased muscle. The number of satellite cells is decreased due to the changing niche during ageing, resulting in attenuation of muscle regeneration capacity. Additionally, in Duchenne muscular dystrophy (DMD) patients, the loss of satellite cell regenerative capacity and decreased satellite cell number due to continuous needs for satellite cells lead to progressive muscle weakness with chronic degeneration. Thus, it is necessary to replenish muscle satellite cells continuously. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is crucial for maintenance of satellite cells as a muscle stem cell pool throughout life. In addition, we discuss roles in the stem cell niche for satellite cell maintenance, as well as related cell therapies for approaching treatment of DMD. PMID:25364710

Muscle Satellite Cell Heterogeneity and Self-Renewal

Directory of Open Access Journals (Sweden)

Norio eMotohashi

2014-01-01

Full Text Available Adult skeletal muscle possesses extraordinary regeneration capacities. After muscle injury or exercise, large numbers of newly formed muscle fibers are generated within a week as a result of expansion and differentiation of a self-renewing pool of muscle stem cells termed muscle satellite cells. Normally, satellite cells are mitotically quiescent and reside beneath the basal lamina of muscle fibers. Upon regeneration, satellite cells are activated, and give rise to daughter myogenic precursor cells. After several rounds of proliferation, these myogenic precursor cells contribute to the formation of new muscle fibers. During cell division, a minor population of myogenic precursor cells returns to quiescent satellite cells as a self-renewal process. Currently, accumulating evidence has revealed the essential roles of satellite cells in muscle regeneration and the regulatory mechanisms, while it still remains to be elucidated how satellite cell self-renewal is molecularly regulated and how satellite cells are important in aging and diseased muscle. The number of satellite cells is decreased due to the changing niche during ageing, resulting in attenuation of muscle regeneration capacity. Additionally, in Duchenne muscular dystrophy (DMD patients, the loss of satellite cell regenerative capacity and decreased satellite cell number due to continuous needs for satellite cells lead to progressive muscle weakness with chronic degeneration. Thus, it is necessary to replenish muscle satellite cells continuously. This review outlines recent findings regarding satellite cell heterogeneity, asymmetric division and molecular mechanisms in satellite cell self-renewal which is crucial for maintenance of satellite cells as a muscle stem cell pool throughout life. In addition, we discuss roles in the stem cell niche for satellite cell maintenance, as well as related cell therapies for approaching treatment of DMD.

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.

Muscle Satellite Cell Protein Teneurin‐4 Regulates Differentiation During Muscle Regeneration

Science.gov (United States)

Ishii, Kana; Suzuki, Nobuharu; Mabuchi, Yo; Ito, Naoki; Kikura, Naomi; Fukada, So‐ichiro; Okano, Hideyuki; Takeda, Shin'ichi

2015-01-01

Abstract Satellite cells are maintained in an undifferentiated quiescent state, but during muscle regeneration they acquire an activated stage, and initiate to proliferate and differentiate as myoblasts. The transmembrane protein teneurin‐4 (Ten‐4) is specifically expressed in the quiescent satellite cells; however, its cellular and molecular functions remain unknown. We therefore aimed to elucidate the function of Ten‐4 in muscle satellite cells. In the tibialis anterior (TA) muscle of Ten‐4‐deficient mice, the number and the size of myofibers, as well as the population of satellite cells, were reduced with/without induction of muscle regeneration. Furthermore, we found an accelerated activation of satellite cells in the regenerated Ten‐4‐deficient TA muscle. The cell culture analysis using primary satellite cells showed that Ten‐4 suppressed the progression of myogenic differentiation. Together, our findings revealed that Ten‐4 functions as a crucial player in maintaining the quiescence of muscle satellite cells. Stem Cells 2015;33:3017–3027 PMID:26013034

Aberrant Muscle Antigen Exposure in Mice Is Sufficient to Cause Myositis in a Treg Cell–Deficient Milieu

Science.gov (United States)

Young, Nicholas A; Sharma, Rahul; Friedman, Alexandra K; Kaffenberger, Benjamin H; Bolon, Brad; Jarjour, Wael N

2013-01-01

Objective Myositis is associated with muscle-targeted inflammation and is observed in some Treg cell–deficient mouse models. Because an autoimmune pathogenesis has been strongly implicated, the aim of this study was to investigate the hypothesis that abnormal exposure to muscle antigens, as observed in muscle injury, can induce autoimmune-mediated myositis in susceptible hosts. Methods FoxP3 mutant (scurfy) mice were mated to synaptotagmin VII (Syt VII) mutant mice, which resulted in a new mouse strain that combines impaired membrane resealing with Treg cell deficiency. Lymphocyte preparations from double-mutant mice were adoptively transferred intraperitoneally, with or without purified Treg cells, into recombination-activating gene 1 (RAG-1)–null recipients. Lymph node cells from mice with the FoxP3 mutation were transferred into RAG-1–null mice either 1) intraperitoneally in conjunction with muscle homogenate or purified myosin protein or 2) intramuscularly with or without cotransfer of purified Treg cells. Results FoxP3-deficient mouse lymph node cells transferred in conjunction with myosin protein or muscle homogenate induced robust skeletal muscle inflammation. The infiltrates consisted predominantly of CD4+ and CD8+ T cells, a limited number of macrophages, and no B cells. Significant inflammation was also seen in similar experiments using lymph node cells from FoxP3/Syt VII double-mutant mice but was absent in experiments using adoptive transfer of FoxP3 mutant mouse cells alone. The cotransfer of Treg cells completely suppressed myositis. Conclusion These data, derived from a new, reproducible model, demonstrate the critical roles of Treg cell deficiency and aberrant muscle antigen exposure in the priming of autoreactive cells to induce myositis. This mouse system has multifaceted potential for examining the interplay in vivo between tissue injury and autoimmunity. PMID:24022275

Studies with muscle cells from controls and a patient with the cerebro-hepato-renal (Zellweger) syndrome

NARCIS (Netherlands)

Wanders, R.J.A.; Barth, P.G.; Roermund, C.W.T. van; Ofman, R.; Wolterman, R.; Schutgens, R.B.H.; Tager, J.M.; Bosch, H. van den; Bolhuis, P.A.

In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of

A Modified Homogeneous Balance Method and Its Applications

International Nuclear Information System (INIS)

Liu Chunping

2011-01-01

A modified homogeneous balance method is proposed by improving some key steps in the homogeneous balance method. Bilinear equations of some nonlinear evolution equations are derived by using the modified homogeneous balance method. Generalized Boussinesq equation, KP equation, and mKdV equation are chosen as examples to illustrate our method. This approach is also applicable to a large variety of nonlinear evolution equations. (general)

Muscle Satellite Cell Protein Teneurin-4 Regulates Differentiation During Muscle Regeneration.

Science.gov (United States)

Ishii, Kana; Suzuki, Nobuharu; Mabuchi, Yo; Ito, Naoki; Kikura, Naomi; Fukada, So-Ichiro; Okano, Hideyuki; Takeda, Shin'ichi; Akazawa, Chihiro

2015-10-01

Satellite cells are maintained in an undifferentiated quiescent state, but during muscle regeneration they acquire an activated stage, and initiate to proliferate and differentiate as myoblasts. The transmembrane protein teneurin-4 (Ten-4) is specifically expressed in the quiescent satellite cells; however, its cellular and molecular functions remain unknown. We therefore aimed to elucidate the function of Ten-4 in muscle satellite cells. In the tibialis anterior (TA) muscle of Ten-4-deficient mice, the number and the size of myofibers, as well as the population of satellite cells, were reduced with/without induction of muscle regeneration. Furthermore, we found an accelerated activation of satellite cells in the regenerated Ten-4-deficient TA muscle. The cell culture analysis using primary satellite cells showed that Ten-4 suppressed the progression of myogenic differentiation. Together, our findings revealed that Ten-4 functions as a crucial player in maintaining the quiescence of muscle satellite cells. © 2015 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

The Skeletal Muscle Satellite Cell

Science.gov (United States)

2011-01-01

The skeletal muscle satellite cell was first described and named based on its anatomic location between the myofiber plasma and basement membranes. In 1961, two independent studies by Alexander Mauro and Bernard Katz provided the first electron microscopic descriptions of satellite cells in frog and rat muscles. These cells were soon detected in other vertebrates and acquired candidacy as the source of myogenic cells needed for myofiber growth and repair throughout life. Cultures of isolated myofibers and, subsequently, transplantation of single myofibers demonstrated that satellite cells were myogenic progenitors. More recently, satellite cells were redefined as myogenic stem cells given their ability to self-renew in addition to producing differentiated progeny. Identification of distinctively expressed molecular markers, in particular Pax7, has facilitated detection of satellite cells using light microscopy. Notwithstanding the remarkable progress made since the discovery of satellite cells, researchers have looked for alternative cells with myogenic capacity that can potentially be used for whole body cell-based therapy of skeletal muscle. Yet, new studies show that inducible ablation of satellite cells in adult muscle impairs myofiber regeneration. Thus, on the 50th anniversary since its discovery, the satellite cell’s indispensable role in muscle repair has been reaffirmed. PMID:22147605

Response of turkey muscle satellite cells to thermal challenge. I. transcriptome effects in proliferating cells.

Science.gov (United States)

Reed, Kent M; Mendoza, Kristelle M; Abrahante, Juan E; Barnes, Natalie E; Velleman, Sandra G; Strasburg, Gale M

2017-05-06

Climate change poses a multi-dimensional threat to food and agricultural systems as a result of increased risk to animal growth, development, health, and food product quality. This study was designed to characterize transcriptional changes induced in turkey muscle satellite cells cultured under cold or hot thermal challenge to better define molecular mechanisms by which thermal stress alters breast muscle ultrastructure. Satellite cells isolated from the pectoralis major muscle of 7-weeks-old male turkeys from two breeding lines (16 weeks body weight-selected and it's randombred control) were proliferated in culture at 33 °C, 38 °C or 43 °C for 72 h. Total RNA was isolated and 12 libraries subjected to RNAseq analysis. Statistically significant differences in gene expression were observed among treatments and between turkey lines with a greater number of genes altered by cold treatment than by hot and fewer differences observed between lines than between temperatures. Pathway analysis found that cold treatment resulted in an overrepresentation of genes involved in cell signaling/signal transduction and cell communication/cell signaling as compared to control (38 °C). Heat-treated muscle satellite cells showed greater tendency towards expression of genes related to muscle system development and differentiation. This study demonstrates significant transcriptome effects on turkey skeletal muscle satellite cells exposed to thermal challenge. Additional effects on gene expression could be attributed to genetic selection for 16 weeks body weight (muscle mass). New targets are identified for further research on the differential control of satellite cell proliferation in poultry.

Oxytocin receptors expressed and coupled to Ca2+ signalling in a human vascular smooth muscle cell line.

Science.gov (United States)

Yazawa, H; Hirasawa, A; Horie, K; Saita, Y; Iida, E; Honda, K; Tsujimoto, G

1996-03-01

1. In a human vascular smooth muscle cell line (HVSMC), binding experiments with [3H]-arginine8-vasopressin (AVP) have shown the existence of a homogeneous population of binding sites with affinity (Kd value) of 0.65 nM and a maximum number of binding sites (Bmax) of 122 fmol mg-1 protein. 2. Nonlabelled compounds compete for [3H]-AVP binding in the HVSMC membrane with an order of potency of oxytocin > lyspressin > or = AVP > Thr4, Gly7-oxytocin > (beta-mercapto-beta-beta-cyclopentamethylenepropionyl-O-Me Tyr2, Arg8) vasopressin > desmopressin > OPC21268 > OPC31260. This order was markedly different from that observed in rat vascular smooth muscle cells (A10), a well-established V1A receptor system. 3. In HVSMC both oxytocin and AVP increased inositol 1,4,5-trisphosphate (IP3) production and [Ca2+]i response, but the efficacy of the responses was greater for oxytocin than AVP. 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay detected only oxytocin receptor but not V1A or V2 receptors in HVSMC, whereas only V1A receptors were found in A10 cells. 5. In conclusion, in HVSMC only oxytocin receptors are expressed among the vasopressin receptor family, and they coupled to phosphatidyl inositol (PI) turnover/Ca2+ signalling. This unexpected observation should provide new insight into the functional role of the oxytocin receptor in a human vascular smooth muscle cell line.

Satellite cells in human skeletal muscle plasticity

Directory of Open Access Journals (Sweden)

Tim eSnijders

2015-10-01

Full Text Available Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodelling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodelling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodelling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

Satellite cells in human skeletal muscle plasticity.

Science.gov (United States)

Snijders, Tim; Nederveen, Joshua P; McKay, Bryon R; Joanisse, Sophie; Verdijk, Lex B; van Loon, Luc J C; Parise, Gianni

2015-01-01

Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

Homogenization on Multi-Materials’ Elements: Application to Printed Circuit Boards and Warpage Analysis

Directory of Open Access Journals (Sweden)

Araújo Manuel

2016-01-01

Full Text Available Multi-material domains are often found in industrial applications. Modelling them can be computationally very expensive due to meshing requirements. The finite element properties comprising different materials are hardly accurate. In this work, a new homogenization method that simplifies the computation of the homogenized Young modulus, Poisson ratio and thermal expansion coefficient is proposed, and applied to composite-like material on a printed circuit board. The results show a good properties correspondence between the homogenized domain and the real geometry simulation.

Regulatory T cells and skeletal muscle regeneration.

Science.gov (United States)

Schiaffino, Stefano; Pereira, Marcelo G; Ciciliot, Stefano; Rovere-Querini, Patrizia

2017-02-01

Skeletal muscle regeneration results from the activation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Inflammatory and immune cells have a crucial role in the regeneration process. Acute muscle injury causes an immediate transient wave of neutrophils followed by a more persistent infiltration of M1 (proinflammatory) and M2 (anti-inflammatory/proregenerative) macrophages. New studies show that injured muscle is also infiltrated by a specialized population of regulatory T (Treg) cells, which control both the inflammatory response, by promoting the M1-to-M2 switch, and the activation of satellite cells. Treg cells accumulate in injured muscle in response to specific cytokines, such as IL-33, and promote muscle growth by releasing growth factors, such as amphiregulin. Muscle repair during aging is impaired due to reduced number of Treg cells and can be enhanced by IL-33 supplementation. Migration of Treg cells could also contribute to explain the effect of heterochronic parabiosis, whereby muscle regeneration of aged mice can be improved by a parabiotically linked young partners. In mdx dystrophin-deficient mice, a model of human Duchenne muscular dystrophy, muscle injury, and inflammation is mitigated by expansion of the Treg-cell population but exacerbated by Treg-cell depletion. These findings support the notion that immunological mechanisms are not only essential in the response to pathogenic microbes and tumor cells but also have a wider homeostatic role in tissue repair, and open new perspectives for boosting muscle growth in chronic muscle disease and during aging. © 2016 Federation of European Biochemical Societies.

Oxytocin receptors expressed and coupled to Ca2+ signalling in a human vascular smooth muscle cell line.

OpenAIRE

Yazawa, H.; Hirasawa, A.; Horie, K.; Saita, Y.; Iida, E.; Honda, K.; Tsujimoto, G.

1996-01-01

1. In a human vascular smooth muscle cell line (HVSMC), binding experiments with [3H]-arginine8-vasopressin (AVP) have shown the existence of a homogeneous population of binding sites with affinity (Kd value) of 0.65 nM and a maximum number of binding sites (Bmax) of 122 fmol mg-1 protein. 2. Nonlabelled compounds compete for [3H]-AVP binding in the HVSMC membrane with an order of potency of oxytocin > lyspressin > or = AVP > Thr4, Gly7-oxytocin > (beta-mercapto-beta-beta-cyclopentamethylenep...

Loss of niche-satellite cell interactions in syndecan-3 null mice alters muscle progenitor cell homeostasis improving muscle regeneration.

Science.gov (United States)

Pisconti, Addolorata; Banks, Glen B; Babaeijandaghi, Farshad; Betta, Nicole Dalla; Rossi, Fabio M V; Chamberlain, Jeffrey S; Olwin, Bradley B

2016-01-01

The skeletal muscle stem cell niche provides an environment that maintains quiescent satellite cells, required for skeletal muscle homeostasis and regeneration. Syndecan-3, a transmembrane proteoglycan expressed in satellite cells, supports communication with the niche, providing cell interactions and signals to maintain quiescent satellite cells. Syndecan-3 ablation unexpectedly improves regeneration in repeatedly injured muscle and in dystrophic mice, accompanied by the persistence of sublaminar and interstitial, proliferating myoblasts. Additionally, muscle aging is improved in syndecan-3 null mice. Since syndecan-3 null myofiber-associated satellite cells downregulate Pax7 and migrate away from the niche more readily than wild type cells, syxndecan-3 appears to regulate satellite cell homeostasis and satellite cell homing to the niche. Manipulating syndecan-3 provides a promising target for development of therapies to enhance muscle regeneration in muscular dystrophies and in aged muscle.

Oral Gingival Cell Cigarette Smoke Exposure Induces Muscle Cell Metabolic Disruption

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Andrea C. Baeder

2016-01-01

Full Text Available Cigarette smoke exposure compromises health through damaging multiple physiological systems, including disrupting metabolic function. The purpose of this study was to determine the role of oral gingiva in mediating the deleterious metabolic effects of cigarette smoke exposure on skeletal muscle metabolic function. Using an in vitro conditioned medium cell model, skeletal muscle cells were incubated with medium from gingival cells treated with normal medium or medium containing suspended cigarette smoke extract (CSE. Following incubation of muscle cells with gingival cell conditioned medium, muscle cell mitochondrial respiration and insulin signaling and action were determined as an indication of overall muscle metabolic health. Skeletal muscle cells incubated with conditioned medium of CSE-treated gingival cells had a profound reduction in mitochondrial respiration and respiratory control. Furthermore, skeletal muscle cells had a greatly reduced response in insulin-stimulated Akt phosphorylation and glycogen synthesis. Altogether, these results provide a novel perspective on the mechanism whereby cigarette smoke affects systemic metabolic function. In conclusion, we found that oral gingival cells treated with CSE create an altered milieu that is sufficient to both disrupted skeletal muscle cell mitochondrial function and insulin sensitivity.

The role of satellite cells in muscle hypertrophy.

Science.gov (United States)

Blaauw, Bert; Reggiani, Carlo

2014-02-01

The role of satellite cells in muscle hypertrophy has long been a debated issue. In the late 1980s it was shown that proteins remain close to the myonucleus responsible for its synthesis, giving rise to the idea of a nuclear domain. This, together with the observation that during various models of muscle hypertrophy there is an activation of the muscle stem cells, i.e. satellite cells, lead to the idea that satellite cell activation is required for muscle hypertrophy. Thus, satellite cells are not only responsible for muscle repair and regeneration, but also for hypertrophic growth. Further support for this line of thinking was obtained after studies showing that irradiation of skeletal muscle, and therefore elimination of all satellite cells, completely prevented overload-induced hypertrophy. Recently however, using different transgenic approaches, it has become clear that muscle hypertrophy can occur without a contribution of satellite cells, even though in most situations of muscle hypertrophy satellite cells are activated. In this review we will discuss the contribution of satellite cells, and other muscle-resident stem cells, to muscle hypertrophy both in mice as well as in humans.

Response of Turkey Muscle Satellite Cells to Thermal Challenge. II. Transcriptome Effects in Differentiating Cells

Directory of Open Access Journals (Sweden)

Kent M. Reed

2017-11-01

Full Text Available Background: Exposure of poultry to extreme temperatures during the critical period of post-hatch growth can seriously affect muscle development and thus compromise subsequent meat quality. This study was designed to characterize transcriptional changes induced in turkey muscle satellite cells by thermal challenge during differentiation. Our goal is to better define how thermal stress alters breast muscle ultrastructure and subsequent development.Results: Skeletal muscle satellite cells previously isolated from the Pectoralis major muscle of 7-wk-old male turkeys (Meleagris gallopavo from two breeding lines: the F-line (16 wk body weight-selected and RBC2 (randombred control line were used in this study. Cultured cells were induced to differentiate at 38°C (control or thermal challenge temperatures of 33 or 43°C. After 48 h of differentiation, cells were harvested and total RNA was isolated for RNAseq analysis. Analysis of 39.9 Gb of sequence found 89% mapped to the turkey genome (UMD5.0, annotation 101 with average expression of 18,917 genes per library. In the cultured satellite cells, slow/cardiac muscle isoforms are generally present in greater abundance than fast skeletal isoforms. Statistically significant differences in gene expression were observed among treatments and between turkey lines, with a greater number of genes affected in the F-line cells following cold treatment whereas more differentially expressed (DE genes were observed in the RBC2 cells following heat treatment. Many of the most significant pathways involved signaling, consistent with ongoing cellular differentiation. Regulation of Ca2+ homeostasis appears to be significantly affected by temperature treatment, particularly cold treatment.Conclusions: Satellite cell differentiation is directly influenced by temperature at the level of gene transcription with greater effects attributed to selection for fast growth. At lower temperature, muscle-associated genes in the

Epiblast cells that express MyoD recruit pluripotent cells to the skeletal muscle lineage

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Gerhart, Jacquelyn; Neely, Christine; Stewart, Benjamin; Perlman, Jordanna; Beckmann, David; Wallon, Margaretha; Knudsen, Karen; George-Weinstein, Mindy

2004-01-01

Embryonic stem cells are derived from the epiblast. A subpopulation of epiblast cells expresses MyoD mRNA and the G8 antigen in vivo. G8 positive (G8pos) and G8 negative (G8neg) populations were isolated by magnetic cell sorting. Nearly all G8pos cells switched from E- to N-cadherin and differentiated into skeletal muscle in culture. G8neg cells were impaired in their ability to switch cadherins and few formed skeletal muscle. Medium conditioned by G8pos cells stimulated skeletal myogenesis and N-cadherin synthesis in G8neg cultures. The effect of conditioned medium from G8pos cultures was inhibited by bone morphogenetic protein (BMP) 4. Treatment of G8neg cells with a soluble form of the BMP receptor-IA or Noggin promoted N-cadherin synthesis and skeletal myogenesis. These results demonstrate that MyoD-positive epiblast cells recruit pluripotent cells to the skeletal muscle lineage. The mechanism of recruitment involves blocking the BMP signaling pathway. PMID:14981095

Vessel-associated stem cells from skeletal muscle: From biology to future uses in cell therapy.

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Sancricca, Cristina; Mirabella, Massimiliano; Gliubizzi, Carla; Broccolini, Aldobrando; Gidaro, Teresa; Morosetti, Roberta

2010-06-26

Over the last years, the existence of different stem cells with myogenic potential has been widely investigated. Besides the classical skeletal muscle progenitors represented by satellite cells, numerous multipotent and embryologically unrelated progenitors with a potential role in muscle differentiation and repair have been identified. In order to conceive a therapeutic approach for degenerative muscle disorders, it is of primary importance to identify an ideal stem cell endowed with all the features for a possible use in vivo. Among all emerging populations, vessel-associated stem cells are a novel and promising class of multipotent progenitors of mesodermal origin and with high myogenic potential which seem to best fit all the requirements for a possible cell therapy. In vitro and in vivostudies have already tested the effectiveness and safety of vessel-associated stem cells in animal models. This leads to the concrete possibility in the future to start pilot human clinical trials, hopefully opening the way to a turning point in the treatment of genetic and acquired muscle disorders.

Enhanced photovoltaic properties of perovskite solar cells by TiO2 homogeneous hybrid structure.

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Su, Pengyu; Fu, Wuyou; Yao, Huizhen; Liu, Li; Ding, Dong; Feng, Fei; Feng, Shuang; Xue, Yebin; Liu, Xizhe; Yang, Haibin

2017-10-01

In this paper, we fabricated a TiO 2 homogeneous hybrid structure for application in perovskite solar cells (PSCs) under ambient conditions. Under the standard air mass 1.5 global (AM 1.5G) illumination, PSCs based on homogeneous hybrid structure present a maximum power conversion efficiency of 5.39% which is higher than that of pure TiO 2 nanosheets. The enhanced properties can be explained by the better contact of TiO 2 nanosheets/nanoparticles with CH 3 NH 3 PbI 3 and fewer pinholes in electron transport materials. The advent of such unique structure opens up new avenues for the future development of high-efficiency photovoltaic cells.

The extraocular muscle stem cell niche is resistant to ageing and disease

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Luigi eFormicola

2014-12-01

Full Text Available Specific muscles are spared in many degenerative myopathies. Most notably, the extraocular muscles (EOMs do not show clinical signs of late stage myopathies including the accumulation of fibrosis and fat. It has been proposed that an altered stem cell niche underlies the resistance of EOMs in these pathologies, however, to date, no reports have provided a detailed characterization of the EOM stem cell niche. PW1/Peg3 is expressed in progenitor cells in all adult tissues including satellite cells and a subset of interstitial non-satellite cell progenitors in muscle. These PW1-positive interstitial cells (PICs include a fibroadipogenic progenitor population (FAPs that give rise to fat and fibrosis in late stage myopathies. PICs/FAPs are mobilized following injury and FAPs exert a promyogenic role upon myoblasts in vitro but require the presence of a minimal population of satellite cells in vivo. We and others recently described that FAPs express promyogenic factors while satellite cells express antimyogenic factors suggesting that PICs/FAPs act as support niche cells in skeletal muscle through paracrine interactions. We analyzed the EOM stem cell niche in young adult and aged wild-type mice and found that the balance between PICs and satellite cells within the EOM stem cell niche is maintained throughout life. Moreover, in the adult mdx mouse model for Duchenne muscular dystrophy, the EOM stem cell niche is unperturbed compared to normal mice, in contrast to Tibialis Anterior (TA muscle, which displays signs of ongoing degeneration/regeneration. Regenerating mdx TA shows increased levels of both PICs and satellite cells, comparable to normal unaffected EOMs. We propose that the increase in PICs that we observe in normal EOMs contributes to preserving the integrity of the myofibers and satellite cells. Our data suggest that molecular cues regulating muscle regeneration are intrinsic properties of EOMs.

Muscle Stem Cells: A Model System for Adult Stem Cell Biology.

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Cornelison, Ddw; Perdiguero, Eusebio

2017-01-01

Skeletal muscle stem cells, originally termed satellite cells for their position adjacent to differentiated muscle fibers, are absolutely required for the process of skeletal muscle repair and regeneration. In the last decade, satellite cells have become one of the most studied adult stem cell systems and have emerged as a standard model not only in the field of stem cell-driven tissue regeneration but also in stem cell dysfunction and aging. Here, we provide background in the field and discuss recent advances in our understanding of muscle stem cell function and dysfunction, particularly in the case of aging, and the potential involvement of muscle stem cells in genetic diseases such as the muscular dystrophies.

Muscle Satellite Cells: Exploring the Basic Biology to Rule Them.

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Almeida, Camila F; Fernandes, Stephanie A; Ribeiro Junior, Antonio F; Keith Okamoto, Oswaldo; Vainzof, Mariz

2016-01-01

Adult skeletal muscle is a postmitotic tissue with an enormous capacity to regenerate upon injury. This is accomplished by resident stem cells, named satellite cells, which were identified more than 50 years ago. Since their discovery, many researchers have been concentrating efforts to answer questions about their origin and role in muscle development, the way they contribute to muscle regeneration, and their potential to cell-based therapies. Satellite cells are maintained in a quiescent state and upon requirement are activated, proliferating, and fusing with other cells to form or repair myofibers. In addition, they are able to self-renew and replenish the stem pool. Every phase of satellite cell activity is highly regulated and orchestrated by many molecules and signaling pathways; the elucidation of players and mechanisms involved in satellite cell biology is of extreme importance, being the first step to expose the crucial points that could be modulated to extract the optimal response from these cells in therapeutic strategies. Here, we review the basic aspects about satellite cells biology and briefly discuss recent findings about therapeutic attempts, trying to raise questions about how basic biology could provide a solid scaffold to more successful use of these cells in clinics.

Extracellular matrix components direct porcine muscle stem cell behavior

International Nuclear Information System (INIS)

Wilschut, Karlijn J.; Haagsman, Henk P.; Roelen, Bernard A.J.

2010-01-01

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

Extracellular matrix components direct porcine muscle stem cell behavior

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Wilschut, Karlijn J. [Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht (Netherlands); Haagsman, Henk P. [Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht (Netherlands); Roelen, Bernard A.J., E-mail: b.a.j.roelen@uu.nl [Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 104, 3584 CM, Utrecht (Netherlands)

2010-02-01

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

HEXIM1 controls satellite cell expansion after injury to regulate skeletal muscle regeneration

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Hong, Peng; Chen, Kang; Huang, Bihui; Liu, Min; Cui, Miao; Rozenberg, Inna; Chaqour, Brahim; Pan, Xiaoyue; Barton, Elisabeth R.; Jiang, Xian-Cheng; Siddiqui, M.A.Q.

2012-01-01

The native capacity of adult skeletal muscles to regenerate is vital to the recovery from physical injuries and dystrophic diseases. Currently, the development of therapeutic interventions has been hindered by the complex regulatory network underlying the process of muscle regeneration. Using a mouse model of skeletal muscle regeneration after injury, we identified hexamethylene bisacetamide inducible 1 (HEXIM1, also referred to as CLP-1), the inhibitory component of the positive transcription elongation factor b (P-TEFb) complex, as a pivotal regulator of skeletal muscle regeneration. Hexim1-haplodeficient muscles exhibited greater mass and preserved function compared with those of WT muscles after injury, as a result of enhanced expansion of satellite cells. Transplanted Hexim1-haplodeficient satellite cells expanded and improved muscle regeneration more effectively than WT satellite cells. Conversely, HEXIM1 overexpression restrained satellite cell proliferation and impeded muscle regeneration. Mechanistically, dissociation of HEXIM1 from P-TEFb and subsequent activation of P-TEFb are required for satellite cell proliferation and the prevention of early myogenic differentiation. These findings suggest a crucial role for the HEXIM1/P-TEFb pathway in the regulation of satellite cell–mediated muscle regeneration and identify HEXIM1 as a potential therapeutic target for degenerative muscular diseases. PMID:23023707

Functional heterogeneity of side population cells in skeletal muscle

International Nuclear Information System (INIS)

Uezumi, Akiyoshi; Ojima, Koichi; Fukada, So-ichiro; Ikemoto, Madoka; Masuda, Satoru; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi

2006-01-01

Skeletal muscle regeneration has been exclusively attributed to myogenic precursors, satellite cells. A stem cell-rich fraction referred to as side population (SP) cells also resides in skeletal muscle, but its roles in muscle regeneration remain unclear. We found that muscle SP cells could be subdivided into three sub-fractions using CD31 and CD45 markers. The majority of SP cells in normal non-regenerating muscle expressed CD31 and had endothelial characteristics. However, CD31 - CD45 - SP cells, which are a minor subpopulation in normal muscle, actively proliferated upon muscle injury and expressed not only several regulatory genes for muscle regeneration but also some mesenchymal lineage markers. CD31 - CD45 - SP cells showed the greatest myogenic potential among three SP sub-fractions, but indeed revealed mesenchymal potentials in vitro. These SP cells preferentially differentiated into myofibers after intramuscular transplantation in vivo. Our results revealed the heterogeneity of muscle SP cells and suggest that CD31 - CD45 - SP cells participate in muscle regeneration

Muscle satellite cells are activated after exercise to exhaustion in Thoroughbred horses.

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Kawai, M; Aida, H; Hiraga, A; Miyata, H

2013-07-01

Although satellite cells are well known as muscle stem cells capable of adding myonuclei during muscle repair and hypertrophy, the response of satellite cells in horse muscles to a run to exhaustion is still unknown. To investigate the time course of satellite cell activation in Thoroughbred horse muscle after running to exhaustion. We hypothesised that this type of intense exercise would induce satellite cell activation in skeletal muscle similar to a resistance exercise. Nine de-trained Thoroughbred horses (6 geldings and 3 mares) aged 3-6 years were studied. Biopsy samples were taken from the gluteus medius muscle of the horses before and 1 min, 3 h, 1 day, 3 days, 1 week and 2 weeks after a treadmill run to exhaustion. The numbers of satellite cells for each fibre type were determined by using immunofluorescence staining. Total RNA was extracted from these samples, and the expressions of interleukin (IL)-6, paired box transcriptional factor (Pax) 7, myogenic differentiation 1 (MyoD), myogenin, proliferating cell nuclear antigen (PCNA), insulin-like growth factor (IGF)-I and hepatocyte growth factor (HGF) mRNA were analysed using real-time reverse transcription-PCR. The numbers of satellite cells were significantly increased in type I and IIa fibres at 1 week and in type IIa/x fibre at 2 weeks post exercise. The expression of IL-6 mRNA increased significantly by 3 h post exercise. The expression of PCNA mRNA also increased by 1 day after running, indicating that running can initiate satellite cell proliferation. The expression of Pax7, MyoD, myogenin, IGF-I and HGF mRNA peaked at 1 week post exercise. Satellite cell activation and proliferation could be enhanced after a run to exhaustion without detectable injury as assessed by the histochemical analysis. Understanding the response of satellite cell activation to running exercise provides fundamental information about the skeletal muscle adaptation in Thoroughbred horses. © 2012 EVJ Ltd.

Selection of suitable prodrug candidates for in vivo studies via in vitro studies; the correlation of prodrug stability in between cell culture homogenates and human tissue homogenates.

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Tsume, Yasuhiro; Amidon, Gordon L

2012-01-01

To determine the correlations/discrepancies of drug stabilities between in the homogenates of human culture cells and of human tissues. Amino acid/dipeptide monoester prodrugs of floxuridine were chosen as the model drugs. The stabilities (half-lives) of floxuridine prodrugs in human tissues (pancreas, liver, and small intestine) homogenates were obtained and compared with ones in cell culture homogenates (AcPC-1, Capan-2, and Caco-2 cells) as well as human liver microsomes. The correlations of prodrug stability in human small bowel tissue homogenate vs. Caco-2 cell homogenate, human liver tissue homogenate vs. human liver microsomes, and human pancreatic tissue homogenate vs. pancreatic cell, AsPC-1 and Capan-2, homogenates were examined. The stabilities of floxuridine prodrugs in human small bowel homogenate exhibited the great correlation to ones in Caco-2 cell homogenate (slope = 1.0-1.3, r2 = 0.79-0.98). The stability of those prodrugs in human pancreas tissue homogenate also exhibited the good correlations to ones in AsPC-1 and Capan-2 cells homogenates (slope = 0.5-0.8, r2 = 0.58-0.79). However, the correlations of prodrug stabilities between in human liver tissue homogenates and in human liver microsomes were weaker than others (slope = 1.3-1.9, r2 = 0.07-0.24). The correlations of drug stabilities in cultured cell homogenates and in human tissue homogenates were compared. Those results exhibited wide range of correlations between in cell homogenate and in human tissue homogenate (r2 = 0.07 - 0.98). Those in vitro studies in cell homogenates would be good tools to predict drug stabilities in vivo and to select drug candidates for further developments. In the series of experiments, 5'-O-D-valyl-floxuridine and 5'-O-L-phenylalanyl-L-tyrosyl-floxuridine would be selected as candidates of oral drug targeting delivery for cancer chemotherapy due to their relatively good stabilities compared to other tested prodrugs.

Cultured smooth muscle cells of the human vesical sphincter are more sensitive to histamine than are detrusor smooth muscle cells.

Science.gov (United States)

Neuhaus, Jochen; Oberbach, Andreas; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

2006-05-01

To compare histamine receptor expression in cultured smooth muscle cells from the human detrusor and internal sphincter using receptor-specific agonists. Smooth muscle cells from the bladder dome and internal sphincter were cultured from 5 male patients undergoing cystectomy for bladder cancer therapy. Calcium transients in cells stimulated with carbachol, histamine, histamine receptor 1 (H1R)-specific heptanecarboxamide (HTMT), dimaprit (H2R), and R-(alpha)-methylhistamine (H3R) were measured by calcium imaging. Histamine receptor proteins were detected by Western blot analysis and immunocytochemistry. H1R, H2R, and H3R expression was found in tissue and cultured cells. Carbachol stimulated equal numbers of detrusor and sphincter cells (60% and 51%, respectively). Histamine stimulated significantly more cells than carbachol in detrusor (100%) and sphincter (99.34%) cells. Calcium responses to carbachol in detrusor and sphincter cells were comparable and did not differ from those to histamine in detrusor cells. However, histamine and specific agonists stimulated more sphincter cells than did carbachol (P <0.001), and the calcium increase was greater in sphincter cells than in detrusor cells. Single cell analysis revealed comparable H2R responses in detrusor and sphincter cells, but H1R and H3R-mediated calcium reactions were significantly greater in sphincter cells. Histamine very effectively induces calcium release in smooth muscle cells. In sphincter cells, histamine is even more effective than carbachol regarding the number of reacting cells and the intracellular calcium increase. Some of the variability in the outcome of antihistaminic interstitial cystitis therapies might be caused by the ineffectiveness of the chosen antihistaminic or unintentional weakening of sphincteric function.

Skeletal-muscle CT, with special reference to polymyositis and myasthenia gravis

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Higashi, Yasuto; Ono, Shimato; Yasuda, Takeshi; Morimoto, Kenji; Terao, Akira; Shirabe, Teruo; Yokobayashi, Tsuneo (Kawasaki Medical School, Kurashiki, Okayama (Japan))

1984-10-01

We here report on skeletal-muscle CT at the thigh level as studied using a whole-body CT scanner, with special reference to polymyositis (PM) and myasthenia gravis (MG). Early diseased muscles appeared homogenous and were likely to be almost normal. The first sign of muscular atrophy was the appearance of small, patchy or linear, low-density tissues in several muscles. These low-density tissues gradually increased in number until finally the diseased muscles were totally replaced by low-density tissue. These pathological findings were more severe in PM than in MG. There was a maldistribution of low-density tissue in several cases of PM. According to these findings, skeletal-muscle CT was thought to be of great help for the recognition of the general condition of muscles and for the follow-up on the patients. We think skeletal-muscle CT has a very practical application for the better selection of suitable muscular biopsy and EMG sites and for the better clinical interpretation of these findings.

Skeletal-muscle CT, with special reference to polymyositis and myasthenia gravis

International Nuclear Information System (INIS)

Higashi, Yasuto; Ono, Shimato; Yasuda, Takeshi; Morimoto, Kenji; Terao, Akira; Shirabe, Teruo; Yokobayashi, Tsuneo

1984-01-01

We here report on skeletal-muscle CT at the thigh level as studied using a whole-body CT scanner, with special reference to polymyositis (PM) and myasthenia gravis (MG). Early diseased muscles appeared homogenous and were likely to be almost normal. The first sign of muscular atrophy was the apperance of small, patchy or linear, low-density tissues in several muscles. These low-density tissues gradually increased in number until finally the diseased muscles were totally replaced by low-density tissue. These pathological findings were more severe in PM than in MG. There was a maldistribution of low-density tissue in several cases of PM. According to these findings, skeletal-muscle CT was thought to be of great help for the recognition of the general condition of muscles and for the follow-up on the patients. We think skeletal-muscle CT has a very practical application for the better selection of suitable muscular biopsy and EMG sites and for the better clinical interpretation of these findings. (author)

Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification.

Science.gov (United States)

Moretti, Alessandra; Caron, Leslie; Nakano, Atsushi; Lam, Jason T; Bernshausen, Alexandra; Chen, Yinhong; Qyang, Yibing; Bu, Lei; Sasaki, Mika; Martin-Puig, Silvia; Sun, Yunfu; Evans, Sylvia M; Laugwitz, Karl-Ludwig; Chien, Kenneth R

2006-12-15

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

Catechins activate muscle stem cells by Myf5 induction and stimulate muscle regeneration.

Science.gov (United States)

Kim, A Rum; Kim, Kyung Min; Byun, Mi Ran; Hwang, Jun-Ha; Park, Jung Il; Oh, Ho Taek; Kim, Hyo Kyeong; Jeong, Mi Gyeong; Hwang, Eun Sook; Hong, Jeong-Ho

2017-07-22

Muscle weakness is one of the most common symptoms in aged individuals and increases risk of mortality. Thus, maintenance of muscle mass is important for inhibiting aging. In this study, we investigated the effect of catechins, polyphenol compounds in green tea, on muscle regeneration. We found that (-)-epicatechin gallate (ECG) and (-)-epigallocatechin-3-gallate (EGCG) activate satellite cells by induction of Myf5 transcription factors. For satellite cell activation, Akt kinase was significantly induced after ECG treatment and ECG-induced satellite cell activation was blocked in the presence of Akt inhibitor. ECG also promotes myogenic differentiation through the induction of myogenic markers, including Myogenin and Muscle creatine kinase (MCK), in satellite and C2C12 myoblast cells. Finally, EGCG administration to mice significantly increased muscle fiber size for regeneration. Taken together, the results suggest that catechins stimulate muscle stem cell activation and differentiation for muscle regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Homogeneous forming technology of composite materials and its application to dispersion nuclear fuel

International Nuclear Information System (INIS)

Hong, Soon Hyun; Ryu, Ho Jin; Sohn, Woong Hee; Kim, Chang Kyu

1997-01-01

Powder metallurgy processing technique of metal matrix composites is reviewed and its application to process homogeneous dispersion nuclear fuel is considered. The homogeneous mixing of reinforcement with matrix powders is very important step to process metal matrix composites. The reinforcement with matrix powders is very important step to process metal matrix composites. The reinforcement can be ceramic particles, whiskers or chopped fibers having high strength and high modulus. The blended powders are consolidated into billets and followed by various deformation processing, such as extrusion, forging, rolling or spinning into final usable shapes. Dispersion nuclear fuel is a class of metal matrix composite consisted of dispersed U-compound fuel particles and metallic matrix. Dispersion nuclear fuel is fabricated by powder metallurgy process such as hot pressing followed by hot extrusion, which is similar to that of SiC/Al metal matrix composite. The fabrication of homogeneous dispersion nuclear fuel is very difficult mainly due to the inhomogeneous mixing characteristics of the powders from quite different densities between uranium alloy powders and aluminum powders. In order to develop homogeneous dispersion nuclear fuel, it is important to investigate the effect of powder characteristics and mixing techniques on homogeneity of dispersion nuclear fuel. An new quantitative analysis technique of homogeneity is needed to be developed for more accurate analysis of homogeneity in dispersion nuclear fuel. (author). 28 refs., 7 figs., 1tab

Homogeneity spoil spectroscopy

International Nuclear Information System (INIS)

Hennig, J.; Boesch, C.; Martin, E.; Grutter, R.

1987-01-01

One of the problems of in vivo MR spectroscopy of P-31 is spectra localization. Surface coil spectroscopy, which is the method of choice for clinical applications, suffers from the high-intensity signal from subcutaneous muscle tissue, which masks the spectrum of interest from deeper structures. In order to suppress this signal while maintaining the simplicity of surface coil spectroscopy, the authors introduced a small sheet of ferromagnetically dotted plastic between the surface coil and the body. This sheet destroys locally the field homogeneity and therefore all signal from structures around the coil. The very high reproducibility of the simple experimental procedure allows long-term studies important for monitoring tumor therapy

"Known Unknowns": Current Questions in Muscle Satellite Cell Biology.

Science.gov (United States)

Cornelison, Ddw

2018-01-01

Our understanding of satellite cells, now known to be the obligate stem cells of skeletal muscle, has increased dramatically in recent years due to the introduction of new molecular, genetic, and technical resources. In addition to their role in acute repair of damaged muscle, satellite cells are of interest in the fields of aging, exercise, neuromuscular disease, and stem cell therapy, and all of these applications have driven a dramatic increase in our understanding of the activity and potential of satellite cells. However, many fundamental questions of satellite cell biology remain to be answered, including their emergence as a specific lineage, the degree and significance of heterogeneity within the satellite cell population, the roles of their interactions with other resident and infiltrating cell types during homeostasis and regeneration, and the relative roles of intrinsic vs extrinsic factors that may contribute to satellite cell dysfunction in the context of aging or disease. This review will address the current state of these open questions in satellite cell biology. © 2018 Elsevier Inc. All rights reserved.

Preliminary development of thermal nuclear cell homogenization code

International Nuclear Information System (INIS)

Su'ud, Z.; Shafii, M. A.; Yudha, S. P.; Waris, A.; Rijal, K.

2012-01-01

Nuclear fuel cell homogenization for thermal reactors usually include three main parts, i.e., fast energy resonance part which usually adopt narrow resonance approximation to treat the resonance, low (intermediate) energy region in which the resonance can not be treated accurately using NR approximation and therefore we should use intermediate resonance treatment, and thermal energy region (very low) in which the effect of thermal must be treated properly. In n this study the application of the intermediate resonance approximation treatment for low energy nuclear resonance is discussed. The method is iterative based. As a sample the method is applied in U-235 low lying resonance and the result is presented and discussed.

Novel method to ascertain chromatin accessibility at specific genomic loci from frozen brain homogenates and laser capture microdissected defined cells.

Science.gov (United States)

Delvaux, Elaine; Mastroeni, Diego; Nolz, Jennifer; Coleman, Paul D

2016-06-01

We describe a novel method for assessing the "open" or "closed" state of chromatin at selected locations within the genome. This method combines the use of Benzonase, which can digest DNA in the presence of actin, with qPCR to define digested regions. We demonstrate the application of this method in brain homogenates and laser captured cells. We also demonstrate application to selected sites within more than one gene and multiple sites within one gene. We demonstrate the validity of the method by treating cells with valproate, known to render chromatin more permissive, and by comparison with classical digestion with DNase I in an in vitro preparation. Although we demonstrate the use of this method in brain tissue we also recognize its applicability to other tissue types.

Muscle type-specific responses to NAD+ salvage biosynthesis promote muscle function in Caenorhabditis elegans.

Science.gov (United States)

Vrablik, Tracy L; Wang, Wenqing; Upadhyay, Awani; Hanna-Rose, Wendy

2011-01-15

Salvage biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) from nicotinamide (NAM) lowers NAM levels and replenishes the critical molecule NAD(+) after it is hydrolyzed. This pathway is emerging as a regulator of multiple biological processes. Here we probe the contribution of the NAM-NAD(+) salvage pathway to muscle development and function using Caenorhabditis elegans. C. elegans males with mutations in the nicotinamidase pnc-1, which catalyzes the first step of this NAD(+) salvage pathway, cannot mate due to a spicule muscle defect. Multiple muscle types are impaired in the hermaphrodites, including body wall muscles, pharyngeal muscles and vulval muscles. An active NAD(+) salvage pathway is required for optimal function of each muscle cell type. However, we found surprising muscle-cell-type specificity in terms of both the timing and relative sensitivity to perturbation of NAD(+) production or NAM levels. Active NAD(+) biosynthesis during development is critical for function of the male spicule protractor muscles during adulthood, but these muscles can surprisingly do without salvage biosynthesis in adulthood under the conditions examined. The body wall muscles require ongoing NAD(+) salvage biosynthesis both during development and adulthood for maximum function. The vulval muscles do not function in the presence of elevated NAM concentrations, but NAM supplementation is only slightly deleterious to body wall muscles during development or upon acute application in adults. Thus, the pathway plays distinct roles in different tissues. As NAM-NAD(+) biosynthesis also impacts muscle differentiation in vertebrates, we propose that similar complexities may be found among vertebrate muscle cell types. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

Application of the principles of systems biology and Wiener's cybernetics for analysis of regulation of energy fluxes in muscle cells in vivo.

Science.gov (United States)

Guzun, Rita; Saks, Valdur

2010-03-08

The mechanisms of regulation of respiration and energy fluxes in the cells are analyzed based on the concepts of systems biology, non-equilibrium steady state kinetics and applications of Wiener's cybernetic principles of feedback regulation. Under physiological conditions cardiac function is governed by the Frank-Starling law and the main metabolic characteristic of cardiac muscle cells is metabolic homeostasis, when both workload and respiration rate can be changed manifold at constant intracellular level of phosphocreatine and ATP in the cells. This is not observed in skeletal muscles. Controversies in theoretical explanations of these observations are analyzed. Experimental studies of permeabilized fibers from human skeletal muscle vastus lateralis and adult rat cardiomyocytes showed that the respiration rate is always an apparent hyperbolic but not a sigmoid function of ADP concentration. It is our conclusion that realistic explanations of regulation of energy fluxes in muscle cells require systemic approaches including application of the feedback theory of Wiener's cybernetics in combination with detailed experimental research. Such an analysis reveals the importance of limited permeability of mitochondrial outer membrane for ADP due to interactions of mitochondria with cytoskeleton resulting in quasi-linear dependence of respiration rate on amplitude of cyclic changes in cytoplasmic ADP concentrations. The system of compartmentalized creatine kinase (CK) isoenzymes functionally coupled to ANT and ATPases, and mitochondrial-cytoskeletal interactions separate energy fluxes (mass and energy transfer) from signalling (information transfer) within dissipative metabolic structures - intracellular energetic units (ICEU). Due to the non-equilibrium state of CK reactions, intracellular ATP utilization and mitochondrial ATP regeneration are interconnected by the PCr flux from mitochondria. The feedback regulation of respiration occurring via cyclic fluctuations of

Single molecular image of cytosolic free Ca2+ of skeletal muscle cells in rats pre- and post-exercise-induced fatigue

Science.gov (United States)

Liu, Yi; Zhang, Heming; Zhao, Yanping; Liu, Zhiming

2009-08-01

A growing body of literature indicated the cytosolic free Ca2+ concentration of skeletal muscle cells changes significantly during exercise-induced fatigue. But it is confusing whether cytosolic free Ca2+ concentration increase or decrease. Furthermore, current researches mainly adopt muscle tissue homogenate as experiment material, but the studies based on cellular and subcellular level is seldom. This study is aimed to establish rat skeletal muscle cell model of exercise-induced fatigue, and confirm the change of cytosolic free Ca2+ concentration of skeletal muscle cells in rats preand post- exercise-induced fatigue. In this research, six male Wistar rats were randomly divided into two groups: control group (n=3) and exercise-induced fatigue group (n=3). The former group were allowed to freely move and the latter were forced to loaded swimming to exhaustive. Three days later, all the rats were sacrificed, the muscle tissue from the same site of skeletal muscle were taken out and digested to cells. After primary culture of the two kinds of skeletal muscle cells from tissue, a fluorescent dye-Fluo-3 AM was used to label the cytosolic free Ca2+. The fluorescent of Ca2+ was recorded by confocal laser scanning microscopy. The results indicated that, the Ca2+ fluorescence intensity of cells from the rat of exercise-induced fatigue group was significantly higher than those in control group. In conclusion, cytosolic free Ca2+ concentration of skeletal muscle cells has a close relation with exercise-induced fatigue, and the increase of cytosolic free Ca2+ concentration may be one of the important factors of exercise-induced fatigue.

Aortic smooth muscle cell proteoglycan synthesis in relation to atherosclerosis

International Nuclear Information System (INIS)

Edwards, I.J.

1989-01-01

Proteoglycans (PG) are implicated in atherogenesis by their effects on tissue permeability and cell proliferation and their interaction with plasma low density lipoproteins. Using the pigeon model in which an atherosclerosis-susceptible (WC) and -resistant (SR) breed can be compared, PG synthesis by cultured aortic smooth muscle cells was examined by the use of [ 35 S]-sodium sulfate and [ 3 H]-serine or [ 3 H]-glucosamine as labeling precursors. In both SR and WC cells, the majority of newly synthesized PG were secreted into the media. Chondroitin sulfate (CS) PG and dermatan sulfate (DS) PG were the major PG produced. Total PG production was consistently lower in WC compared to SR cultures due in part to reduce PG synthesis but also to degradation of newly synthesized PG. Since increased DS-PG accompanines atherosclerosis progression, experiments were designed to test the hypothesis that macrophages modulate smooth muscle cell metabolism to cause increase DS-PG production. Cultured WC aortic smooth muscle cells were exposed to the media of cholesteryl ester-loaded pigeon peritoneal macrophages or a macrophage cell line P388D1 and the production of PG examined. Increasing concentration of conditioned media from both types of macrophages caused increased incorporation of 35 S-sulfate into secreted PG, but no change in cell-associated PG. Lipopolysaccharide activation of P388D1 cells enhanced the effect

Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells.

Science.gov (United States)

Kreuzer, J; Denger, S; Reifers, F; Beisel, C; Haack, K; Gebert, J; Kübler, W

1996-07-01

Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and

Neuromuscular electrical stimulation as a method to maximize the beneficial effects of muscle stem cells transplanted into dystrophic skeletal muscle.

Directory of Open Access Journals (Sweden)

Giovanna Distefano

Full Text Available Cellular therapy is a potential approach to improve the regenerative capacity of damaged or diseased skeletal muscle. However, its clinical use has often been limited by impaired donor cell survival, proliferation and differentiation following transplantation. Additionally, functional improvements after transplantation are all-too-often negligible. Because the host microenvironment plays an important role in the fate of transplanted cells, methods to modulate the microenvironment and guide donor cell behavior are warranted. The purpose of this study was to investigate whether the use of neuromuscular electrical stimulation (NMES for 1 or 4 weeks following muscle-derived stem cell (MDSC transplantation into dystrophic skeletal muscle can modulate the fate of donor cells and enhance their contribution to muscle regeneration and functional improvements. Animals submitted to 4 weeks of NMES after transplantation demonstrated a 2-fold increase in the number of dystrophin+ myofibers as compared to control transplanted muscles. These findings were concomitant with an increased vascularity in the MDSC+NMES group when compared to non-stimulated counterparts. Additionally, animals subjected to NMES (with or without MDSC transplantation presented an increased maximal specific tetanic force when compared to controls. Although cell transplantation and/or the use of NMES resulted in no changes in fatigue resistance, the combination of both MDSC transplantation and NMES resulted in a faster recovery from fatigue, when compared to non-injected and non-stimulated counterparts. We conclude that NMES is a viable method to improve MDSC engraftment, enhance dystrophic muscle strength, and, in combination with MDSC transplantation, improve recovery from fatigue. These findings suggest that NMES may be a clinically-relevant adjunct approach for cell transplantation into skeletal muscle.

Tissue-specific stem cells: Lessons from the skeletal muscle satellite cell

Science.gov (United States)

Brack, Andrew S.; Rando, Thomas A.

2012-01-01

In 1961, the satellite cell was first identified when electron microscopic examination of skeletal muscle demonstrated a cell wedged between the plasma membrane of the muscle fiber and the basement membrane. In recent years it has been conclusively demonstrated that the satellite cell is the primary cellular source for muscle regeneration and is equipped with the potential to self renew, thus functioning as a bone fide skeletal muscle stem cell (MuSC). As we move past the 50th anniversary of the satellite cell, we take this opportunity to discuss the current state of the art and dissect the unknowns in the MuSC field. PMID:22560074

Novel method to ascertain chromatin accessibility at specific genomic loci from frozen brain homogenates and laser capture microdissected defined cells

Directory of Open Access Journals (Sweden)

Elaine Delvaux

2016-06-01

Full Text Available We describe a novel method for assessing the “open” or “closed” state of chromatin at selected locations within the genome. This method combines the use of Benzonase, which can digest DNA in the presence of actin, with quantitative polymerase chain reaction to define digested regions. We demonstrate the application of this method in brain homogenates and laser captured cells. We also demonstrate application to selected sites within more than 1 gene and multiple sites within 1 gene. We demonstrate the validity of the method by treating cells with valproate, known to render chromatin more permissive, and by comparison with classical digestion with DNase I in an in vitro preparation. Although we demonstrate the use of this method in brain tissue, we also recognize its applicability to other tissue types.

Engineered matrices for skeletal muscle satellite cell engraftment and function.

Science.gov (United States)

Han, Woojin M; Jang, Young C; García, Andrés J

2017-07-01

Regeneration of traumatically injured skeletal muscles is severely limited. Moreover, the regenerative capacity of skeletal muscle declines with aging, further exacerbating the problem. Recent evidence supports that delivery of muscle satellite cells to the injured muscles enhances muscle regeneration and reverses features of aging, including reduction in muscle mass and regenerative capacity. However, direct delivery of satellite cells presents a challenge at a translational level due to inflammation and donor cell death, motivating the need to develop engineered matrices for muscle satellite cell delivery. This review will highlight important aspects of satellite cell and their niche biology in the context of muscle regeneration, and examine recent progresses in the development of engineered cell delivery matrices designed for skeletal muscle regeneration. Understanding the interactions of muscle satellite cells and their niche in both native and engineered systems is crucial to developing muscle pathology-specific cell- and biomaterial-based therapies. Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

Science.gov (United States)

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F.

2017-01-01

ABSTRACT Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. PMID:28188264

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

Directory of Open Access Journals (Sweden)

Ludovic Arandel

2017-04-01

Full Text Available Myotonic dystrophy type 1 (DM1 and type 2 (DM2 are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations.

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.

Science.gov (United States)

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F; Furling, Denis

2017-04-01

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. © 2017. Published by The Company of Biologists Ltd.

Quercetin inhibits adipogenesis of muscle progenitor cells in vitro

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Tomoko Funakoshi

2018-03-01

Full Text Available Muscle satellite cells are committed myogenic progenitors capable of contributing to myogenesis to maintain adult muscle mass and function. Several experiments have demonstrated that muscle satellite cells can differentiate into adipocytes in vitro, supporting the mesenchymal differentiation potential of these cells. Moreover, muscle satellite cells may be a source of ectopic muscle adipocytes, explaining the lipid accumulation often observed in aged skeletal muscle (sarcopenia and in muscles of patients` with diabetes. Quercetin, a polyphenol, is one of the most abundant flavonoids distributed in edible plants, such as onions and apples, and possesses antioxidant, anticancer, and anti-inflammatory properties. In this study, we examined whether quercetin inhibited the adipogenesis of muscle satellite cells in vitro with primary cells from rat limbs by culture in the presence of quercetin under adipogenic conditions. Morphological observations, Oil Red-O staining results, triglyceride content analysis, and quantitative reverse transcription polymerase chain reaction revealed that quercetin was capable of inhibiting the adipogenic induction of muscle satellite cells into adipocytes in a dose-dependent manner by suppressing the transcript levels of adipogenic markers, such as peroxisome proliferator-activated receptor-γ and fatty acid binding protein 4. Our results suggested that quercetin inhibited the adipogenesis of muscle satellite cells in vitro by suppressing the transcription of adipogenic markers. Keywords: Quercetin, Muscle satellite cell, Differentiation, Intramuscular lipid

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.

Immunohistochemical detection of interleukin-6 in human skeletal muscle fibers following exercise

DEFF Research Database (Denmark)

Penkowa, Milena; Keller, Charlotte; Keller, Pernille

2003-01-01

individuals. The IL-6 immunostainings of skeletal muscle cells were homogeneous and without difference between muscle fiber types. The IL-6 mRNA peaked immediately after the exercise, and, in accordance, the IL-6 protein expression within muscle cells was most pronounced around 3 h post-exercise. However......, the finding that plasma IL-6 concentration peaked in the end of exercise indicates a high turnover of muscle-derived IL-6. In conclusion, the finding of marked IL-6 protein expression exclusively within skeletal muscle fibers following exercise demonstrates that skeletal muscle fibers of all types...

Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

DEFF Research Database (Denmark)

Hellsten, Ylva; Frandsen, Ulrik

1997-01-01

1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P Skeletal muscle cells were...... in the extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P contracted, but not in the moderately contracted muscle cells...

A comparison of maximal bioenergetic enzyme activities obtained with commonly used homogenization techniques.

Science.gov (United States)

Grace, M; Fletcher, L; Powers, S K; Hughes, M; Coombes, J

1996-12-01

Homogenization of tissue for analysis of bioenergetic enzyme activities is a common practice in studies examining metabolic properties of skeletal muscle adaptation to disease, aging, inactivity or exercise. While numerous homogenization techniques are in use today, limited information exists concerning the efficacy of specific homogenization protocols. Therefore, the purpose of this study was to compare the efficacy of four commonly used approaches to homogenizing skeletal muscle for analysis of bioenergetic enzyme activity. The maximal enzyme activity (Vmax) of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured from homogenous muscle samples (N = 48 per homogenization technique) and used as indicators to determine which protocol had the highest efficacy. The homogenization techniques were: (1) glass-on-glass pestle; (2) a combination of a mechanical blender and a teflon pestle (Potter-Elvehjem); (3) a combination of the mechanical blender and a biological detergent; and (4) the combined use of a mechanical blender and a sonicator. The glass-on-glass pestle homogenization protocol produced significantly higher (P pestle homogenization protocol is the technique of choice for studying bioenergetic enzyme activity in skeletal muscle.

Hamster thecal cells express muscle characteristics

International Nuclear Information System (INIS)

Self, D.A.; Schroeder, P.C.; Gown, A.M.

1988-01-01

Contraction of the follicular wall about the time of ovulation appears to be a coordinated event; however, the cells that mediate it remain poorly studied. We examined the theca externa cells in the wall of hamster follicles for the presence of a functional actomyosin system, both in developing follicles and in culture. We used a monoclonal antibody (HHF35) that recognizes the alpha and gamma isoelectric variants of actin normally found in muscle, but not the beta variant associated with non-muscle sources, to evaluate large preovulatory follicles for actin content and composition. Antibody staining of sectioned ovaries showed intense circumferential reactivity in the outermost wall of developing follicles. Immunoblots from two-dimensional gels of theca externa lysates demonstrated the presence of the two muscle-specific isozymes of actin. Immunofluorescence of cultured follicular cells pulse-labeled with [3H] thymidine (for autoradiographic detection of DNA replication) revealed the presence, in many dividing cells, of actin filaments aligned primarily along the longitudinal axis of the cells. In cultures exposed to the calcium ionophore A23187 (10(-4) M) for varying periods (5 min to 1 h), contraction of many individual muscle-actin-positive cells was observed. Immunofluorescence of these cells, fixed immediately after ionophore-induced contraction, revealed compaction of the actin filaments. Our findings demonstrate that the cells of the theca externa contain muscle actins from an early stage and that these cells are capable of contraction even while proliferating in subconfluent cultures. They suggest that follicular growth may include a naturally occurring developmental sequence in which a contractile cell type proliferates in the differentiated state

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.

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

Application of the Principles of Systems Biology and Wiener’s Cybernetics for Analysis of Regulation of Energy Fluxes in Muscle Cells in Vivo

Science.gov (United States)

Guzun, Rita; Saks, Valdur

2010-01-01

The mechanisms of regulation of respiration and energy fluxes in the cells are analyzed based on the concepts of systems biology, non-equilibrium steady state kinetics and applications of Wiener’s cybernetic principles of feedback regulation. Under physiological conditions cardiac function is governed by the Frank-Starling law and the main metabolic characteristic of cardiac muscle cells is metabolic homeostasis, when both workload and respiration rate can be changed manifold at constant intracellular level of phosphocreatine and ATP in the cells. This is not observed in skeletal muscles. Controversies in theoretical explanations of these observations are analyzed. Experimental studies of permeabilized fibers from human skeletal muscle vastus lateralis and adult rat cardiomyocytes showed that the respiration rate is always an apparent hyperbolic but not a sigmoid function of ADP concentration. It is our conclusion that realistic explanations of regulation of energy fluxes in muscle cells require systemic approaches including application of the feedback theory of Wiener’s cybernetics in combination with detailed experimental research. Such an analysis reveals the importance of limited permeability of mitochondrial outer membrane for ADP due to interactions of mitochondria with cytoskeleton resulting in quasi-linear dependence of respiration rate on amplitude of cyclic changes in cytoplasmic ADP concentrations. The system of compartmentalized creatine kinase (CK) isoenzymes functionally coupled to ANT and ATPases, and mitochondrial-cytoskeletal interactions separate energy fluxes (mass and energy transfer) from signalling (information transfer) within dissipative metabolic structures – intracellular energetic units (ICEU). Due to the non-equilibrium state of CK reactions, intracellular ATP utilization and mitochondrial ATP regeneration are interconnected by the PCr flux from mitochondria. The feedback regulation of respiration occurring via cyclic fluctuations

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle

Science.gov (United States)

McCarthy, John J.; Mula, Jyothi; Miyazaki, Mitsunori; Erfani, Rod; Garrison, Kelcye; Farooqui, Amreen B.; Srikuea, Ratchakrit; Lawson, Benjamin A.; Grimes, Barry; Keller, Charles; Van Zant, Gary; Campbell, Kenneth S.; Esser, Karyn A.; Dupont-Versteegden, Esther E.; Peterson, Charlotte A.

2011-01-01

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca2+ sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells. PMID:21828094

Cell homogenization methods for pin-by-pin core calculations tested in slab geometry

International Nuclear Information System (INIS)

Yamamoto, Akio; Kitamura, Yasunori; Yamane, Yoshihiro

2004-01-01

In this paper, performances of spatial homogenization methods for fuel or non-fuel cells are compared in slab geometry in order to facilitate pin-by-pin core calculations. Since the spatial homogenization methods were mainly developed for fuel assemblies, systematic study of their performance for the cell-level homogenization has not been carried out. Importance of cell-level homogenization is recently increasing since the pin-by-pin mesh core calculation in actual three-dimensional geometry, which is less approximate approach than current advanced nodal method, is getting feasible. Four homogenization methods were investigated in this paper; the flux-volume weighting, the generalized equivalence theory, the superhomogenization (SPH) method and the nonlinear iteration method. The last one, the nonlinear iteration method, was tested as the homogenization method for the first time. The calculations were carried out in simplified colorset assembly configurations of PWR, which are simulated by slab geometries, and homogenization performances were evaluated through comparison with the reference cell-heterogeneous calculations. The calculation results revealed that the generalized equivalence theory showed best performance. Though the nonlinear iteration method can significantly reduce homogenization error, its performance was not as good as that of the generalized equivalence theory. Through comparison of the results obtained by the generalized equivalence theory and the superhomogenization method, important byproduct was obtained; deficiency of the current superhomogenization method, which could be improved by incorporating the 'cell-level discontinuity factor between assemblies', was clarified

Bone marrow mesenchymal cells improve muscle function in a skeletal muscle re-injury model.

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Bruno M Andrade

Full Text Available Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively. Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model.

The muscle stem cell niche : regulation of satellite cells during regeneration

NARCIS (Netherlands)

Boonen, K.J.M.; Post, M.J.

2008-01-01

Satellite cells are considered to be adult skeletal muscle stem cells. Their ability to regenerate large muscle defects is highly dependent on their specific niche. When these cells are cultured in vitro, the loss of this niche leads to a loss of proliferative capacity and defective regeneration

Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

Science.gov (United States)

Starkey, Jessica D.; Yamamoto, Masakazu; Yamamoto, Shoko; Goldhamer, David J.

2011-01-01

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoDiCre/+;R26REYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate. PMID:21339173

An α-smooth muscle actin (acta2/αsma zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells.

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Thomas R Whitesell

Full Text Available Mural cells of the vascular system include vascular smooth muscle cells (SMCs and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is α smooth muscle actin (acta2; αsma, which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels.

Human lung mast cells modulate the functions of airway smooth muscle cells in asthma.

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Alkhouri, H; Hollins, F; Moir, L M; Brightling, C E; Armour, C L; Hughes, J M

2011-09-01

Activated mast cell densities are increased on the airway smooth muscle in asthma where they may modulate muscle functions and thus contribute to airway inflammation, remodelling and airflow obstruction. To determine the effects of human lung mast cells on the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma. Freshly isolated human lung mast cells were stimulated with IgE/anti-IgE. Culture supernatants were collected after 2 and 24 h and the mast cells lysed. The supernatants/lysates were added to serum-deprived, subconfluent airway smooth muscle cells for up to 48 h. Released chemokines and extracellular matrix were measured by ELISA, proliferation was quantified by [(3) H]-thymidine incorporation and cell counting, and intracellular signalling by phospho-arrays. Mast cell 2-h supernatants reduced CCL11 and increased CXCL8 and fibronectin production from both asthmatic and nonasthmatic muscle cells. Leupeptin reversed these effects. Mast cell 24-h supernatants and lysates reduced CCL11 release from both muscle cell types but increased CXCL8 release by nonasthmatic cells. The 24-h supernatants also reduced asthmatic, but not nonasthmatic, muscle cell DNA synthesis and asthmatic cell numbers over 5 days through inhibiting extracellular signal-regulated kinase (ERK) and phosphatidylinositol (PI3)-kinase pathways. However, prostaglandins, thromboxanes, IL-4 and IL-13 were not involved in reducing the proliferation. Mast cell proteases and newly synthesized products differentially modulated the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma. Thus, mast cells may modulate their own recruitment and airway smooth muscle functions locally in asthma. © 2011 John Wiley & Sons A/S.

CD133+ cells derived from skeletal muscles of Duchenne muscular dystrophy patients have a compromised myogenic and muscle regenerative capability.

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Meng, Jinhong; Muntoni, Francesco; Morgan, Jennifer

2018-05-12

Cell-mediated gene therapy is a possible means to treat muscular dystrophies like Duchenne muscular dystrophy. Autologous patient stem cells can be genetically-corrected and transplanted back into the patient, without causing immunorejection problems. Regenerated muscle fibres derived from these cells will express the missing dystrophin protein, thus improving muscle function. CD133+ cells derived from normal human skeletal muscle contribute to regenerated muscle fibres and form muscle stem cells after their intra-muscular transplantation into an immunodeficient mouse model. But it is not known whether CD133+ cells derived from DMD patient muscles have compromised muscle regenerative function. To test this, we compared CD133+ cells derived from DMD and normal human muscles. DMD CD133+ cells had a reduced capacity to undergo myogenic differentiation in vitro compared with CD133+ cells derived from normal muscle. In contrast to CD133+ cells derived from normal human muscle, those derived from DMD muscle formed no satellite cells and gave rise to significantly fewer muscle fibres of donor origin, after their intra-muscular transplantation into an immunodeficient, non-dystrophic, mouse muscle. DMD CD133+ cells gave rise to more clones of smaller size and more clones that were less myogenic than did CD133+ cells derived from normal muscle. The heterogeneity of the progeny of CD133+ cells, combined with the reduced proliferation and myogenicity of DMD compared to normal CD133+ cells, may explain the reduced regenerative capacity of DMD CD133+ cells. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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

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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.

Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

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Zhang, Meixiang; Ngo, Justine; Pirozzi, Filomena; Sun, Ying-Pu; Wynshaw-Boris, Anthony

2018-03-15

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically

Application of multi-pass high pressure homogenization under variable temperature regimes to induce autolysis of wine yeasts.

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Comuzzo, Piergiorgio; Calligaris, Sonia; Iacumin, Lucilla; Ginaldi, Federica; Voce, Sabrina; Zironi, Roberto

2017-06-01

The effects of the number of passes and processing temperature management (controlled vs. uncontrolled) were investigated during high pressure homogenization-induced autolysis of Saccharomyces bayanus wine yeasts, treated at 150MPa. Both variables were able to affect cell viability, and the release of soluble molecules (free amino acids, proteins and glucidic colloids), but the effect of temperature was more important. S. bayanus cells were completely inactivated in 10 passes without temperature control (corresponding to a processing temperature of 75°C). The two processing variables also affected the volatile composition of the autolysates produced: higher temperatures led to a lower concentration of volatile compounds. The management of the operating conditions may allow the compositional characteristics of the products to be modulated, making them suitable for different winemaking applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Advancements in stem cells treatment of skeletal muscle wasting

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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.

Engineered CHO cells for production of diverse, homogeneous glycoproteins

DEFF Research Database (Denmark)

Yang, Zhang; Wang, Shengjun; Halim, Adnan

2015-01-01

Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferas...

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

Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression.

Science.gov (United States)

Woodruff, Prescott G; Dolganov, Gregory M; Ferrando, Ronald E; Donnelly, Samantha; Hays, Steven R; Solberg, Owen D; Carter, Roderick; Wong, Hofer H; Cadbury, Peggy S; Fahy, John V

2004-05-01

Bronchial hyperresponsiveness in mild to moderate asthma may result from airway smooth muscle cell proliferation or acquisition of a hypercontractile phenotype. Because these cells have not been well characterized in mild to moderate asthma, we examined the morphometric and gene expression characteristics of smooth muscle cells in this subgroup of patients with asthma. Using bronchial biopsies from 14 subjects with mild to moderate asthma and 15 control subjects, we quantified smooth muscle cell morphology by stereology and the expression of a panel of genes related to a hypercontractile phenotype of airway smooth muscle, using laser microdissection and two-step real-time polymerase chain reaction. We found that airway smooth muscle cell size was similar in both groups, but cell number was nearly twofold higher in subjects with asthma (p = 0.03), and the amount of smooth muscle in the submucosa was increased 50-83% (p 0.1). We conclude that airway smooth muscle proliferation is a pathologic characteristic of subjects with mild to moderate asthma. However, smooth muscle cells in mild to moderate asthma do not show hypertrophy or gene expression changes of a hypercontractile phenotype observed in vitro.

Muscle-Derived Cells for Treatment of Iatrogenic Sphincter Damage and Urinary Incontinence in Men

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H. Gerullis

2012-01-01

Full Text Available Introduction. Aim of this study was to assess the safety and efficacy of injection of autologous muscle-derived cells into the urinary sphincter for treatment of postprostatectomy urinary incontinence in men and to characterize the injected cells prior to transplantation. Methods. 222 male patients with stress urinary incontinence and sphincter damage after uroloical procedures were treated with transurethral injection of autologous muscle-derived cells. The transplanted cells were investigated after cultivation and prior to application by immunocytochemistry using different markers of myogenic differentiation. Feasibility and functionality assessment was achieved with a follow-up of at least 12 months. Results. Follow-up was at least 12 months. Of the 222 treated patients, 120 responded to therapy of whom 26 patients (12% were continent, and 94 patients (42% showed improvement. In 102 (46% patients, the therapy was ineffective. Clinical improvement was observed on average 4.7 months after transplantation and continued in all improved patients. The cells injected into the sphincter were at least ~50% of myogenic origin and representative for early stages of muscle cell differentiation. Conclusions. Transurethral injection of muscle-derived cells into the damaged urethral sphincter of male patients is a safe procedure. Transplanted cells represent different phases of myogenic differentiation.

A population of Pax7-expressing muscle progenitor cells show differential responses to muscle injury dependent on developmental stage and injury extent

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Stefanie eKnappe

2015-08-01

Full Text Available Muscle regeneration in vertebrates occurs by the activation of quiescent progenitor cells that express pax7 and replace and repair damaged fibers. We have developed a mechanical injury paradigm in zebrafish to determine whether developmental stage and injury size affect the regeneration dynamics of damaged muscle. We found that both small, focal injuries and large injuries affecting the entire myotome lead to the expression of myf5 and myogenin. Their expression was prolonged in older larvae, indicating a slower process of regeneration. We characterized the endogenous behavior of a population of muscle-resident Pax7-expressing cells using a pax7a:eGFP transgenic line and found that GFP+ cell migration in the myotome dramatically declined between 5 and 7 days post fertilization (dpf. Following a small injury, we observed that GFP+ cells responded by extending processes, before migrating to the injured fibers. Furthermore, these cells responded more rapidly to injury in 4dpf larvae compared to 7dpf. Interestingly, we did not see GFP+ fibers after repair of small injuries, indicating that pax7a-expressing cells did not contribute to fiber formation in this injury context. On the contrary, numerous GFP+ fibers could be observed after a large single myotome injury. Both injury models were accompanied by an increased number of proliferating GFP+ cells, which was more pronounced in larvae injured at 4dpf than 7dpf, This indicates intriguing developmental differences, even at these relatively early ages. Our data also suggests an interesting disparity in the role that pax7a-expressing muscle progenitor cells play during muscle regeneration, which may reflect the extent of muscle damage.

Injectable biomimetic liquid crystalline scaffolds enhance muscle stem cell transplantation

Science.gov (United States)

Sleep, Eduard; McClendon, Mark T.; Preslar, Adam T.; Chen, Charlotte H.; Sangji, M. Hussain; Pérez, Charles M. Rubert; Haynes, Russell D.; Meade, Thomas J.; Blau, Helen M.; Stupp, Samuel I.

2017-01-01

Muscle stem cells are a potent cell population dedicated to efficacious skeletal muscle regeneration, but their therapeutic utility is currently limited by mode of delivery. We developed a cell delivery strategy based on a supramolecular liquid crystal formed by peptide amphiphiles (PAs) that encapsulates cells and growth factors within a muscle-like unidirectionally ordered environment of nanofibers. The stiffness of the PA scaffolds, dependent on amino acid sequence, was found to determine the macroscopic degree of cell alignment templated by the nanofibers in vitro. Furthermore, these PA scaffolds support myogenic progenitor cell survival and proliferation and they can be optimized to induce cell differentiation and maturation. We engineered an in vivo delivery system to assemble scaffolds by injection of a PA solution that enabled coalignment of scaffold nanofibers with endogenous myofibers. These scaffolds locally retained growth factors, displayed degradation rates matching the time course of muscle tissue regeneration, and markedly enhanced the engraftment of muscle stem cells in injured and noninjured muscles in mice. PMID:28874575

Effects of extracts of denervated muscles on the morphology of cultured muscle cells

NARCIS (Netherlands)

Hooisma, J.; Krijger, J.de; Groot, D.M.G. de

1981-01-01

Previously tropic effects of extracts from whole chick embryos and from innervated muscles on cultured muscle cells were described. The present study demonstrated similar effects of extracts from 10-days denervated chick muscles. Extracts from innervated as well as from denervated muscles

Applications of a systematic homogenization theory for nodal diffusion methods

International Nuclear Information System (INIS)

Zhang, Hong-bin; Dorning, J.J.

1992-01-01

The authors recently have developed a self-consistent and systematic lattice cell and fuel bundle homogenization theory based on a multiple spatial scales asymptotic expansion of the transport equation in the ratio of the mean free path to the reactor characteristics dimension for use with nodal diffusion methods. The mathematical development leads naturally to self-consistent analytical expressions for homogenized diffusion coefficients and cross sections and flux discontinuity factors to be used in nodal diffusion calculations. The expressions for the homogenized nuclear parameters that follow from the systematic homogenization theory (SHT) are different from those for the traditional flux and volume-weighted (FVW) parameters. The calculations summarized here show that the systematic homogenization theory developed recently for nodal diffusion methods yields accurate values for k eff and assembly powers even when compared with the results of a fine mesh transport calculation. Thus, it provides a practical alternative to equivalence theory and GET (Ref. 3) and to simplified equivalence theory, which requires auxiliary fine-mesh calculations for assemblies embedded in a typical environment to determine the discontinuity factors and the equivalent diffusion coefficient for a homogenized assembly

FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration

Science.gov (United States)

Castiglioni, Alessandra; Basso, Veronica; Vezzoli, Michela; Monno, Antonella; Almada, Albert E.; Mondino, Anna; Wagers, Amy J.; Manfredi, Angelo A.; Rovere-Querini, Patrizia

2015-01-01

Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2-/- γ-chain-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4+CD25+FOXP3+ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue. PMID:26039259

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...

Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice

International Nuclear Information System (INIS)

Bhagavati, Satyakam; Xu Weimin

2005-01-01

Embryonic stem (ES) cells have great therapeutic potential because of their capacity to proliferate extensively and to form any fully differentiated cell of the body, including skeletal muscle cells. Successful generation of skeletal muscle in vivo, however, requires selective induction of the skeletal muscle lineage in cultures of ES cells and following transplantation, integration of appropriately differentiated skeletal muscle cells with recipient muscle. Duchenne muscular dystrophy (DMD), a severe progressive muscle wasting disease due to a mutation in the dystrophin gene and the mdx mouse, an animal model for DMD, are characterized by the absence of the muscle membrane associated protein, dystrophin. Here, we show that co-culturing mouse ES cells with a preparation from mouse muscle enriched for myogenic stem and precursor cells, followed by injection into mdx mice, results occasionally in the formation of normal, vascularized skeletal muscle derived from the transplanted ES cells. Study of this phenomenon should provide valuable insights into skeletal muscle development in vivo from transplanted ES cells

Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

Science.gov (United States)

Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

2012-03-01

Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion. Copyright © 2011 AlphaMed Press.

Enhanced elastin synthesis and maturation in human vascular smooth muscle tissue derived from induced-pluripotent stem cells.

Science.gov (United States)

Eoh, Joon H; Shen, Nian; Burke, Jacqueline A; Hinderer, Svenja; Xia, Zhiyong; Schenke-Layland, Katja; Gerecht, Sharon

2017-04-01

Obtaining vascular smooth muscle tissue with mature, functional elastic fibers is a key obstacle in tissue-engineered blood vessels. Poor elastin secretion and organization leads to a loss of specialization in contractile smooth muscle cells, resulting in over proliferation and graft failure. In this study, human induced-pluripotent stem cells (hiPSCs) were differentiated into early smooth muscle cells, seeded onto a hybrid poly(ethylene glycol) dimethacrylate/poly (l-lactide) (PEGdma-PLA) scaffold and cultured in a bioreactor while exposed to pulsatile flow, towards maturation into contractile smooth muscle tissue. We evaluated the effects of pulsatile flow on cellular organization as well as elastin expression and assembly in the engineered tissue compared to a static control through immunohistochemistry, gene expression and functionality assays. We show that culturing under pulsatile flow resulted in organized and functional hiPSC derived smooth muscle tissue. Immunohistochemistry analysis revealed hiPSC-smooth muscle tissue with robust, well-organized cells and elastic fibers and the supporting microfibril proteins necessary for elastic fiber assembly. Through qRT-PCR analysis, we found significantly increased expression of elastin, fibronectin, and collagen I, indicating the synthesis of necessary extracellular matrix components. Functionality assays revealed that hiPSC-smooth muscle tissue cultured in the bioreactor had an increased calcium signaling and contraction in response to a cholinergic agonist, significantly higher mature elastin content and improved mechanical properties in comparison to the static control. The findings presented here detail an effective approach to engineering elastic human vascular smooth muscle tissue with the functionality necessary for tissue engineering and regenerative medicine applications. Obtaining robust, mature elastic fibers is a key obstacle in tissue-engineered blood vessels. Human induced-pluripotent stem cells have

Stimulation of aortic smooth muscle cell mitogenesis by serotonin

International Nuclear Information System (INIS)

Nemecek, G.M.; Coughlin, S.R.; Handley, D.A.; Moskowitz, M.A.

1986-01-01

Bovine aortic smooth muscle cells in vitro responded to 1 nM to 10 μM serotonin with increased incorporation of [ 3 H]thymidine into DNA. The mitogenic effect of serotonin was half-maximal at 80 nM and maximal above 1 μM. At a concentration of 1 μM, serotonin stimulated smooth muscle cell mitogenesis to the same extent as human platelet-derived growth factor (PDGF) at 12 ng/ml. Tryptamine was ≅ 1/10th as potent as serotonin as a mitogen for smooth muscle cells. Other indoles that are structurally related to serotonin (D- and L-tryptophan, 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, melatonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol) and quipazine were inactive. The stimulatory effect of serotonin on smooth muscle cell DNA synthesis required prolonged (20-24 hr) exposure to the agonist and was attenuated in the presence of serotonin D receptor antagonists. When smooth muscle cells were incubated with submaximal concentrations of serotonin and PDGF, synergistic rather than additive mitogenic responses were observed. These data indicate that serotonin has a significant mitogenic effect on smooth muscle cells in vitro, which appears to be mediated by specific plasma membrane receptors

Fetal stem cells and skeletal muscle regeneration: a therapeutic approach

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Michela ePozzobon

2014-08-01

Full Text Available More than 40% of the body mass is represented by muscle tissue, which possesses the innate ability to regenerate after damage through the activation of muscle specific stem cell, namely satellite cells. Muscle diseases, in particular chronic degenerative state of skeletal muscle such as dystrophies, lead to a perturbation of the regenerative process, which causes the premature exhaustion of satellite cell reservoir due to continue cycles of degeneration/regeneration. Nowadays, the research is focused on different therapeutic approaches, ranging from gene and cell to pharmacological therapy, but still there is not a definitive cure in particular for genetic muscle disease. Taking this in mind, in this article we will give special consideration to muscle diseases and the use of fetal derived stem cells as new approach for therapy. Cells of fetal origin, from cord blood to placenta and amniotic fluid, can be easily obtained without ethical concern, expanded and differentiated in culture, and possess immunemodulatory properties. The in vivo approach in animal models can be helpful to study the mechanism underneath the operating principle of the stem cell reservoir, namely the niche, which holds great potential to understand the onset of muscle pathologies.

Skeletal muscle lymphoma: observations at MR imaging

International Nuclear Information System (INIS)

Eustace, S.; Winalski, C.S.; McGowen, A.; Lan, H.; Dorfman, D.

1996-01-01

We present the MR appearances of three patients with biopsy-proven primary lymphoma of skeletal muscle. In each case lymphoma resulted in bulky expansion of the involved muscle, homogeneously isointense to skeletal muscle on T1-weighted images, homogeneously hyperintense to skeletal muscle on T2-weighted images and diffusely enhancing following intravenous administration of gadopentate dimeglumine. (orig.)

BMP signaling regulates satellite cell-dependent postnatal muscle growth.

Science.gov (United States)

Stantzou, Amalia; Schirwis, Elija; Swist, Sandra; Alonso-Martin, Sonia; Polydorou, Ioanna; Zarrouki, Faouzi; Mouisel, Etienne; Beley, Cyriaque; Julien, Anaïs; Le Grand, Fabien; Garcia, Luis; Colnot, Céline; Birchmeier, Carmen; Braun, Thomas; Schuelke, Markus; Relaix, Frédéric; Amthor, Helge

2017-08-01

Postnatal growth of skeletal muscle largely depends on the expansion and differentiation of resident stem cells, the so-called satellite cells. Here, we demonstrate that postnatal satellite cells express components of the bone morphogenetic protein (BMP) signaling machinery. Overexpression of noggin in postnatal mice (to antagonize BMP ligands), satellite cell-specific knockout of Alk3 (the gene encoding the BMP transmembrane receptor) or overexpression of inhibitory SMAD6 decreased satellite cell proliferation and accretion during myofiber growth, and ultimately retarded muscle growth. Moreover, reduced BMP signaling diminished the adult satellite cell pool. Abrogation of BMP signaling in satellite cell-derived primary myoblasts strongly diminished cell proliferation and upregulated the expression of cell cycle inhibitors p21 and p57 In conclusion, these results show that BMP signaling defines postnatal muscle development by regulating satellite cell-dependent myofiber growth and the generation of the adult muscle stem cell pool. © 2017. Published by The Company of Biologists Ltd.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

KAUST Repository

Castagnetti, Francesco

2017-06-13

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and agerelated muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

Muscle Stem Cell Fate Is Controlled by the Cell-Polarity Protein Scrib

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Yusuke Ono

2015-02-01

Full Text Available Satellite cells are resident skeletal muscle stem cells that supply myonuclei for homeostasis, hypertrophy, and repair in adult muscle. Scrib is one of the major cell-polarity proteins, acting as a potent tumor suppressor in epithelial cells. Here, we show that Scrib also controls satellite-cell-fate decisions in adult mice. Scrib is undetectable in quiescent cells but becomes expressed during activation. Scrib is asymmetrically distributed in dividing daughter cells, with robust accumulation in cells committed to myogenic differentiation. Low Scrib expression is associated with the proliferative state and preventing self-renewal, whereas high Scrib levels reduce satellite cell proliferation. Satellite-cell-specific knockout of Scrib in mice causes a drastic and insurmountable defect in muscle regeneration. Thus, Scrib is a regulator of tissue stem cells, controlling population expansion and self-renewal with Scrib expression dynamics directing satellite cell fate.

α-smooth muscle actin is not a marker of fibrogenic cell activity in skeletal muscle fibrosis.

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Wanming Zhao

Full Text Available α-Smooth muscle actin (α-SMA is used as a marker for a subset of activated fibrogenic cells, myofibroblasts, which are regarded as important effector cells of tissue fibrogenesis. We address whether α-SMA-expressing myofibroblasts are detectable in fibrotic muscles of mdx5cv mice, a mouse model for Duchenne muscular dystrophy (DMD, and whether the α-SMA expression correlates with the fibrogenic function of intramuscular fibrogenic cells. α-SMA immunostaining signal was not detected in collagen I (GFP-expressing cells in fibrotic muscles of ColI-GFP/mdx5cv mice, but it was readily detected in smooth muscle cells lining intramuscular blood vessel walls. α-SMA expression was detected by quantitative RT-PCR and Western blot in fibrogenic cells sorted from diaphragm and quadriceps muscles of the ColI-GFP/mdx5cv mice. Consistent with the more severe fibrosis in the ColI-GFP/mdx5cv diaphragm, the fibrogenic cells in the diaphragm exerted a stronger fibrogenic function than the fibrogenic cells in the quadriceps as gauged by their extracellular matrix gene expression. However, both gene and protein expression of α-SMA was lower in the diaphragm fibrogenic cells than in the quadriceps fibrogenic cells in the ColI-GFP/mdx5cv mice. We conclude that myofibroblasts are present in fibrotic skeletal muscles, but their expression of α-SMA is not detectable by immunostaining. The level of α-SMA expression by intramuscular fibrogenic cells does not correlate positively with the level of collagen gene expression or the severity of skeletal muscle fibrosis in the mdx5cv mice. α-SMA is not a functional marker of fibrogenic cells in skeletal muscle fibrosis associated with muscular dystrophy.

Polyvinylpyrrolidone-Based Bio-Ink Improves Cell Viability and Homogeneity during Drop-On-Demand Printing

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Wei Long Ng

2017-02-01

Full Text Available Drop-on-demand (DOD bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP macromolecules. Different PVP-based bio-inks (0%–3% w/v were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh, which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v. Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process.

Homogeneity evaluation of mesenchymal stem cells based on electrotaxis analysis

OpenAIRE

Kim, Min Sung; Lee, Mi Hee; Kwon, Byeong-Ju; Kim, Dohyun; Koo, Min-Ah; Seon, Gyeung Mi; Park, Jong-Chul

2017-01-01

Stem cell therapy that can restore function to damaged tissue, avoid host rejection and reduce inflammation throughout body without use of immunosuppressive drugs. The established methods were used to identify and to isolate specific stem cell markers by FACS or by immunomagnetic cell separation. The procedures for distinguishing population of stem cells took a time and needed many preparations. Here we suggest an electrotaxis analysis as a new method to evaluate the homogeneity of mesenchyma...

Proliferation Rates of Bovine Primary Muscle Cells Relate to Liveweight and Carcase Weight in Cattle

Science.gov (United States)

Coles, Chantal A.; Wadeson, Jenny; Leyton, Carolina P.; Siddell, Jason P.; Greenwood, Paul L.; White, Jason D.; McDonagh, Matthew B.

2015-01-01

Muscling in cattle is largely influenced by genetic background, ultimately affecting beef yield and is of major interest to the beef industry. This investigation aimed to determine whether primary skeletal muscle cells isolated from different breeds of cattle with a varying genetic potential for muscling differ in their myogenic proliferative capacity. Primary skeletal muscle cells were isolated and cultured from the Longissimus muscle (LM) of 6 month old Angus, Hereford and Wagyu X Angus cattle. Cells were assessed for rate of proliferation and gene expression of PAX7, MYOD, MYF5, and MYOG. Proliferation rates were found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5–20 hours in culture) in vitro (P cattle (P cattle (P cattle. PMID:25875203

Cluster-cell calculation using the method of generalized homogenization

International Nuclear Information System (INIS)

Laletin, N.I.; Boyarinov, V.F.

1988-01-01

The generalized-homogenization method (GHM), used for solving the neutron transfer equation, was applied to calculating the neutron distribution in the cluster cell with a series of cylindrical cells with cylindrically coaxial zones. Single-group calculations of the technological channel of the cell of an RBMK reactor were performed using GHM. The technological channel was understood to be the reactor channel, comprised of the zirconium rod, the water or steam-water mixture, the uranium dioxide fuel element, and the zirconium tube, together with the adjacent graphite layer. Calculations were performed for channels with no internal sources and with unit incoming current at the external boundary as well as for channels with internal sources and zero current at the external boundary. The PRAKTINETs program was used to calculate the symmetric neutron distributions in the microcell and in channels with homogenized annular zones. The ORAR-TsM program was used to calculate the antisymmetric distribution in the microcell. The accuracy of the calculations were compared for the two channel versions

Vascular wall-resident CD44+ multipotent stem cells give rise to pericytes and smooth muscle cells and contribute to new vessel maturation.

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Diana Klein

Full Text Available Here, we identify CD44(+CD90(+CD73(+CD34(-CD45(- cells within the adult human arterial adventitia with properties of multipotency which were named vascular wall-resident multipotent stem cells (VW-MPSCs. VW-MPSCs exhibit typical mesenchymal stem cell characteristics including cell surface markers in immunostaining and flow cytometric analyses, and differentiation into adipocytes, chondrocytes and osteocytes under culture conditions. Particularly, TGFß1 stimulation up-regulates smooth muscle cell markers in VW-MPSCs. Using fluorescent cell labelling and co-localisation studies we show that VW-MPSCs differentiate to pericytes/smooth muscle cells which cover the wall of newly formed endothelial capillary-like structures in vitro. Co-implantation of EGFP-labelled VW-MPSCs and human umbilical vein endothelial cells into SCID mice subcutaneously via Matrigel results in new vessels formation which were covered by pericyte- or smooth muscle-like cells generated from implanted VW-MPSCs. Our results suggest that VW-MPSCs are of relevance for vascular morphogenesis, repair and self-renewal of vascular wall cells and for local capacity of neovascularization in disease processes.

Adipogenic Differentiation of Muscle Derived Cells is Repressed by Inhibition of GSK-3 Activity

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Zoe Redshaw

2018-06-01

Full Text Available Intramuscular fat is important in large animal livestock species in regard to meat quality and in humans is of clinical significance in particular in relation to insulin resistance. The canonical Wnt signalling pathway has been implicated at a whole body level in regulating relative levels of adiposity versus lean body mass. Previously we have shown that pig muscle cells can undergo adipogenic differentiation to a degree that is dependent upon the specific muscle source. In this work we examine the role of the canonical Wnt pathway which acts through inactivation of glycogen synthase kinase-3 (GSK-3 in the regulation of adipogenic differentiation in muscle cells derived from the pig semimembranosus muscle.The application of lithium chloride to muscle derived cells significantly increased the phosphorylation of GSK-3β and thus inhibited its activity thus mimicking Wnt signaling. This was associated with a significant decrease in the expression of the adipogenic transcription factor PPARγ and an almost complete inhibition of adipogenesis in the cells. The data also suggest that GSK-3α plays, at most, a small role in this process.Studies in vivo have suggested that the Wnt pathway is a major regulator of whole body adiposity. In this study we have shown that the ability of cells derived from porcine skeletal muscle to differentiate along an adipogenic lineage, in vitro, is severely impaired by mimicking the action of this pathway. This was done by inactivation of GSK-3β by the use of Lithium Chloride.

On homogenization of stokes flow in slowly varying media with applications to fluid–structure interaction

KAUST Repository

Brown, Donald L.

2011-09-11

In this paper we establish corrector estimates for Stokes flow in slowly varying perforated media via two scale asymptotic analysis. Current methods and techniques are often not able to deal with changing geometries prevalent in applied problems. For example, in a deformable porous medium environment, the geometry does not remain periodic under mechanical deformation and if slow variation in the geometry occurs. For such problems, one cannot use classical homogenization results directly and new homogenization results and estimates are needed. Our work uses asymptotic techniques of Marusic-Paloka and Mikelic (Bollettino U. M. I 7:661-671, 1996) where the authors constructed a downscaled velocity which converges to the fine-scale velocity at a rate of ε1/6 where ε is the characteristic length scale. We assume a slowly varying porous medium and study homogenization and corrector estimates for the Stokes equations. Slowly varying media arise, e. g., in fluid-structure interaction (FSI) problems (Popov et al. in Iterative upscaling of flows in deformable porous media, 2008), carbonation of porous concrete (Peter in C. R. Mecanique 335:357-362, 2007a; C. R. Mecanique 335:679-684, 2007b), and various other multiphysics processes. To homogenize Stokes flows in such media we restate the cell problems of Marusic-Paloka and Mikelic (Bollettino U. M. I 7:661-671, 1996) in a moving RVE framework. Further, to recover the same convergence properties it is necessary to solve an additional cell problem and add one more corrector term to the downscaled velocity. We further extend the framework of Marusic-Paloka and Mikelic (Bollettino U. M. I 7:661-671, 1996) to three spatial dimensions in both periodic and variable pore-space cases. Next, we also propose an efficient algorithm for computing the correctors by solving a limited number of cell problems at selected spatial locations. We present two computational examples: one for a constructed medium of elliptical perforations, and

Molecular aging and rejuvenation of human muscle stem cells

DEFF Research Database (Denmark)

Carlson, Morgan E; Suetta, Charlotte; Conboy, Michael J

2009-01-01

. Our findings establish key evolutionarily conserved mechanisms of human stem cell aging. We find that satellite cells are maintained in aged human skeletal muscle, but fail to activate in response to muscle attrition, due to diminished activation of Notch compounded by elevated transforming growth...... factor beta (TGF-beta)/phospho Smad3 (pSmad3). Furthermore, this work reveals that mitogen-activated protein kinase (MAPK)/phosphate extracellular signal-regulated kinase (pERK) signalling declines in human muscle with age, and is important for activating Notch in human muscle stem cells. This molecular......Very little remains known about the regulation of human organ stem cells (in general, and during the aging process), and most previous data were collected in short-lived rodents. We examined whether stem cell aging in rodents could be extrapolated to genetically and environmentally variable humans...

Skeletal muscle stem cells from animals I. Basic cell biology

Science.gov (United States)

Skeletal muscle stem cells from food-producing animals have been of interest to agricultural life scientists seeking to develop a better understanding of the molecular regulation of lean tissue (skeletal muscle protein hypertrophy) and intramuscular fat (marbling) development. Enhanced understanding...

Protein Availability and Satellite Cell Dynamics in Skeletal Muscle.

Science.gov (United States)

Shamim, Baubak; Hawley, John A; Camera, Donny M

2018-06-01

Human skeletal muscle satellite cells are activated in response to both resistance and endurance exercise. It was initially proposed that satellite cell proliferation and differentiation were only required to support resistance exercise-induced hypertrophy. However, satellite cells may also play a role in muscle fibre remodelling after endurance-based exercise and extracellular matrix regulation. Given the importance of dietary protein, particularly branched chain amino acids, in supporting myofibrillar and mitochondrial adaptations to both resistance and endurance-based training, a greater understanding of how protein intake impacts satellite cell activity would provide further insight into the mechanisms governing skeletal muscle remodelling with exercise. While many studies have investigated the capacity for protein ingestion to increase post-exercise rates of muscle protein synthesis, few investigations have examined the role for protein ingestion to modulate satellite cell activity. Here we review the molecular mechanisms controlling the activation of satellite cells in response to mechanical stress and protein intake in both in vitro and in vivo models. We provide a mechanistic framework that describes how protein ingestion may enhance satellite activity and promote exercise adaptations in human skeletal muscle.

Advanced maturation by electrical stimulation : differences in response between C2C12 and primary muscle progenitor cells

NARCIS (Netherlands)

Langelaan, M.L.P.; Boonen, K.J.M.; Rosaria-Chak, K.Y.; Schaft, van der D.W.J.; Post, M.J.; Baaijens, F.P.T.

2011-01-01

Skeletal muscle tissue engineering still does not result in the desired functional properties and texture as preferred for regenerative medicine and meat production applications. Electrical stimulation has been appropriately used as a tool to advance muscle cell maturation in vitro, thereby

Could a functional artificial skeletal muscle be useful in muscle wasting?

Science.gov (United States)

Fuoco, Claudia; Cannata, Stefano; Gargioli, Cesare

2016-05-01

Regardless of the underlying cause, skeletal muscle wasting is detrimental for a person's life quality, leading to impaired strength, locomotion, and physiological activity. Here, we propose a series of studies presenting tissue engineering-based approaches to reconstruct artificial muscle in vitro and in vivo. Skeletal muscle tissue engineering is attracting more and more attention from scientists, clinicians, patients, and media, thanks to the promising results obtained in the last decade with animal models of muscle wasting. The use of novel and refined biomimetic scaffolds mimicking three-dimensional muscle environment, thus supporting cell survival and differentiation, in combination with well characterized myogenic stem/progenitor cells, revealed the noteworthy potential of these technologies for creating artificial skeletal muscle tissue. In vitro, the production of three-dimensional muscle structures offer the possibility to generate a drug-screening platform for patient-specific pharmacological treatment, opening new frontiers in the development of new compounds with specific therapeutic actions. In vivo, three-dimensional artificial muscle biomimetic constructs offer the possibility to replace, in part or entirely, wasted muscle by means of straight reconstruction and/or by enhancing endogenous regeneration. Reports of tissue engineering approaches for artificial muscle building appeared in large numbers in the specialized press lately, advocating the suitability of this technology for human application upon scaling up and a near future applicability for medical care of muscle wasting. http://links.lww.com/COCN/A9

Insights into skeletal muscle development and applications in regenerative medicine.

Science.gov (United States)

Tran, T; Andersen, R; Sherman, S P; Pyle, A D

2013-01-01

Embryonic and postnatal development of skeletal muscle entails highly regulated processes whose complexity continues to be deconstructed. One key stage of development is the satellite cell, whose niche is composed of multiple cell types that eventually contribute to terminally differentiated myotubes. Understanding these developmental processes will ultimately facilitate treatments of myopathies such as Duchenne muscular dystrophy (DMD), a disease characterized by compromised cell membrane structure, resulting in severe muscle wasting. One theoretical approach is to use pluripotent stem cells in a therapeutic setting to help replace degenerated muscle tissue. This chapter discusses key myogenic developmental stages and their regulatory pathways; artificial myogenic induction in pluripotent stem cells; advantages and disadvantages of DMD animal models; and therapeutic approaches targeting DMD. Furthermore, skeletal muscle serves as an excellent paradigm for understanding general cell fate decisions throughout development. Copyright © 2013 Elsevier Inc. All rights reserved.

Development of an in vitro potency assay for human skeletal muscle derived cells.

Science.gov (United States)

Thurner, Marco; Asim, Faheem; Garczarczyk-Asim, Dorota; Janke, Katrin; Deutsch, Martin; Margreiter, Eva; Troppmair, Jakob; Marksteiner, Rainer

2018-01-01

Potency is a quantitative measure of the desired biological function of an advanced therapy medicinal product (ATMP) and is a prerequisite for market approval application (MAA). To assess the potency of human skeletal muscle-derived cells (SMDCs), which are currently investigated in clinical trials for the regeneration of skeletal muscle defects, we evaluated acetylcholinesterase (AChE), which is expressed in skeletal muscle and nervous tissue of all mammals. CD56+ SMDCs were separated from CD56- SMDCs by magnetic activated cell sorting (MACS) and both differentiated in skeletal muscle differentiation medium. AChE activity of in vitro differentiated SMDCs was correlated with CD56 expression, fusion index, cell number, cell doubling numbers, differentiation markers and compared to the clinical efficacy in patients treated with SMDCs against fecal incontinence. CD56- SMDCs did not form multinucleated myotubes and remained low in AChE activity during differentiation. CD56+ SMDCs generated myotubes and increased in AChE activity during differentiation. AChE activity was found to accurately reflect the number of CD56+ SMDCs in culture, their fusion competence, and cell doubling number. In patients with fecal incontinence responding to SMDCs treatment, the improvement of clinical symptoms was positively linked with the AChE activity of the SMDCs injected. AChE activity was found to truly reflect the in vitro differentiation status of SMDCs and to be superior to the mere use of surface markers as it reflects not only the number of myogenic SMDCs in culture but also their fusion competence and population doubling number, thus combining cell quality and quantification of the expected mode of action (MoA) of SMDCs. Moreover, the successful in vitro validation of the assay proves its suitability for routine use. Most convincingly, our results demonstrate a link between clinical efficacy and the AChE activity of the SMDCs preparations used for the treatment of fecal

Non-homogeneous Markov process models with informative observations with an application to Alzheimer's disease.

Science.gov (United States)

Chen, Baojiang; Zhou, Xiao-Hua

2011-05-01

Identifying risk factors for transition rates among normal cognition, mildly cognitive impairment, dementia and death in an Alzheimer's disease study is very important. It is known that transition rates among these states are strongly time dependent. While Markov process models are often used to describe these disease progressions, the literature mainly focuses on time homogeneous processes, and limited tools are available for dealing with non-homogeneity. Further, patients may choose when they want to visit the clinics, which creates informative observations. In this paper, we develop methods to deal with non-homogeneous Markov processes through time scale transformation when observation times are pre-planned with some observations missing. Maximum likelihood estimation via the EM algorithm is derived for parameter estimation. Simulation studies demonstrate that the proposed method works well under a variety of situations. An application to the Alzheimer's disease study identifies that there is a significant increase in transition rates as a function of time. Furthermore, our models reveal that the non-ignorable missing mechanism is perhaps reasonable. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differentiation and Application of Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells.

Science.gov (United States)

Maguire, Eithne Margaret; Xiao, Qingzhong; Xu, Qingbo

2017-11-01

Vascular smooth muscle cells (VSMCs) play a role in the development of vascular disease, for example, neointimal formation, arterial aneurysm, and Marfan syndrome caused by genetic mutations in VSMCs, but little is known about the mechanisms of the disease process. Advances in induced pluripotent stem cell technology have now made it possible to derive VSMCs from several different somatic cells using a selection of protocols. As such, researchers have set out to delineate key signaling processes involved in triggering VSMC gene expression to grasp the extent of gene regulatory networks involved in phenotype commitment. This technology has also paved the way for investigations into diseases affecting VSMC behavior and function, which may be treatable once an identifiable culprit molecule or gene has been repaired. Moreover, induced pluripotent stem cell-derived VSMCs are also being considered for their use in tissue-engineered blood vessels as they may prove more beneficial than using autologous vessels. Finally, while several issues remains to be clarified before induced pluripotent stem cell-derived VSMCs can become used in regenerative medicine, they do offer both clinicians and researchers hope for both treating and understanding vascular disease. In this review, we aim to update the recent progress on VSMC generation from stem cells and the underlying molecular mechanisms of VSMC differentiation. We will also explore how the use of induced pluripotent stem cell-derived VSMCs has changed the game for regenerative medicine by offering new therapeutic avenues to clinicians, as well as providing researchers with a new platform for modeling of vascular disease. © 2017 American Heart Association, Inc.

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.

Skeletal Muscle-derived Hematopoietic Stem Cells: Muscular Dystrophy Therapy by Bone Marrow Transplantation

OpenAIRE

Asakura, Atsushi

2012-01-01

For postnatal growth and regeneration of skeletal muscle, satellite cells, a self-renewing pool of muscle stem cells, give rise to daughter myogenic precursor cells that contribute to the formation of new muscle fibers. In addition to this key myogenic cell class, adult skeletal muscle also contains hematopoietic stem cell and progenitor cell populations which can be purified as a side population (SP) fraction or as a hematopoietic marker CD45-positive cell population. These muscle-derived he...

Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting

Directory of Open Access Journals (Sweden)

Jin Yipeng

2017-07-01

Full Text Available Smooth muscle differentiated human adipose derived stem cells (hADSCs provide a crucial stem cell source for urinary tissue engineering, but the induction of hADSCs for smooth muscle differentiation still has several issues to overcome, including a relatively long induction time and equipment dependence, which limits access to abundant stem cells within a short period of time for further application. Three-dimensional (3D bioprinting holds great promise in regenerative medicine due to its controllable construction of a designed 3D structure. When evenly mixed with bioink, stem cells can be spatially distributed within a bioprinted 3D structure, thus avoiding drawbacks such as, stem cell detachment in a conventional cell-scaffold strategy. Notwithstanding the advantages mentioned above, cell viability is often compromised during 3D bioprinting, which is often due to pressure during the bioprinting process. The objective of our study was to improve the efficiency of hADSC smooth muscle differentiation and cell viability of a 3D bioprinted structure. Here, we employed the hanging-drop method to generate hADSC microtissues in a smooth muscle inductive medium containing human transforming growth factor β1 and bioprinted the induced microtissues onto a 3D structure. After 3 days of smooth muscle induction, the expression of α-smooth muscle actin and smoothelin was higher in microtissues than in their counterpart monolayer cultured hADSCs, as confirmed by immunofluorescence and western blotting analysis. The semi-quantitative assay showed that the expression of α-smooth muscle actin (α-SMA was 0.218 ± 0.077 in MTs and 0.082 ± 0.007 in Controls; smoothelin expression was 0.319 ± 0.02 in MTs and 0.178 ± 0.06 in Controls. Induced MTs maintained their phenotype after the bioprinting process. Live/dead and cell count kit 8 assays showed that cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time

The role of Six1 in muscle progenitor cells and the establishment of fast-twitch muscle fibres

OpenAIRE

Nord, Hanna

2014-01-01

Myogenesis is the process of skeletal muscle tissue formation where committed muscle progenitor cells differentiate into skeletal muscle fibres. Depending on the instructive cues the muscle progenitor cells receive they will differentiate into specific fibre types with different properties. The skeletal muscle fibres can be broadly classified as fast-twitch fibres or slow-twitch fibres, based on their contractile speed. However, subgroups of fast- and slow-twitch fibres with different metabol...

Muscle satellite cells are functionally impaired in myasthenia gravis: consequences on muscle regeneration.

Science.gov (United States)

Attia, Mohamed; Maurer, Marie; Robinet, Marieke; Le Grand, Fabien; Fadel, Elie; Le Panse, Rozen; Butler-Browne, Gillian; Berrih-Aknin, Sonia

2017-12-01

Myasthenia gravis (MG) is a neuromuscular disease caused in most cases by anti-acetyl-choline receptor (AChR) autoantibodies that impair neuromuscular signal transmission and affect skeletal muscle homeostasis. Myogenesis is carried out by muscle stem cells called satellite cells (SCs). However, myogenesis in MG had never been explored. The aim of this study was to characterise the functional properties of myasthenic SCs as well as their abilities in muscle regeneration. SCs were isolated from muscle biopsies of MG patients and age-matched controls. We first showed that the number of Pax7+ SCs was increased in muscle sections from MG and its experimental autoimmune myasthenia gravis (EAMG) mouse model. Myoblasts isolated from MG muscles proliferate and differentiate more actively than myoblasts from control muscles. MyoD and MyoG were expressed at a higher level in MG myoblasts as well as in MG muscle biopsies compared to controls. We found that treatment of control myoblasts with MG sera or monoclonal anti-AChR antibodies increased the differentiation and MyoG mRNA expression compared to control sera. To investigate the functional ability of SCs from MG muscle to regenerate, we induced muscle regeneration using acute cardiotoxin injury in the EAMG mouse model. We observed a delay in maturation evidenced by a decrease in fibre size and MyoG mRNA expression as well as an increase in fibre number and embryonic myosin heavy-chain mRNA expression. These findings demonstrate for the first time the altered function of SCs from MG compared to control muscles. These alterations could be due to the anti-AChR antibodies via the modulation of myogenic markers resulting in muscle regeneration impairment. In conclusion, the autoimmune attack in MG appears to have unsuspected pathogenic effects on SCs and muscle regeneration, with potential consequences on myogenic signalling pathways, and subsequently on clinical outcome, especially in the case of muscle stress.

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.

Expression of uncoupling protein 1 in bovine muscle cells.

Science.gov (United States)

Abd Eldaim, M A; Hashimoto, O; Ohtsuki, H; Yamada, T; Murakami, M; Onda, K; Sato, R; Kanamori, Y; Qiao, Y; Tomonaga, S; Matsui, T; Funaba, M

2016-12-01

Uncoupling protein 1 (Ucp1) is predominantly expressed in brown/beige adipocytes in mammals. Although myogenic cells have been suggested to commit to a brown adipocyte lineage through the induction of Prdm16 expression, Prdm16 is also expressed in skeletal muscle. Thus, we examined expression of Ucp1 in bovine myogenic cells. Considering that Ucp1 is a principle molecule that induces energy expenditure in brown/beige adipocytes, expression of Ucp1 is not preferable in beef cattle because of potential decrease in energy (fattening) efficiency. The RT-PCR analyses revealed the expression of Ucp1 in the skeletal muscle of cattle; expression levels were markedly lower than those in the brown fat of calves. Immunohistochemical analyses showed that Ucp1 surrounded muscle fibers, but not adipocytes residing in skeletal muscle. Myosatellite cells cultured in myogenic medium showed an increase in the expression levels of myogenic regulatory factors ( levels were greater in cells after myogenic culture for 12 d than in those after myogenic culture for 6 d ( bovine skeletal muscle, which suggests the necessity for further studies on Ucp1-mediated energy expenditure in bovine skeletal muscle.

Satellite cell depletion prevents fiber hypertrophy in skeletal muscle.

Science.gov (United States)

Egner, Ingrid M; Bruusgaard, Jo C; Gundersen, Kristian

2016-08-15

The largest mammalian cells are the muscle fibers, and they have multiple nuclei to support their large cytoplasmic volumes. During hypertrophic growth, new myonuclei are recruited from satellite stem cells into the fiber syncytia, but it was recently suggested that such recruitment is not obligatory: overload hypertrophy after synergist ablation of the plantaris muscle appeared normal in transgenic mice in which most of the satellite cells were abolished. When we essentially repeated these experiments analyzing the muscles by immunohistochemistry and in vivo and ex vivo imaging, we found that overload hypertrophy was prevented in the satellite cell-deficient mice, in both the plantaris and the extensor digitorum longus muscles. We attribute the previous findings to a reliance on muscle mass as a proxy for fiber hypertrophy, and to the inclusion of a significant number of regenerating fibers in the analysis. We discuss that there is currently no model in which functional, sustainable hypertrophy has been unequivocally demonstrated in the absence of satellite cells; an exception is re-growth, which can occur using previously recruited myonuclei without addition of new myonuclei. © 2016. Published by The Company of Biologists Ltd.

The quasi-parallel lives of satellite cells and atrophying muscle

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Stefano eBiressi

2015-07-01

Full Text Available Skeletal muscle atrophy or wasting accompanies various chronic illnesses and the aging process, thereby reducing muscle function. One of the most important components contributing to effective muscle repair in postnatal organisms, the satellite cells, have recently become the focus of several studies examining factors participating in the atrophic process. We critically examine here the experimental evidence linking satellite cell function with muscle loss in connection with various diseases as well as aging, and in the subsequent recovery process. Several recent reports have investigated the changes in satellite cells in terms of their differentiation and proliferative capacity in response to various atrophic stimuli. In this regard, we review the molecular changes within satellite cells that contribute to their dysfunctional status in atrophy, with the intention of shedding light on novel potential pharmacological targets to counteract the loss of muscle mass.

Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention.

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Mingming Yang

Full Text Available Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter.

Cell fate determination in zebrafish embryonic and adult muscle development

NARCIS (Netherlands)

Tee, J.M.

2010-01-01

We are interested in how the genetic basis of muscle precursor cells determines the outcome of the muscle cell fate, and thus leading to disruption in muscle formation and maintenance. We utilized the zebrafish carrying mutations in both Axin1 and Apc1, resulting in overactivation of the

Inducible satellite cell depletion attenuates skeletal muscle regrowth following a scald-burn injury.

Science.gov (United States)

Finnerty, Celeste C; McKenna, Colleen F; Cambias, Lauren A; Brightwell, Camille R; Prasai, Anesh; Wang, Ye; El Ayadi, Amina; Herndon, David N; Suman, Oscar E; Fry, Christopher S

2017-11-01

Severe burns result in significant skeletal muscle cachexia that impedes recovery. Activity of satellite cells, skeletal muscle stem cells, is altered following a burn injury and likely hinders regrowth of muscle. Severe burn injury induces satellite cell proliferation and fusion into myofibres with greater activity in muscles proximal to the injury site. Conditional depletion of satellite cells attenuates recovery of myofibre area and volume following a scald burn injury in mice. Skeletal muscle regrowth following a burn injury requires satellite cell activity, underscoring the therapeutic potential of satellite cells in the prevention of prolonged frailty in burn survivors. Severe burns result in profound skeletal muscle atrophy; persistent muscle atrophy and weakness are major complications that hamper recovery from burn injury. Many factors contribute to the erosion of muscle mass following burn trauma, and we have previously shown concurrent activation and apoptosis of muscle satellite cells following a burn injury in paediatric patients. To determine the necessity of satellite cells during muscle recovery following a burn injury, we utilized a genetically modified mouse model (Pax7 CreER -DTA) that allows for the conditional depletion of satellite cells in skeletal muscle. Additionally, mice were provided 5-ethynyl-2'-deoxyuridine to determine satellite cell proliferation, activation and fusion. Juvenile satellite cell-wild-type (SC-WT) and satellite cell-depleted (SC-Dep) mice (8 weeks of age) were randomized to sham or burn injury consisting of a dorsal scald burn injury covering 30% of total body surface area. Both hindlimb and dorsal muscles were studied at 7, 14 and 21 days post-burn. SC-Dep mice had >93% depletion of satellite cells compared to SC-WT (P satellite cell proliferation and fusion. Depletion of satellite cells impaired post-burn recovery of both muscle fibre cross-sectional area and volume (P satellite cells in the aetiology of lean

Forward Programming of Cardiac Stem Cells by Homogeneous Transduction with MYOCD plus TBX5.

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Elisa Belian

Full Text Available Adult cardiac stem cells (CSCs express many endogenous cardiogenic transcription factors including members of the Gata, Hand, Mef2, and T-box family. Unlike its DNA-binding targets, Myocardin (Myocd-a co-activator not only for serum response factor, but also for Gata4 and Tbx5-is not expressed in CSCs. We hypothesised that its absence was a limiting factor for reprogramming. Here, we sought to investigate the susceptibility of adult mouse Sca1+ side population CSCs to reprogramming by supplementing the triad of GATA4, MEF2C, and TBX5 (GMT, and more specifically by testing the effect of the missing co-activator, Myocd. Exogenous factors were expressed via doxycycline-inducible lentiviral vectors in various combinations. High throughput quantitative RT-PCR was used to test expression of 29 cardiac lineage markers two weeks post-induction. GMT induced more than half the analysed cardiac transcripts. However, no protein was detected for the induced sarcomeric genes Actc1, Myh6, and Myl2. Adding MYOCD to GMT affected only slightly the breadth and level of gene induction, but, importantly, triggered expression of all three proteins examined (α-cardiac actin, atrial natriuretic peptide, sarcomeric myosin heavy chains. MYOCD + TBX was the most effective pairwise combination in this system. In clonal derivatives homogenously expressing MYOCD + TBX at high levels, 93% of cardiac transcripts were up-regulated and all five proteins tested were visualized.(1 GMT induced cardiac genes in CSCs, but not cardiac proteins under the conditions used. (2 Complementing GMT with MYOCD induced cardiac protein expression, indicating a more complete cardiac differentiation program. (3 Homogeneous transduction with MYOCD + TBX5 facilitated the identification of differentiating cells and the validation of this combinatorial reprogramming strategy. Together, these results highlight the pivotal importance of MYOCD in driving CSCs toward a cardiac muscle fate.

Vinpocetine Attenuates the Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

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Yun-Yun Ma

Full Text Available Vascular calcification is an active process of osteoblastic differentiation of vascular smooth muscle cells; however, its definite mechanism remains unknown. Vinpocetine, a derivative of the alkaloid vincamine, has been demonstrated to inhibit the high glucose-induced proliferation of vascular smooth muscle cells; however, it remains unknown whether vinpocetine can affect the osteoblastic differentiation of vascular smooth muscle cells. We hereby investigated the effect of vinpocetine on vascular calcification using a beta-glycerophosphate-induced cell model. Our results showed that vinpocetine significantly reduced the osteoblast-like phenotypes of vascular smooth muscle cells including ALP activity, osteocalcin, collagen type I, Runx2 and BMP-2 expression as well as the formation of mineralized nodule. Vinpocetine, binding to translocation protein, induced phosphorylation of extracellular signal-related kinase and Akt and thus inhibited the translocation of nuclear factor-kappa B into the nucleus. Silencing of translocator protein significantly attenuated the inhibitory effect of vinpocetine on osteoblastic differentiation of vascular smooth muscle cells. Taken together, vinpocetine may be a promising candidate for the clinical therapy of vascular calcification.

Satellite Cells CD44 Positive Drive Muscle Regeneration in Osteoarthritis Patients

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Scimeca, Manuel; Bonanno, Elena; Piccirilli, Eleonora; Baldi, Jacopo; Mauriello, Alessandro; Orlandi, Augusto; Tancredi, Virginia; Gasbarra, Elena; Tarantino, Umberto

2015-01-01

Age-related bone diseases, such as osteoarthritis and osteoporosis, are strongly associated with sarcopenia and muscle fiber atrophy. In this study, we analyzed muscle biopsies in order to demonstrate that, in osteoarthritis patients, both osteophytes formation and regenerative properties of muscle stem cells are related to the same factors. In particular, thanks to immunohistochemistry, transmission electron microscopy, and immunogold labeling we investigated the role of BMP-2 in muscle stem cells activity. In patients with osteoarthritis both immunohistochemistry and transmission electron microscopy allowed us to note a higher number of CD44 positive satellite muscle cells forming syncytium. Moreover, the perinuclear and cytoplasmic expression of BMP-2 assessed by in situ molecular characterization of satellite cells syncytia suggest a very strict correlation between BMP-2 expression and muscle regeneration capability. Summing up, the higher BMP-2 expression in osteoarthritic patients could explain the increased bone mineral density as well as decreased muscle atrophy in osteoarthrosic patients. In conclusion, our results suggest that the control of physiological BMP-2 balance between bone and muscle tissues may be considered as a potential pharmacological target in bone-muscle related pathology. PMID:26101529

Satellite Cells CD44 Positive Drive Muscle Regeneration in Osteoarthritis Patients

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Manuel Scimeca

2015-01-01

Full Text Available Age-related bone diseases, such as osteoarthritis and osteoporosis, are strongly associated with sarcopenia and muscle fiber atrophy. In this study, we analyzed muscle biopsies in order to demonstrate that, in osteoarthritis patients, both osteophytes formation and regenerative properties of muscle stem cells are related to the same factors. In particular, thanks to immunohistochemistry, transmission electron microscopy, and immunogold labeling we investigated the role of BMP-2 in muscle stem cells activity. In patients with osteoarthritis both immunohistochemistry and transmission electron microscopy allowed us to note a higher number of CD44 positive satellite muscle cells forming syncytium. Moreover, the perinuclear and cytoplasmic expression of BMP-2 assessed by in situ molecular characterization of satellite cells syncytia suggest a very strict correlation between BMP-2 expression and muscle regeneration capability. Summing up, the higher BMP-2 expression in osteoarthritic patients could explain the increased bone mineral density as well as decreased muscle atrophy in osteoarthrosic patients. In conclusion, our results suggest that the control of physiological BMP-2 balance between bone and muscle tissues may be considered as a potential pharmacological target in bone-muscle related pathology.

Effects of cyclic stretch on proliferation of mesenchymal stem cells and their differentiation to smooth muscle cells

International Nuclear Information System (INIS)

Ghazanfari, Samane; Tafazzoli-Shadpour, Mohammad; Shokrgozar, Mohammad Ali

2009-01-01

Bone marrow mesenchymal stem cells (MSCs) are capable of differentiating into a variety of cell types such as vascular smooth muscle cells (SMCs). In this study, we investigated influence of cyclic stretch on proliferation of hMSCs for different loading conditions, alignment of actin filaments, and consequent differentiation to SMCs. Isolated cells from bone marrow were exposed to cyclic stretch utilizing a customized device. Cell proliferation was examined by MTT assay, alignment of actin fibers by a designed image processing code, and cell differentiation by fluorescence staining. Results indicated promoted proliferation of hMSCs by cyclic strain, enhanced by elevated strain amplitude and number of cycles. Such loading regulated smooth muscle α-actin, and reoriented actin fibers. Cyclic stretch led to differentiation of hMSCs to SMCs without addition of growth factor. It was concluded that applying appropriate loading treatment on hMSCs could enhance proliferation capability, and produce functional SMCs for engineered tissues.

Smooth muscle-like tissue constructs with circumferentially oriented cells formed by the cell fiber technology.

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Hsiao, Amy Y; Okitsu, Teru; Onoe, Hiroaki; Kiyosawa, Mahiro; Teramae, Hiroki; Iwanaga, Shintaroh; Kazama, Tomohiko; Matsumoto, Taro; Takeuchi, Shoji

2015-01-01

The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.

Muscle atrophy reversed by growth factor activation of satellite cells in a mouse muscle atrophy model.

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Simon Hauerslev

Full Text Available Muscular dystrophies comprise a large group of inherited disorders that lead to progressive muscle wasting. We wanted to investigate if targeting satellite cells can enhance muscle regeneration and thus increase muscle mass. We treated mice with hepatocyte growth factor and leukemia inhibitory factor under three conditions: normoxia, hypoxia and during myostatin deficiency. We found that hepatocyte growth factor treatment led to activation of the Akt/mTOR/p70S6K protein synthesis pathway, up-regulation of the myognic transcription factors MyoD and myogenin, and subsequently the negative growth control factor, myostatin and atrophy markers MAFbx and MuRF1. Hypoxia-induced atrophy was partially restored by hepatocyte growth factor combined with leukemia inhibitory factor treatment. Dividing satellite cells were three-fold increased in the treatment group compared to control. Finally, we demonstrated that myostatin regulates satellite cell activation and myogenesis in vivo following treatment, consistent with previous findings in vitro. Our results suggest, not only a novel in vivo pharmacological treatment directed specifically at activating the satellite cells, but also a myostatin dependent mechanism that may contribute to the progressive muscle wasting seen in severely affected patients with muscular dystrophy and significant on-going regeneration. This treatment could potentially be applied to many conditions that feature muscle wasting to increase muscle bulk and strength.

Molecular decompostition of anisotropic homogeneous mixed-norm spaces with applications to the boundedness of operators

DEFF Research Database (Denmark)

Cleanthous, Galatia; Georgiadis, Athanasios; Nielsen, Morten

2018-01-01

. Molecular decompositions for all the considered spaces are derived with the help of the algebra of almost diagonal operators. As an application, we obtain boundedness results on the considered spaces for Fourier multipliers and for pseudodifferential operators with suitable adapted homogeneous symbols using...

Skeletal muscle aging: stem cell function and tissue homeostasis

OpenAIRE

Victor, Pedro Sousa

2012-01-01

Muscle aging, in particular, is characterized by the reduction of tissue mass and function, which are particularly prominent in geriatric individuals undergoing sarcopenia. The age-associated muscle wasting is also associated with a decline in regenerative ability and a reduction in resident muscle stem cell (satellite cell) number and function. Although sarcopenia is one of the major contributors to the general loss of physiological function, the mechanisms involved in age-related loss of mu...

Human skeletal muscle-derived stem cells retain stem cell properties after expansion in myosphere culture

International Nuclear Information System (INIS)

Wei, Yan; Li, Yuan; Chen, Chao; Stoelzel, Katharina; Kaufmann, Andreas M.; Albers, Andreas E.

2011-01-01

Human skeletal muscle contains an accessible adult stem-cell compartment in which differentiated myofibers are maintained and replaced by a self-renewing stem cell pool. Previously, studies using mouse models have established a critical role for resident stem cells in skeletal muscle, but little is known about this paradigm in human muscle. Here, we report the reproducible isolation of a population of cells from human skeletal muscle that is able to proliferate for extended periods of time as floating clusters of rounded cells, termed 'myospheres' or myosphere-derived progenitor cells (MDPCs). The phenotypic characteristics and functional properties of these cells were determined using reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunocytochemistry. Our results showed that these cells are clonogenic, express skeletal progenitor cell markers Pax7, ALDH1, Myod, and Desmin and the stem cell markers Nanog, Sox2, and Oct3/4 significantly elevated over controls. They could be maintained proliferatively active in vitro for more than 20 weeks and passaged at least 18 times, despite an average donor-age of 63 years. Individual clones (4.2%) derived from single cells were successfully expanded showing clonogenic potential and sustained proliferation of a subpopulation in the myospheres. Myosphere-derived cells were capable of spontaneous differentiation into myotubes in differentiation media and into other mesodermal cell lineages in induction media. We demonstrate here that direct culture and expansion of stem cells from human skeletal muscle is straightforward and reproducible with the appropriate technique. These cells may provide a viable resource of adult stem cells for future therapies of disease affecting skeletal muscle or mesenchymal lineage derived cell types.

MASTR directs MyoD-dependent satellite cell differentiation during skeletal muscle regeneration

OpenAIRE

Mokalled, Mayssa H.; Johnson, Aaron N.; Creemers, Esther E.; Olson, Eric N.

2012-01-01

Muscle repair is regulated by satellite cells, adult skeletal muscle stem cells that control muscle regeneration by proliferating and fusing with injured myofibers. MyoD is required for muscle regeneration; however, the mechanisms regulating MyoD expression in satellite cells are unclear. In this study, Olson and colleagues have demonstrated that deletion of MASTR and MRTF-A, two members of the Myocardin family of transcription factors, leads to skeletal muscle regeneration defects and down-r...

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes.

Science.gov (United States)

Subasinghe, Supundi; Unabia, Sharon; Barrow, Colin J; Mok, Su San; Aguilar, Marie-Isabel; Small, David H

2003-02-01

Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.

Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy

Science.gov (United States)

Fry, Christopher S.; Lee, Jonah D.; Jackson, Janna R.; Kirby, Tyler J.; Stasko, Shawn A.; Liu, Honglu; Dupont-Versteegden, Esther E.; McCarthy, John J.; Peterson, Charlotte A.

2014-01-01

Our aim in the current study was to determine the necessity of satellite cells for long-term muscle growth and maintenance. We utilized a transgenic Pax7-DTA mouse model, allowing for the conditional depletion of > 90% of satellite cells with tamoxifen treatment. Synergist ablation surgery, where removal of synergist muscles places functional overload on the plantaris, was used to stimulate robust hypertrophy. Following 8 wk of overload, satellite cell-depleted muscle demonstrated an accumulation of extracellular matrix (ECM) and fibroblast expansion that resulted in reduced specific force of the plantaris. Although the early growth response was normal, an attenuation of hypertrophy measured by both muscle wet weight and fiber cross-sectional area occurred in satellite cell-depleted muscle. Isolated primary myogenic progenitor cells (MPCs) negatively regulated fibroblast ECM mRNA expression in vitro, suggesting a novel role for activated satellite cells/MPCs in muscle adaptation. These results provide evidence that satellite cells regulate the muscle environment during growth.—Fry, C. S., Lee, J. D., Jackson, J. R., Kirby, T. J., Stasko, S. A., Liu, H., Dupont-Versteegden, E. E., McCarthy, J. J., Peterson, C. A. Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy. PMID:24376025

Aging, metabolism and stem cells: Spotlight on muscle stem cells.

Science.gov (United States)

García-Prat, Laura; Muñoz-Cánoves, Pura

2017-04-15

All tissues and organs undergo a progressive regenerative decline as they age. This decline has been mainly attributed to loss of stem cell number and/or function, and both stem cell-intrinsic changes and alterations in local niches and/or systemic environment over time are known to contribute to the stem cell aging phenotype. Advancing in the molecular understanding of the deterioration of stem cell cells with aging is key for targeting the specific causes of tissue regenerative dysfunction at advanced stages of life. Here, we revise exciting recent findings on why stem cells age and the consequences on tissue regeneration, with a special focus on regeneration of skeletal muscle. We also highlight newly identified common molecular pathways affecting diverse types of aging stem cells, such as altered proteostasis, metabolism, or senescence entry, and discuss the questions raised by these findings. Finally, we comment on emerging stem cell rejuvenation strategies, principally emanating from studies on muscle stem cells, which will surely burst tissue regeneration research for future benefit of the increasing human aging population. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Increased Stiffness in Aged Skeletal Muscle Impairs Muscle Progenitor Cell Proliferative Activity.

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Grégory Lacraz

Full Text Available Skeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs. Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM, which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential.We showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers.These findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.

Chiral Orientation of Skeletal Muscle Cells Requires Rigid Substrate

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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.

Artificial Muscles: Mechanisms, Applications, and Challenges.

Science.gov (United States)

Mirvakili, Seyed M; Hunter, Ian W

2018-02-01

The area of artificial muscle is a highly interdisciplinary field of research that has evolved rapidly in the last 30 years. Recent advances in nanomaterial fabrication and characterization, specifically carbon nanotubes and nanowires, have had major contributions in the development of artificial muscles. However, what can artificial muscles really do for humans? This question is considered here by first examining nature's solutions to this design problem and then discussing the structure, actuation mechanism, applications, and limitations of recently developed artificial muscles, including highly oriented semicrystalline polymer fibers; nanocomposite actuators; twisted nanofiber yarns; thermally activated shape-memory alloys; ionic-polymer/metal composites; dielectric-elastomer actuators; conducting polymers; stimuli-responsive gels; piezoelectric, electrostrictive, magnetostrictive, and photostrictive actuators; photoexcited actuators; electrostatic actuators; and pneumatic actuators. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Muscle Tissue Engineering Using Gingival Mesenchymal Stem Cells Encapsulated in Alginate Hydrogels Containing Multiple Growth Factors.

Science.gov (United States)

Ansari, Sahar; Chen, Chider; Xu, Xingtian; Annabi, Nasim; Zadeh, Homayoun H; Wu, Benjamin M; Khademhosseini, Ali; Shi, Songtao; Moshaverinia, Alireza

2016-06-01

Repair and regeneration of muscle tissue following traumatic injuries or muscle diseases often presents a challenging clinical situation. If a significant amount of tissue is lost the native regenerative potential of skeletal muscle will not be able to grow to fill the defect site completely. Dental-derived mesenchymal stem cells (MSCs) in combination with appropriate scaffold material, present an advantageous alternative therapeutic option for muscle tissue engineering in comparison to current treatment modalities available. To date, there has been no report on application of gingival mesenchymal stem cells (GMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of the current study were to develop an injectable 3D RGD-coupled alginate scaffold with multiple growth factor delivery capacity for encapsulating GMSCs, and to evaluate the capacity of encapsulated GMSCs to differentiate into myogenic tissue in vitro and in vivo where encapsulated GMSCs were transplanted subcutaneously into immunocompromised mice. The results demonstrate that after 4 weeks of differentiation in vitro, GMSCs as well as the positive control human bone marrow mesenchymal stem cells (hBMMSCs) exhibited muscle cell-like morphology with high levels of mRNA expression for gene markers related to muscle regeneration (MyoD, Myf5, and MyoG) via qPCR measurement. Our quantitative PCR analyzes revealed that the stiffness of the RGD-coupled alginate regulates the myogenic differentiation of encapsulated GMSCs. Histological and immunohistochemical/fluorescence staining for protein markers specific for myogenic tissue confirmed muscle regeneration in subcutaneous transplantation in our in vivo animal model. GMSCs showed significantly greater capacity for myogenic regeneration in comparison to hBMMSCs (p alginate hydrogel with multiple growth factor delivery capacity is a promising candidate for muscle tissue engineering.

Dense-body aggregates as plastic structures supporting tension in smooth muscle cells.

Science.gov (United States)

Zhang, Jie; Herrera, Ana M; Paré, Peter D; Seow, Chun Y

2010-11-01

The wall of hollow organs of vertebrates is a unique structure able to generate active tension and maintain a nearly constant passive stiffness over a large volume range. These properties are predominantly attributable to the smooth muscle cells that line the organ wall. Although smooth muscle is known to possess plasticity (i.e., the ability to adapt to large changes in cell length through structural remodeling of contractile apparatus and cytoskeleton), the detailed structural basis for the plasticity is largely unknown. Dense bodies, one of the most prominent structures in smooth muscle cells, have been regarded as the anchoring sites for actin filaments, similar to the Z-disks in striated muscle. Here, we show that the dense bodies and intermediate filaments formed cable-like structures inside airway smooth muscle cells and were able to adjust the cable length according to cell length and tension. Stretching the muscle cell bundle in the relaxed state caused the cables to straighten, indicating that these intracellular structures were connected to the extracellular matrix and could support passive tension. These plastic structures may be responsible for the ability of smooth muscle to maintain a nearly constant tensile stiffness over a large length range. The finding suggests that the structural plasticity of hollow organs may originate from the dense-body cables within the smooth muscle cells.

Cellular location of rat muscle ferritins and their preferential loss during cell isolation.

Science.gov (United States)

Linder, M C; Roboz, M; McKown, M J; Pardridge, W M; Zak, R

1984-04-10

Heart and other muscles of the rat contain two forms of ferritin separable in polyacrylamide gel electrophoresis. The cellular location of the fast- and slow-migrating ferritins was investigated using primary cultures of hindlimb skeletal muscle, and isolated myocardial cell populations. Muscle and non-muscle cells were isolated in good yield from hearts of adult rats pretreated with large doses of iron to increase their ferritin content. In virtually all cases, the isolated muscle cells contained traces only of the fast-migrating species and the non-muscle cells contained small amounts of the slow-migrating ferritin. During cell isolation, 90-100% of both ferritins was lost and could be recovered in the perfusates and solutions employed, while one third of the total tissue protein, and a larger percentage of creatine phosphokinase, was recovered in the isolated cells. Primary cultures of thigh muscle from adult rats which had differentiated into multi-nucleated myotubes, were incubated for 1-3 days with chelated iron. These cells contained substantial amounts of the electrophoretically fast migrating ferritin, with its characteristic larger Stokes' radius (determined by quantitative polyacrylamide gel electrophoresis). None of the slow-migrating ferritin species was detected, although hindlimb muscle from iron-treated rats contained both forms. It is concluded that the fast-migrating ferritin of muscle, which is much larger and more asymmetric than other ferritins, is confined to the muscle cell population, while the other form is predominantly or exclusively in the non-muscle cells. Both ferritins are lost preferentially over other proteins during procedures which injure muscle tissue.

Low density lipoprotein uptake by an endothelial-smooth muscle cell bilayer

International Nuclear Information System (INIS)

Alexander, J.J.; Miguel, R.; Graham, D.

1991-01-01

To study the interaction of endothelial and smooth muscle cells, and the means by which such interaction may affect lipid permeability of the arterial wall, cell bilayers were established by use of a transwell culture system. After confluent growth of both cell types had been achieved, iodine 125 bound to low-density lipoprotein (10 ng protein/ml) was added to the media of the upper well. After a 3-hour incubation period, the iodine 125-bound low-density lipoprotein content of the upper and lower media demonstrated an impedance to lipoprotein movement across the endothelial cell monolayer as compared to the bare porous polycarbonate filter of the transwell (p less than 10(-6)). The presence of smooth muscle cells in the bottom well significantly enhanced the permeability of the endothelial cell layer (p less than 10(-60)). This effect remained unchanged over a 9-day time course. Membrane binding and cellular uptake of low-density lipoprotein by endothelial cells was not altered by smooth muscle cells, indicating that this change in permeability could not be easily attributed to changes in receptor-mediated transport or transcytosis. Membrane binding (p less than 0.02) and cellular uptake (p less than 10(-6)) of low-density lipoprotein by smooth muscle cells in the bilayer, when adjusted for counts available in the smooth muscle cell media, were both reduced in the early incubation period as compared to isolated smooth muscle cells. The disproportionate reduction in uptake as compared to binding would suggest that this was not entirely a receptor-dependent process

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

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

Mac-1low early myeloid cells in the bone marrow-derived SP fraction migrate into injured skeletal muscle and participate in muscle regeneration

International Nuclear Information System (INIS)

Ojima, Koichi; Uezumi, Akiyoshi; Miyoshi, Hiroyuki; Masuda, Satoru; Morita, Yohei; Fukase, Akiko; Hattori, Akihito; Nakauchi, Hiromitsu; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi

2004-01-01

Recent studies have shown that bone marrow (BM) cells, including the BM side population (BM-SP) cells that enrich hematopoietic stem cells (HSCs), are incorporated into skeletal muscle during regeneration, but it is not clear how and what kinds of BM cells contribute to muscle fiber regeneration. We found that a large number of SP cells migrated from BM to muscles following injury in BM-transplanted mice. These BM-derived SP cells in regenerating muscles expressed different surface markers from those of HSCs and could not reconstitute the mouse blood system. BM-derived SP/Mac-1 low cells increased in number in regenerating muscles following injury. Importantly, our co-culture studies with activated satellite cells revealed that this fraction carried significant potential for myogenic differentiation. By contrast, mature inflammatory (Mac-1 high ) cells showed negligible myogenic activities. Further, these BM-derived SP/Mac-1 low cells gave rise to mononucleate myocytes, indicating that their myogenesis was not caused by stochastic fusion with host myogenic cells, although they required cell-to-cell contact with myogenic cells for muscle differentiation. Taken together, our data suggest that neither HSCs nor mature inflammatory cells, but Mac-1 low early myeloid cells in the BM-derived SP fraction, play an important role in regenerating skeletal muscles

Local myogenic pulp-derived cell injection enhances craniofacial muscle regeneration in vivo.

Science.gov (United States)

Jung, J E; Song, M J; Shin, S; Choi, Y J; Kim, K H; Chung, C J

2017-02-01

To enhance myogenic differentiation in pulp cells isolated from extracted premolars by epigenetic modification using a DNA demethylation agent, 5-aza-2'-deoxycytidine (5-Aza), and to evaluate the potent stimulatory effect of 5-Aza-treated pulp cell injection for craniofacial muscle regeneration in vivo. Pulp cells were isolated from premolars extracted for orthodontic purposes from four adults (age range, 18-22.1 years). Levels of myogenic differentiation and functional contraction response in vitro were compared between pulp cells with or without pre-treatment of 5-Aza. Changes in muscle regeneration in response to green fluorescent protein (GFP)-labelled myogenic pulp cell injection in vivo were evaluated using a cardiotoxin (CTX)-induced muscle injury model of the gastrocnemius as well as the masseter muscle in mice. Pre-treatment of 5-Aza in pulp cells stimulated myotube formation, myogenic differentiation in terms of desmin and myogenin expression, and the level of collagen gel contraction. The local injection of 5-Aza pre-treated myogenic pulp cells was engrafted into the host tissue and indicated signs of enhanced muscle regeneration in both the gastrocnemius and the masseter muscles. The epigenetic modification of pulp cells from extracted premolars and the local injection of myogenic pulp cells may stimulate craniofacial muscles regeneration in vivo. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ficus Deltoidea Enhance Glucose Uptake Activity in Cultured Muscle Cells

International Nuclear Information System (INIS)

Zainah Adam; Shafii Khamis; Amin Ismail; Muhajir Hamid

2015-01-01

Ficus deltoidea or locally known as Mas cotek is one of the common medicinal plants used in Malaysia. Our previous studies showed that this plant have blood glucose lowering effect. Glucose uptake into muscle and adipocytes cells is one of the known mechanisms of blood glucose lowering effect. This study was performed to evaluate the effect of Ficus deltoidea on glucose uptake activity into muscle cells. The cells were incubated with Ficus deltoidea extracts either alone or combination with insulin. Amount of glucose uptake by L6 myotubes was determined using glucose tracer, 2-deoxy-(1- 3 H 1 )-glucose. The results showed that Ficus deltoidea extracts at particular doses enhanced basal or insulin-mediated glucose uptake into muscle cells significantly. Hot aqueous extract enhanced glucose uptake at the low concentration (10 μg/ ml) whereas methanolic extract enhanced glucose uptake at low and high concentrations. Methanolic extract also mimicked insulin activity during enhancing glucose uptake into L^ muscle cells. Glucose uptake activity of Ficus deltoidea could be attributed by the phenolic compound presence in the plant. This study had shown that Ficus deltoidea has the ability to enhance glucose uptake into muscle cells which is partly contributed the antidiabetic activity of this plant. (author)

Skeletal Muscle Regeneration, Repair and Remodelling in Aging: The Importance of Muscle Stem Cells and Vascularization.

Science.gov (United States)

Joanisse, Sophie; Nederveen, Joshua P; Snijders, Tim; McKay, Bryon R; Parise, Gianni

2017-01-01

Sarcopenia is the age-related loss of skeletal muscle mass and strength. Ultimately, sarcopenia results in the loss of independence, which imposes a large financial burden on healthcare systems worldwide. A critical facet of sarcopenia is the diminished ability for aged muscle to regenerate, repair and remodel. Over the years, research has focused on elucidating underlying mechanisms of sarcopenia and the impaired ability of muscle to respond to stimuli with aging. Muscle-specific stem cells, termed satellite cells (SC), play an important role in maintaining muscle health throughout the lifespan. It is well established that SC are essential in skeletal muscle regeneration, and it has been hypothesized that a reduction and/or dysregulation of the SC pool, may contribute to accelerated loss of skeletal muscle mass that is observed with advancing age. The preservation of skeletal muscle tissue and its ability to respond to stimuli may be impacted by reduced SC content and impaired function observed with aging. Aging is also associated with a reduction in capillarization of skeletal muscle. We have recently demonstrated that the distance between type II fibre-associated SC and capillaries is greater in older compared to younger adults. The greater distance between SC and capillaries in older adults may contribute to the dysregulation in SC activation ultimately impairing muscle's ability to remodel and, in extreme circumstances, regenerate. This viewpoint will highlight the importance of optimal SC activation in addition to skeletal muscle capillarization to maximize the regenerative potential of skeletal muscle in older adults. © 2016 S. Karger AG, Basel.

Lsd1 regulates skeletal muscle regeneration and directs the fate of satellite cells.

Science.gov (United States)

Tosic, Milica; Allen, Anita; Willmann, Dominica; Lepper, Christoph; Kim, Johnny; Duteil, Delphine; Schüle, Roland

2018-01-25

Satellite cells are muscle stem cells required for muscle regeneration upon damage. Of note, satellite cells are bipotent and have the capacity to differentiate not only into skeletal myocytes, but also into brown adipocytes. Epigenetic mechanisms regulating fate decision and differentiation of satellite cells during muscle regeneration are not yet fully understood. Here, we show that elevated levels of lysine-specific demethylase 1 (Kdm1a, also known as Lsd1) have a beneficial effect on muscle regeneration and recovery after injury, since Lsd1 directly regulates key myogenic transcription factor genes. Importantly, selective Lsd1 ablation or inhibition in Pax7-positive satellite cells, not only delays muscle regeneration, but changes cell fate towards brown adipocytes. Lsd1 prevents brown adipocyte differentiation of satellite cells by repressing expression of the novel pro-adipogenic transcription factor Glis1. Together, downregulation of Glis1 and upregulation of the muscle-specific transcription program ensure physiological muscle regeneration.

DNA Methylation in Skeletal Muscle Stem Cell Specification, Proliferation, and Differentiation

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Rhianna C. Laker

2016-01-01

Full Text Available An unresolved and critically important question in skeletal muscle biology is how muscle stem cells initiate and regulate the genetic program during muscle development. Epigenetic dynamics are essential for cellular development and organogenesis in early life and it is becoming increasingly clear that epigenetic remodeling may also be responsible for the cellular adaptations that occur in later life. DNA methylation of cytosine bases within CpG dinucleotide pairs is an important epigenetic modification that reduces gene expression when located within a promoter or enhancer region. Recent advances in the field suggest that epigenetic regulation is essential for skeletal muscle stem cell identity and subsequent cell development. This review summarizes what is currently known about how skeletal muscle stem cells regulate the myogenic program through DNA methylation, discusses a novel role for metabolism in this process, and addresses DNA methylation dynamics in adult skeletal muscle in response to physical activity.

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.

Muscle Stem Cell Therapy for the Treatment of DMD Associated Cardiomyopathy

Science.gov (United States)

2013-10-01

SUBTITLE Muscle Stem Cell Therapy for the Treatment of DMD Associated Cardiomyopathy 5a. CONTRACT NUMBER Subproject 1: Muscle Stem Cell Therapy...various muscle diseases, including Duchenne muscular dystrophy (DMD), develop progressive cardiomyopathy. Cellular cardiomyoplasty, which involves the

A theoretical study on the influence of the homogeneity of heavy-ion irradiation field on the survival fraction of cells

International Nuclear Information System (INIS)

Wen Xiaoqiong; Li Qiang; Zhou Guangming; Li Wenjian; Wang Jufang; Wei Zengquan

2001-01-01

In order to provide theoretical basis for the homogeneity request of heavy-ion irradiation field, the most important design parameter of the heavy-ion radiotherapy facility planned in IMP (Institute of Modern Physics), the influence of the homogeneity of heavy-ion irradiation field on the survival fraction of cells was investigated theoretically. A formula for survival fraction of cells irradiated by the un-uniform heavy-ion irradiation field was deduced to estimate the influence of the homogeneity of heavy-ion irradiation field on the survival fraction of cells. The results show that the survival fraction of cells irradiation by the un-uniform irradiation field is larger than that of cells irradiated by the uniform irradiation field, and the survival fraction of cells increases as the homogeneity of heavy-ion irradiation field decreasing. Practically, the heavy-ion irradiation field can be treated as uniform irradiation field when its homogeneity is better than 95%. According to these results, design request for the homogeneity of heavy-ion irradiation field should be better than 95%. The present results also show that the agreement of homogeneity of heavy-ion irradiation field must be checked while comparing the survival fraction curves obtained by different laboratory

Ureter smooth muscle cell orientation in rat is predominantly longitudinal.

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Bart Spronck

Full Text Available In ureter peristalsis, the orientation of the contracting smooth muscle cells is essential, yet current descriptions of orientation and composition of the smooth muscle layer in human as well as in rat ureter are inconsistent. The present study aims to improve quantification of smooth muscle orientation in rat ureters as a basis for mechanistic understanding of peristalsis. A crucial step in our approach is to use two-photon laser scanning microscopy and image analysis providing objective, quantitative data on smooth muscle cell orientation in intact ureters, avoiding the usual sectioning artifacts. In 36 rat ureter segments, originating from a proximal, middle or distal site and from a left or right ureter, we found close to the adventitia a well-defined longitudinal smooth muscle orientation. Towards the lamina propria, the orientation gradually became slightly more disperse, yet the main orientation remained longitudinal. We conclude that smooth muscle cell orientation in rat ureter is predominantly longitudinal, though the orientation gradually becomes more disperse towards the proprial side. These findings do not support identification of separate layers. The observed longitudinal orientation suggests that smooth muscle contraction would rather cause local shortening of the ureter, than cause luminal constriction. However, the net-like connective tissue of the ureter wall may translate local longitudinal shortening into co-local luminal constriction, facilitating peristalsis. Our quantitative, minimally invasive approach is a crucial step towards more mechanistic insight into ureter peristalsis, and may also be used to study smooth muscle cell orientation in other tube-like structures like gut and blood vessels.

Ureter smooth muscle cell orientation in rat is predominantly longitudinal.

Science.gov (United States)

Spronck, Bart; Merken, Jort J; Reesink, Koen D; Kroon, Wilco; Delhaas, Tammo

2014-01-01

In ureter peristalsis, the orientation of the contracting smooth muscle cells is essential, yet current descriptions of orientation and composition of the smooth muscle layer in human as well as in rat ureter are inconsistent. The present study aims to improve quantification of smooth muscle orientation in rat ureters as a basis for mechanistic understanding of peristalsis. A crucial step in our approach is to use two-photon laser scanning microscopy and image analysis providing objective, quantitative data on smooth muscle cell orientation in intact ureters, avoiding the usual sectioning artifacts. In 36 rat ureter segments, originating from a proximal, middle or distal site and from a left or right ureter, we found close to the adventitia a well-defined longitudinal smooth muscle orientation. Towards the lamina propria, the orientation gradually became slightly more disperse, yet the main orientation remained longitudinal. We conclude that smooth muscle cell orientation in rat ureter is predominantly longitudinal, though the orientation gradually becomes more disperse towards the proprial side. These findings do not support identification of separate layers. The observed longitudinal orientation suggests that smooth muscle contraction would rather cause local shortening of the ureter, than cause luminal constriction. However, the net-like connective tissue of the ureter wall may translate local longitudinal shortening into co-local luminal constriction, facilitating peristalsis. Our quantitative, minimally invasive approach is a crucial step towards more mechanistic insight into ureter peristalsis, and may also be used to study smooth muscle cell orientation in other tube-like structures like gut and blood vessels.

Plasticity of the Muscle Stem Cell Microenvironment.

Science.gov (United States)

Dinulovic, Ivana; Furrer, Regula; Handschin, Christoph

2017-01-01

Satellite cells (SCs) are adult muscle stem cells capable of repairing damaged and creating new muscle tissue throughout life. Their functionality is tightly controlled by a microenvironment composed of a wide variety of factors, such as numerous secreted molecules and different cell types, including blood vessels, oxygen, hormones, motor neurons, immune cells, cytokines, fibroblasts, growth factors, myofibers, myofiber metabolism, the extracellular matrix and tissue stiffness. This complex niche controls SC biology-quiescence, activation, proliferation, differentiation or renewal and return to quiescence. In this review, we attempt to give a brief overview of the most important players in the niche and their mutual interaction with SCs. We address the importance of the niche to SC behavior under physiological and pathological conditions, and finally survey the significance of an artificial niche both for basic and translational research purposes.

Thermal inactivation profiles of Mycobacterium avium subsp. paratuberculosis in lamb skeletal muscle homogenate fluid.

Science.gov (United States)

Whittington, Richard J; Waldron, Anna; Warne, Darian

2010-01-31

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in livestock and there is a debate about its role in humans in chronic inflammatory bowel disorders such as Crohn's disease, but the relationship remains unproven. Nevertheless livestock health authorities in many countries aim to lower the prevalence of this infection to reduce potential contamination of the human food supply. MAP may occur in bovine milk and data on thermal inactivation suggest pasteurisation is an effective process. Recently MAP has been identified in skeletal muscle of cattle and sheep but there are no data on its thermal inactivation in these substrates. In this study the inactivation of MAP was studied in a fluid homogenate of lamb skeletal muscle at temperatures previously identified as being relevant to cooking processes applied by domestic consumers. A PCR thermocycler was used to ensure accurate temperatures and rapid heat exchange, while radiometric culture was used to ensure sensitive detection of viable MAP for determination of D and z values. Among the two predominant strains of MAP, S and C, D(55) ranged from 56 to 89 min, D(60) was 8 to 11 min, D(65) was 26 to 35s while D(70) was 1.5 to 1.8s. Values for z were 4.21C degrees for the S strain and 4.51C degrees for the C strain. At temperatures of 65-70 degrees C, MAP appeared to be less heat tolerant in skeletal muscle fluid than in previous reports using milk as the medium. The total thermal exposure of MAP during baking of a sample of 16 leg-of-lamb roasts in domestic ovens was determined to result in more than 20 log reductions in most cases, that is the product was microbiologically safe. Based on the models used in this study, there is a low probability of survival of MAP provided that red meat is cooked to recommended standards. Crown Copyright 2009. Published by Elsevier B.V. All rights reserved.

A Wireless sEMG Recording System and Its Application to Muscle Fatigue Detection

Science.gov (United States)

Chang, Kang-Ming; Liu, Shin-Hong; Wu, Xuan-Han

2012-01-01

Surface electromyography (sEMG) is an important measurement for monitoring exercise and fitness. Because if its high sampling frequency requirement, wireless transmission of sEMG data is a challenge. In this article a wireless sEMG measurement system with a sampling frequency of 2 KHz is developed based upon a MSP 430 microcontroller and Bluetooth transmission. Standard isotonic and isometric muscle contraction are clearly represented in the receiving user interface. Muscle fatigue detection is an important application of sEMG. Traditional muscle fatigue is detected from the median frequency of the sEMG power spectrum. The regression slope of the linear regression of median frequency is an important muscle fatigue index. A more negative slope value represents a higher muscle fatigue condition. To test the system performance, muscle fatigue detection was examined by having subjects run on a pedaled-multifunctional elliptical trainer for approximately 30 minutes at three loading levels. Ten subjects underwent a total of 60 exercise sessions to provide the experimental data. Results showed that the regression slope gradually decreases as expected, and there is a significant gender difference. PMID:22368481

Effects of voluntary wheel running on satellite cells in the rat plantaris muscle.

Science.gov (United States)

Kurosaka, Mitsutoshi; Naito, Hisashi; Ogura, Yuji; Kojima, Atsushi; Goto, Katsumasa; Katamoto, Shizuo

2009-01-01

This study investigated the effects of voluntary wheel running on satellite cells in the rat plantaris muscle. Seventeen 5-week-old male Wistar rats were assigned to a control (n = 5) or training (n = 12) group. Each rat in the training group ran voluntarily in a running-wheel cage for 8 weeks. After the training period, the animals were anesthetized, and the plantaris muscles were removed, weighed, and analyzed immunohistochemically and biochemically. Although there were no significant differences in muscle weight or fiber area between the groups, the numbers of satellite cells and myonuclei per muscle fiber, percentage of satellite cells, and citrate synthase activity were significantly higher in the training group compared with the control group (p run in the training group (r = 0.61, p running can induce an increase in the number of satellite cells without changing the mean fiber area in the rat plantaris muscle; this increase in satellite cell content is a function of distance run. Key pointsThere is no study about the effect of voluntary running on satellite cells in the rat plantaris muscle.Voluntary running training causes an increase of citrate synthase activity in the rat plantaris muscle but does not affect muscle weight and mean fiber area in the rat plantaris muscle.Voluntary running can induce an increase in the number of satellite cells without hypertrophy of the rat plantaris muscle.

Myogenic Precursors from iPS Cells for Skeletal Muscle Cell Replacement Therapy

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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.

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

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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.

Myo/Nog cells: targets for preventing the accumulation of skeletal muscle-like cells in the human lens.

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Jacquelyn Gerhart

Full Text Available Posterior capsule opacification (PCO is a vision impairing condition that arises in some patients following cataract surgery. The fibrotic form of PCO is caused by myofibroblasts that may emerge in the lens years after surgery. In the chick embryo lens, myofibroblasts are derived from Myo/Nog cells that are identified by their expression of the skeletal muscle specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin, and the epitope recognized by the G8 monoclonal antibody. The goal of this study was to test the hypothesis that depletion of Myo/Nog cells will prevent the accumulation of myofibroblasts in human lens tissue. Myo/Nog cells were present in anterior, equatorial and bow regions of the human lens, cornea and ciliary processes. In anterior lens tissue removed by capsulorhexis, Myo/Nog cells had synthesized myofibroblast and skeletal muscle proteins, including vimentin, MyoD and sarcomeric myosin. Alpha smooth muscle actin (α-SMA was detected in a subpopulation of Myo/Nog cells. Areas of the capsule denuded of epithelial cells were surrounded by Myo/Nog cells. Some of these cell free areas contained a wrinkle in the capsule. Depletion of Myo/Nog cells eliminated cells expressing skeletal muscle proteins in 5-day cultures but did not affect cells immunoreactive for beaded filament proteins that accumulate in differentiating lens epithelial cells. Transforming growth factor-betas 1 and 2 that mediate an epithelial-mesenchymal transition, did not induce the expression of skeletal muscle proteins in lens cells following Myo/Nog cell depletion. This study demonstrates that Myo/Nog cells in anterior lens tissue removed from cataract patients have undergone a partial differentiation to skeletal muscle. Myo/Nog cells appear to be the source of skeletal muscle-like cells in explants of human lens tissue. Targeting Myo/Nog cells with the G8 antibody during cataract surgery may reduce the incidence of PCO.

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RGD peptide-displaying M13 bacteriophage/PLGA nanofibers as cell-adhesive matrices for smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Shin, Yong Cheol; Lee, Jong Ho; Jin, Oh Seong; Lee, Eun Ji; Jin, Lin Hua; Kim, Chang Seok; Hong, Suck Won; Han, Dong Wook; Kim, Chun Tae; Oh, Jin Woo [Pusan National University, Busan (Korea, Republic of)

2015-01-15

Extracellular matrices (ECMs) are network structures that play an essential role in regulating cellular growth and differentiation. In this study, novel nanofibrous matrices were fabricated by electrospinning M13 bacteriophage and poly(lactic-co-glycolic acid) (PLGA) and were shown to be structurally and functionally similar to natural ECMs. A genetically-engineered M13 bacteriophage was constructed to display Arg-Gly-Asp (RGD) peptides on its surface. The physicochemical properties of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage)/PLGA nanofibers were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. We used immunofluorescence staining to confirm that M13 bacteriophages were homogenously distributed in RGD-M13 phage/PLGA matrices. Furthermore, RGD-M13 phage/PLGA nanofibrous matrices, having excellent biocompatibility, can enhance the behaviors of vascular smooth muscle cells. This result suggests that RGD-M13 phage/PLGA nanofibrous matrices have potentials to serve as tissue engineering scaffolds.

A new numerical scheme for non uniform homogenized problems: Application to the non linear Reynolds compressible equation

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Buscaglia Gustavo C.

2001-01-01

Full Text Available A new numerical approach is proposed to alleviate the computational cost of solving non-linear non-uniform homogenized problems. The article details the application of the proposed approach to lubrication problems with roughness effects. The method is based on a two-parameter Taylor expansion of the implicit dependence of the homogenized coefficients on the average pressure and on the local value of the air gap thickness. A fourth-order Taylor expansion provides an approximation that is accurate enough to be used in the global problem solution instead of the exact dependence, without introducing significant errors. In this way, when solving the global problem, the solution of local problems is simply replaced by the evaluation of a polynomial. Moreover, the method leads naturally to Newton-Raphson nonlinear iterations, that further reduce the cost. The overall efficiency of the numerical methodology makes it feasible to apply rigorous homogenization techniques in the analysis of compressible fluid contact considering roughness effects. Previous work makes use of an heuristic averaging technique. Numerical comparison proves that homogenization-based methods are superior when the roughness is strongly anisotropic and not aligned with the flow direction.

Characterization of two-scale gradient Young measures and application to homogenization

OpenAIRE

Babadjian, Jean-Francois; Baia, Margarida; Santos, Pedro M.

2006-01-01

This work is devoted to the study of two-scale gradient Young measures naturally arising in nonlinear elasticity homogenization problems. Precisely, a characterization of this class of measures is derived and an integral representation formula for homogenized energies, whose integrands satisfy very weak regularity assumptions, is obtained in terms of two-scale gradient Young measures.

Vitamin K2 improves proliferation and migration of bovine skeletal muscle cells in vitro.

Science.gov (United States)

Rønning, Sissel Beate; Pedersen, Mona Elisabeth; Berg, Ragnhild Stenberg; Kirkhus, Bente; Rødbotten, Rune

2018-01-01

Skeletal muscle function is highly dependent on the ability to regenerate, however, during ageing or disease, the proliferative capacity is reduced, leading to loss of muscle function. We have previously demonstrated the presence of vitamin K2 in bovine skeletal muscles, but whether vitamin K has a role in muscle regulation and function is unknown. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to assess a potential effect of vitamin K2 (MK-4) during myogenesis of muscle cells. Cell viability experiments demonstrate that the amount of ATP produced by the cells was unchanged when MK-4 was added, indicating viable cells. Cytotoxicity analysis show that MK-4 reduced the lactate dehydrogenase (LDH) released into the media, suggesting that MK-4 was beneficial to the muscle cells. Cell migration, proliferation and differentiation was characterised after MK-4 incubation using wound scratch analysis, immunocytochemistry and real-time PCR analysis. Adding MK-4 to the cells led to an increased muscle proliferation, increased gene expression of the myogenic transcription factor myod as well as increased cell migration. In addition, we observed a reduction in the fusion index and relative gene expression of muscle differentiation markers, with fewer complex myotubes formed in MK-4 stimulated cells compared to control cells, indicating that the MK-4 plays a significant role during the early phases of muscle proliferation. Likewise, we see the same pattern for the relative gene expression of collagen 1A, showing increased gene expression in proliferating cells, and reduced expression in differentiating cells. Our results also suggest that MK-4 incubation affect low density lipoprotein receptor-related protein 1 (LRP1) and the low-density lipoprotein receptor (LDLR) with a peak in gene expression after 45 min of MK-4 incubation. Altogether, our experiments show that MK-4 has a positive effect on muscle cell migration and

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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.

Cytokine-induced differentiation of multipotent adult progenitor cells into functional smooth muscle cells.

Science.gov (United States)

Ross, Jeffrey J; Hong, Zhigang; Willenbring, Ben; Zeng, Lepeng; Isenberg, Brett; Lee, Eu Han; Reyes, Morayma; Keirstead, Susan A; Weir, E Kenneth; Tranquillo, Robert T; Verfaillie, Catherine M

2006-12-01

Smooth muscle formation and function are critical in development and postnatal life. Hence, studies aimed at better understanding SMC differentiation are of great importance. Here, we report that multipotent adult progenitor cells (MAPCs) isolated from rat, murine, porcine, and human bone marrow demonstrate the potential to differentiate into cells with an SMC-like phenotype and function. TGF-beta1 alone or combined with PDGF-BB in serum-free medium induces a temporally correct expression of transcripts and proteins consistent with smooth muscle development. Furthermore, SMCs derived from MAPCs (MAPC-SMCs) demonstrated functional L-type calcium channels. MAPC-SMCs entrapped in fibrin vascular molds became circumferentially aligned and generated force in response to KCl, the L-type channel opener FPL64176, or the SMC agonists 5-HT and ET-1, and exhibited complete relaxation in response to the Rho-kinase inhibitor Y-27632. Cyclic distention (5% circumferential strain) for 3 weeks increased responses by 2- to 3-fold, consistent with what occurred in neonatal SMCs. These results provide evidence that MAPC-SMCs are phenotypically and functionally similar to neonatal SMCs and that the in vitro MAPC-SMC differentiation system may be an ideal model for the study of SMC development. Moreover, MAPC-SMCs may lend themselves to tissue engineering applications.

Geometric effect of the hydrogel grid structure on in vitro formation of homogeneous MIN6 cell clusters.

Science.gov (United States)

Bae, Chae Yun; Min, Mun-kyeong; Kim, Hail; Park, Je-Kyun

2014-07-07

A microstructure-based hydrogel was employed to study the relationship between spatial specificity and cellular behavior, including cell fate, proliferation, morphology, and insulin secretion in pancreatic β-cells. To effectively form homogeneous cell clusters in vitro, we made cell-containing hydrogel membrane constructs with an adapted grid structure based on a hexagonal micropattern. Homogeneous cell clusters (average diameter: 83.6 ± 14.2 μm) of pancreatic insulinoma (MIN6) cells were spontaneously generated in the floating hydrogel membrane constructs, including a hexagonal grid structure (size of cavity: 100 μm, interval between cavities: 30 μm). Interestingly, 3D clustering of MIN6 cells mimicking the structure of pancreatic islets was coalesced into a merged aggregate attaching to each hexagonal cavity of the hydrogel grid structure. The fate and insulin secretion of homogeneous cell clusters in the hydrogel grid structure were also assessed. The results of these designable hydrogel-cell membrane constructs suggest that facultative in vitro β-cell proliferation and maintenance can be applied to biofunctional assessments.

The costo-uterine muscle of the rat contains a homogeneous population of beta-adrenoceptors.

Science.gov (United States)

Hartley, M. L.; Pennefather, J. N.

1985-01-01

The effects of two selective beta-adrenoceptor antagonists on the inhibitory responses to some sympathomimetic amines of electrically-stimulated preparations of costo-uterine muscle, taken from virgin rats, have been examined quantitatively. pA2 values for the antagonist, atenolol (beta 1-selective) and ICI 118,551 (beta 2-selective) were obtained using as agonists, fenoterol (beta 2-selective agonist) and noradrenaline (alpha- and beta-adrenoceptor agonist, beta 1-selective); and in addition, with ICI 118,551 only, isoprenaline (beta-agonist, non-selective) and adrenaline (alpha- and beta-adrenoceptor agonist, beta 2-selective). Catecholamine uptake mechanisms and alpha-adrenoceptors were not blocked in any of these experiments. Atenolol competitively antagonized the effects of fenoterol and noradrenaline to a similar extent, the pA2 values being 5.4 and 5.7, respectively. ICI 118,551 competitively antagonized the effects of fenoterol, isoprenaline, adrenaline and noradrenaline to a similar extent; pA2 values ranged from 8.7 with noradrenaline to 9.1 with isoprenaline. These results extend our previous observations which indicated that the adrenoceptors mediating inhibition of electrically-evoked contractions of costo-uterine muscle of the virgin rat are homogeneous and of the beta 2-subtype. The potency of the beta 1-selective agonist RO 363 in producing inhibition of electrically-evoked contractions of this tissue was also examined. RO 363 was 200 times less potent than isoprenaline but was a full agonist. This indicates that there is efficient coupling between beta 2-adrenoceptor activation and tissue response in this non-innervated preparation. PMID:2858239

Engineered skeletal muscle tissue for soft robotics: fabrication strategies, current applications, and future challenges.

Science.gov (United States)

Duffy, Rebecca M; Feinberg, Adam W

2014-01-01

Skeletal muscle is a scalable actuator system used throughout nature from the millimeter to meter length scales and over a wide range of frequencies and force regimes. This adaptability has spurred interest in using engineered skeletal muscle to power soft robotics devices and in biotechnology and medical applications. However, the challenges to doing this are similar to those facing the tissue engineering and regenerative medicine fields; specifically, how do we translate our understanding of myogenesis in vivo to the engineering of muscle constructs in vitro to achieve functional integration with devices. To do this researchers are developing a number of ways to engineer the cellular microenvironment to guide skeletal muscle tissue formation. This includes understanding the role of substrate stiffness and the mechanical environment, engineering the spatial organization of biochemical and physical cues to guide muscle alignment, and developing bioreactors for mechanical and electrical conditioning. Examples of engineered skeletal muscle that can potentially be used in soft robotics include 2D cantilever-based skeletal muscle actuators and 3D skeletal muscle tissues engineered using scaffolds or directed self-organization. Integration into devices has led to basic muscle-powered devices such as grippers and pumps as well as more sophisticated muscle-powered soft robots that walk and swim. Looking forward, current, and future challenges include identifying the best source of muscle precursor cells to expand and differentiate into myotubes, replacing cardiomyocytes with skeletal muscle tissue as the bio-actuator of choice for soft robots, and vascularization and innervation to enable control and nourishment of larger muscle tissue constructs. © 2013 Wiley Periodicals, Inc.

Akt1 deficiency diminishes skeletal muscle hypertrophy by reducing satellite cell proliferation.

Science.gov (United States)

Moriya, Nobuki; Miyazaki, Mitsunori

2018-02-14

Skeletal muscle mass is determined by the net dynamic balance between protein synthesis and degradation. Although the Akt/mechanistic target of rapamycin (mTOR)-dependent pathway plays an important role in promoting protein synthesis and subsequent skeletal muscle hypertrophy, the precise molecular regulation of mTOR activity by the upstream protein kinase Akt is largely unknown. In addition, the activation of satellite cells has been indicated as a key regulator of muscle mass. However, the requirement of satellite cells for load-induced skeletal muscle hypertrophy is still under intense debate. In this study, female germline Akt1 knockout (KO) mice were used to examine whether Akt1 deficiency attenuates load-induced skeletal muscle hypertrophy through suppressing mTOR-dependent signaling and satellite cell proliferation. Akt1 KO mice showed a blunted hypertrophic response of skeletal muscle, with a diminished rate of satellite cell proliferation following mechanical overload. In contrast, Akt1 deficiency did not affect the load-induced activation of mTOR signaling and the subsequent enhanced rate of protein synthesis in skeletal muscle. These observations suggest that the load-induced activation of mTOR signaling occurs independently of Akt1 regulation and that Akt1 plays a critical role in regulating satellite cell proliferation during load-induced muscle hypertrophy.

Osteogenic differentiation capacity of human skeletal muscle-derived progenitor cells.

Directory of Open Access Journals (Sweden)

Teruyo Oishi

Full Text Available Heterotopic ossification (HO is defined as the formation of ectopic bone in soft tissue outside the skeletal tissue. HO is thought to result from aberrant differentiation of osteogenic progenitors within skeletal muscle. However, the precise origin of HO is still unclear. Skeletal muscle contains two kinds of progenitor cells, myogenic progenitors and mesenchymal progenitors. Myogenic and mesenchymal progenitors in human skeletal muscle can be identified as CD56(+ and PDGFRα(+ cells, respectively. The purpose of this study was to investigate the osteogenic differentiation potential of human skeletal muscle-derived progenitors. Both CD56(+ cells and PDGFRα(+ cells showed comparable osteogenic differentiation potential in vitro. However, in an in vivo ectopic bone formation model, PDGFRα(+ cells formed bone-like tissue and showed successful engraftment, while CD56(+ cells did not form bone-like tissue and did not adapt to an osteogenic environment. Immunohistological analysis of human HO sample revealed that many PDGFRα(+ cells were localized in proximity to ectopic bone formed in skeletal muscle. MicroRNAs (miRNAs are known to regulate many biological processes including osteogenic differentiation. We investigated the participation of miRNAs in the osteogenic differentiation of PDGFRα(+ cells by using microarray. We identified miRNAs that had not been known to be involved in osteogenesis but showed dramatic changes during osteogenic differentiation of PDGFRα(+ cells. Upregulation of miR-146b-5p and -424 and downregulation of miR-7 during osteogenic differentiation of PDGFRα(+ cells were confirmed by quantitative real-time RT-PCR. Inhibition of upregulated miRNAs, miR-146b-5p and -424, resulted in the suppression of osteocyte maturation, suggesting that these two miRNAs have the positive role in the osteogenesis of PDGFRα(+ cells. Our results suggest that PDGFRα(+ cells may be the major source of HO and that the newly identified mi

Diabetic Myopathy: Impact of Diabetes Mellitus on Skeletal Muscle Progenitor Cells

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Donna M D'Souza

2013-12-01

Full Text Available Diabetes mellitus is defined as a group of metabolic diseases that are associated with the presence of a hyperglycemic state due to impairments in insulin function. While the development of each form of diabetes (Type 1 or Type 2 drastically differs, resultant pathologies often overlap. In each diabetic condition a failure to maintain healthy muscle is often observed, and is termed diabetic myopathy. This significant, but often overlooked, complication is believed to contribute to the progression of additional diabetic pathologies due to the vital importance of skeletal muscle for our physical and metabolic well-being. While studies have investigated the link between changes to skeletal muscle metabolic health following diabetes mellitus onset (particularly Type 2 diabetes mellitus, few have examined the negative impact of diabetes mellitus on the growth and reparative capacities of skeletal muscle that often coincides with disease development. Importantly, evidence is accumulating that the muscle progenitor cell population (particularly the muscle satellite cell population is also negatively affected by the diabetic environment, and as such, likely contributes to the declining skeletal muscle health observed in diabetes mellitus. In this review, we summarize the current knowledge surrounding the influence of diabetes mellitus on skeletal muscle growth and repair, with a particular emphasis on the impact of diabetes mellitus on the progenitor cell population of skeletal muscle.

Increased proinflammatory responses from asthmatic human airway smooth muscle cells in response to rhinovirus infection

Directory of Open Access Journals (Sweden)

King Nicholas JC

2006-05-01

Full Text Available Abstract Background Exacerbations of asthma are associated with viral respiratory tract infections, of which rhinoviruses (RV are the predominant virus type. Airway smooth muscle is important in asthma pathogenesis, however little is known about the potential interaction of RV and human airway smooth muscle cells (HASM. We hypothesised that rhinovirus induction of inflammatory cytokine release from airway smooth muscle is augmented and differentially regulated in asthmatic compared to normal HASM cells. Methods HASM cells, isolated from either asthmatic or non-asthmatic subjects, were infected with rhinovirus. Cytokine production was assayed by ELISA, ICAM-1 cell surface expression was assessed by FACS, and the transcription regulation of IL-6 was measured by luciferase activity. Results RV-induced IL-6 release was significantly greater in HASM cells derived from asthmatic subjects compared to non-asthmatic subjects. This response was RV specific, as 5% serum- induced IL-6 release was not different in the two cell types. Whilst serum stimulated IL-8 production in cells from both subject groups, RV induced IL-8 production in only asthmatic derived HASM cells. The transcriptional induction of IL-6 was differentially regulated via C/EBP in the asthmatic and NF-κB + AP-1 in the non-asthmatic HASM cells. Conclusion This study demonstrates augmentation and differential transcriptional regulation of RV specific innate immune response in HASM cells derived from asthmatic and non-asthmatics, and may give valuable insight into the mechanisms of RV-induced asthma exacerbations.

A Wireless sEMG Recording System and Its Application to Muscle Fatigue Detection

Directory of Open Access Journals (Sweden)

Xuan-Han Wu

2012-01-01

Full Text Available Surface electromyography (sEMG is an important measurement for monitoring exercise and fitness. Because if its high sampling frequency requirement, wireless transmission of sEMG data is a challenge. In this article a wireless sEMG measurement system with a sampling frequency of 2 KHz is developed based upon a MSP 430 microcontroller and Bluetooth transmission. Standard isotonic and isometric muscle contraction are clearly represented in the receiving user interface. Muscle fatigue detection is an important application of sEMG. Traditional muscle fatigue is detected from the median frequency of the sEMG power spectrum. The regression slope of the linear regression of median frequency is an important muscle fatigue index. A more negative slope value represents a higher muscle fatigue condition. To test the system performance, muscle fatigue detection was examined by having subjects run on a pedaled-multifunctional elliptical trainer for approximately 30 minutes at three loading levels. Ten subjects underwent a total of 60 exercise sessions to provide the experimental data. Results showed that the regression slope gradually decreases as expected, and there is a significant gender difference.

Enzymatic cell wall degradation of high-pressure-homogenized tomato puree and its effect on lycopene bioaccessibility.

Science.gov (United States)

Palmero, Paola; Colle, Ines; Lemmens, Lien; Panozzo, Agnese; Nguyen, Tuyen Thi My; Hendrickx, Marc; Van Loey, Ann

2016-01-15

High-pressure homogenization disrupts cell structures, assisting carotenoid release from the matrix and subsequent micellarization. However, lycopene bioaccessibility of tomato puree upon high-pressure homogenization is limited by the formation of a process-induced barrier. In this context, cell wall-degrading enzymes were applied to hydrolyze the formed barrier and enhance lycopene bioaccessibility. The effectiveness of the enzymes in degrading their corresponding substrates was evaluated (consistency, amount of reducing sugars, molar mass distribution and immunolabeling). An in vitro digestion procedure was applied to evaluate the effect of the enzymatic treatments on lycopene bioaccessibility. Enzymatic treatments with pectinases and cellulase were proved to effectively degrade their corresponding cell wall polymers; however, no further significant increase in lycopene bioaccessibility was obtained. A process-induced barrier consisting of cell wall material is not the only factor governing lycopene bioaccessibility upon high-pressure homogenization. © 2015 Society of Chemical Industry.

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.

Influence of exercise contraction mode and protein supplementation on human skeletal muscle satellite cell content and muscle fiber growth

DEFF Research Database (Denmark)

Farup, Jean; Rahbek, Stine Klejs; Riis, Simon

2014-01-01

-specific association between emergence of satellite cells (SCs), muscle growth, and remodeling in response to 12 wk unilateral resistance training performed as eccentric (Ecc) or concentric (Conc) resistance training ± whey protein (Whey, 19.5 g protein + 19.5 g glucose) or placebo (Placebo, 39 g glucose......Skeletal muscle satellite cells (SCs) are involved in remodeling and hypertrophy processes of skeletal muscle. However, little knowledge exists on extrinsic factors that influence the content of SCs in skeletal muscle. In a comparative human study, we investigated the muscle fiber type......) supplementation. Muscle biopsies (vastus lateralis) were analyzed for fiber type-specific SCs, myonuclei, and fiber cross-sectional area (CSA). Following training, SCs increased with Conc in both type I and type II fibers (P

Tenascin-Y, a component of distinctive connective tissues, supports muscle cell growth.

Science.gov (United States)

Hagios, C; Brown-Luedi, M; Chiquet-Ehrismann, R

1999-12-15

Chicken tenascin-Y is an extracellular matrix protein most closely related to the mammalian tenascin-X. It is highly expressed in the connective tissue of skeletal muscle (C. Hagios, M. Koch, J. Spring, M. Chiquet, and R. Chiquet-Ehrismann, 1996, J. Cell Biol. 134, 1499-1512). Here we demonstrate the presence of tenascin-Y in specific areas of the connective tissues in developing lung, kidney, and skin. In skin tenascin-Y shows a complementary expression pattern to tenascin-C, whereas in the lung and kidney the sites of expression are partly overlapping. Tenascin-Y is also present in embryonic skeletal muscle where it is expressed in the developing connective tissue in between the muscle fibers. This connective tissue is also the major site of alpha5 integrin expression. We purified recombinantly expressed tenascin-Y and tested its effect on cell adhesion and its influence on muscle cell growth and differentiation. C2C12 myoblasts were able to adhere to tenascin-Y and showed extensive formation of actin-rich processes without generation of stress fibers. Furthermore, we found that tenascin-Y influenced cell morphology of chick embryo fibroblasts over prolonged times in culture and that it supports primary muscle cell growth and restricts muscle cell differentiation. Copyright 1999 Academic Press.

MASTR directs MyoD-dependent satellite cell differentiation during skeletal muscle regeneration

Science.gov (United States)

Mokalled, Mayssa H.; Johnson, Aaron N.; Creemers, Esther E.; Olson, Eric N.

2012-01-01

In response to skeletal muscle injury, satellite cells, which function as a myogenic stem cell population, become activated, expand through proliferation, and ultimately fuse with each other and with damaged myofibers to promote muscle regeneration. Here, we show that members of the Myocardin family of transcriptional coactivators, MASTR and MRTF-A, are up-regulated in satellite cells in response to skeletal muscle injury and muscular dystrophy. Global and satellite cell-specific deletion of MASTR in mice impairs skeletal muscle regeneration. This impairment is substantially greater when MRTF-A is also deleted and is due to aberrant differentiation and excessive proliferation of satellite cells. These abnormalities mimic those associated with genetic deletion of MyoD, a master regulator of myogenesis, which is down-regulated in the absence of MASTR and MRTF-A. Consistent with an essential role of MASTR in transcriptional regulation of MyoD expression, MASTR activates a muscle-specific postnatal MyoD enhancer through associations with MEF2 and members of the Myocardin family. Our results provide new insights into the genetic circuitry of muscle regeneration and identify MASTR as a central regulator of this process. PMID:22279050

A new formulation for the problem of fuel cell homogenization

International Nuclear Information System (INIS)

Chao, Y.-A.; Martinez, A.S.

1982-01-01

A new homogenization method for reactor cells is described. This new method consists in eliminating the NR approximation for the fuel resonance and the Wigner approximation for the resonance escape probability; the background cross section is then redefined and the problem studied is reanalyzed. (E.G.) [pt

Primary non-Hodgkin lymphoma of skeletal muscle: imaging findings

International Nuclear Information System (INIS)

Zhou Liangping; Peng Weijun; Tang Feng; Mao Jian; Yang Wentao

2006-01-01

Objective: To analyze the imaging manifestations of primary non-Hodgkin lymphoma of skeletal muscle and improve the recognition of this rare disease. Methods: Five cases of primary non- Hodgkin lymphoma of skeletal muscle proved pathologically underwent imaging exam, including MRI and CT in 3 cases, only MRI in 1 case, only CT in 1 case, X-ray in 2 cases and bone scintigraphy in 2 cases. Results: Diffuse enlargements of involved muscle with presentation of overall configuration were observed in all five cases. All 4 cases manifested as homogeneous soft masses, which is isoattenuating to normal muscle on unenhanced CT images. After intravenous injection of contrast media, the masses enhanced homogeneously and slightly (2 cases) or moderately (1 case) on CT images. The lesions were homogenous and had isointense or slightly low signal intensity compared with that of uninvolved muscle on T 1 -weighted images and high signal intensity on T 2 -weighted images. After intravenous injection of contrast media, all 2 cases enhanced homogeneously and moderately with the enhanced signal intensity of involved muscle greatly higher than that of uninvolved muscle on MR images. Two cases of X-ray plain showed no destruction of bone and 2 cases of bone scintigraphy exams showed increased radiotracer uptake of involved muscle with no infiltration of bone marrow. Conclusion: There are several characteristics on the imaging of primary non-Hodgkin lymphoma of skeletal muscle. MRI is the optimal imaging method for the diagnosis of this disease. (authors)

Retained Myogenic Potency of Human Satellite Cells from Torn Rotator Cuff Muscles Despite Fatty Infiltration.

Science.gov (United States)

Koide, Masashi; Hagiwara, Yoshihiro; Tsuchiya, Masahiro; Kanzaki, Makoto; Hatakeyama, Hiroyasu; Tanaka, Yukinori; Minowa, Takashi; Takemura, Taro; Ando, Akira; Sekiguchi, Takuya; Yabe, Yutaka; Itoi, Eiji

2018-01-01

Rotator cuff tears (RCTs) are a common shoulder problem in the elderly that can lead to both muscle atrophy and fatty infiltration due to less physical load. Satellite cells, quiescent cells under the basal lamina of skeletal muscle fibers, play a major role in muscle regeneration. However, the myogenic potency of human satellite cells in muscles with fatty infiltration is unclear due to the difficulty in isolating from small samples, and the mechanism of the progression of fatty infiltration has not been elucidated. The purpose of this study was to analyze the population of myogenic and adipogenic cells in disused supraspinatus (SSP) and intact subscapularis (SSC) muscles of the RCTs from the same patients using fluorescence-activated cell sorting. The microstructure of the muscle with fatty infiltration was observed as a whole mount condition under multi-photon microscopy. Myogenic differentiation potential and gene expression were evaluated in satellite cells. The results showed that the SSP muscle with greater fatty infiltration surrounded by collagen fibers compared with the SSC muscle under multi-photon microscopy. A positive correlation was observed between the ratio of muscle volume to fat volume and the ratio of myogenic precursor to adipogenic precursor. Although no difference was observed in the myogenic potential between the two groups in cell culture, satellite cells in the disused SSP muscle showed higher intrinsic myogenic gene expression than those in the intact SSC muscle. Our results indicate that satellite cells from the disused SSP retain sufficient potential of muscle growth despite the fatty infiltration.

Effect of IR Laser on Myoblasts: Prospects of Application for Counteracting Microgravity-Induced Muscle Atrophy

Science.gov (United States)

Monici, Monica; Cialdai, Francesca; Romano, Giovanni; Corsetto, Paola Antonia; Rizzo, Angela Maria; Caselli, Anna; Ranaldi, Francesco

2013-02-01

Microgravity-induced muscle atrophy is a problem of utmost importance for the impact it may have on the health and performance of astronauts. Therefore, appropriate countermeasures are needed to prevent disuse atrophy and favour muscle recovery. Muscle atrophy is characterized by loss of muscle mass and strength, and a shift in substrate utilization from fat to glucose, that leads to a reduced metabolic efficiency and enhanced fatigability. Laser therapy is already used in physical medicine and rehabilitation to accelerate muscle recovery and in sports medicine to prevent damages produced by metabolic disturbances and inflammatory reactions after heavy exercise. The aim of the research we present was to get insights on possible benefits deriving from the application of an advanced infrared laser system to counteract deficits of muscle energy metabolism and stimulate the recovery of the hypotrophic tissue. The source used was a Multiwave Locked System (MLS) laser, which combines continuous and pulsed emissions at 808 nm and 905 nm, respectively. We studied the effect of MLS treatment on morphology and energy metabolism of C2C12 cells, a widely accepted myoblast model, previously exposed to microgravity conditions modelled by a Random Positioning Machine. The MLS laser treatment was able to restore basal levels of serine/threonine protein phosphatase activity and to counteract cytoskeletal alterations and increase in glycolytic enzymes activity that occurred following the exposure to modelled microgravity. In conclusion, the results provide interesting insights for the application of infrared laser in the treatment of muscle atrophy.

Hypoxia Enhances Differentiation of Adipose Tissue-Derived Stem Cells toward the Smooth Muscle Phenotype

Directory of Open Access Journals (Sweden)

Fang Wang

2018-02-01

Full Text Available Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction (p < 0.01. Cells differentiated in 5% oxygen conditions showed greater contraction effect (p < 0.01. Hypoxia influences differentiation of smooth muscle cells from adipose stem cells and 5% oxygen was the optimal condition to generate smooth muscle cells that contract from adipose stem cells.

Functional dysregulation of stem cells during aging: a focus on skeletal muscle stem cells.

Science.gov (United States)

García-Prat, Laura; Sousa-Victor, Pedro; Muñoz-Cánoves, Pura

2013-09-01

Aging of an organism is associated with the functional decline of tissues and organs, as well as a sharp decline in the regenerative capacity of stem cells. A prevailing view holds that the aging rate of an individual depends on the ratio of tissue attrition to tissue regeneration. Therefore, manipulations that favor the balance towards regeneration may prevent or delay aging. Skeletal muscle is a specialized tissue composed of postmitotic myofibers that contract to generate force. Satellite cells are the adult stem cells responsible for skeletal muscle regeneration. Recent studies on the biology of skeletal muscle and satellite cells in aging have uncovered the critical impact of systemic and niche factors on stem cell functionality and demonstrated the capacity of aged satellite cells to rejuvenate and increase their regenerative potential when exposed to a youthful environment. Here we review the current literature on the coordinated relationship between cell extrinsic and intrinsic factors that regulate the function of satellite cells, and ultimately determine tissue homeostasis and repair during aging, and which encourage the search for new anti-aging strategies. © 2013 The Authors Journal compilation © 2013 FEBS.

7 CFR 58.920 - Homogenization.

Science.gov (United States)

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Homogenization. 58.920 Section 58.920 Agriculture... Procedures § 58.920 Homogenization. Where applicable concentrated products shall be homogenized for the... homogenization and the pressure at which homogenization is accomplished will be that which accomplishes the most...

Transient HIF2A inhibition promotes satellite cell proliferation and muscle regeneration.

Science.gov (United States)

Xie, Liwei; Yin, Amelia; Nichenko, Anna S; Beedle, Aaron M; Call, Jarrod A; Yin, Hang

2018-03-13

The remarkable regeneration capability of skeletal muscle depends on coordinated proliferation and differentiation of satellite cells. The self-renewal of satellite cells is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in satellite cells in vivo remains largely unknown. Here, we report that satellite cells are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of satellite cells by maintaining the quiescence, increasing the self-renewal and blocking the myogenic differentiation of satellite cells. HIF2A stabilization in satellite cells cultured under normoxia augmented their engraftment potential in regenerative muscle. Reversely, HIF2A ablation led to the depletion of satellite cells and the consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerated muscle regeneration by increasing satellite cell proliferation and differentiation. Mechanistically, HIF2A induces the quiescence/self-renewal of satellite cells by binding the promoter of Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in satellite cells and may be therapeutically targeted to improve muscle regeneration.

Application of 31P-NMR spectroscopy to the study of striated muscle metabolism

International Nuclear Information System (INIS)

Meyer, R.A.; Kushmerick, M.J.; Brown, T.R.

1982-01-01

This review presents the principles and limitations of phosphorus nuclear magnetic resonance ( 31 P-NMR) spectroscopy as applied to the study of striated muscle metabolism. Application of the techniques discussed include noninvasive measurement of high-energy phosphate, intracellular pH, intracellular free Mg 2+ , and metabolite compartmentation. In perfused cat biceps (fast-twitch) muscles, but not in soleus (slow-twitch), NMR spectra indicate a substantially lower (1 mM) free inorganic phosphate level than when measured chemically (6 mM). In addition, saturation and inversion spin-transfer methods that enable direct measurement of the unidirectional fluxes through creatine kinase are described. In perfused cat biceps muscle, results suggest that this enzyme and its substrates are in simple chemical equilibrium

Image analysis for the automated estimation of clonal growth and its application to the growth of smooth muscle cells.

Science.gov (United States)

Gavino, V C; Milo, G E; Cornwell, D G

1982-03-01

Image analysis was used for the automated measurement of colony frequency (f) and colony diameter (d) in cultures of smooth muscle cells, Initial studies with the inverted microscope showed that number of cells (N) in a colony varied directly with d: log N = 1.98 log d - 3.469 Image analysis generated the complement of a cumulative distribution for f as a function of d. The number of cells in each segment of the distribution function was calculated by multiplying f and the average N for the segment. These data were displayed as a cumulative distribution function. The total number of colonies (fT) and the total number of cells (NT) were used to calculate the average colony size (NA). Population doublings (PD) were then expressed as log2 NA. Image analysis confirmed previous studies in which colonies were sized and counted with an inverted microscope. Thus, image analysis is a rapid and automated technique for the measurement of clonal growth.

Age-related effect of cell death on fiber morphology and number in tongue muscle.

Science.gov (United States)

Kletzien, Heidi; Hare, Allison J; Leverson, Glen; Connor, Nadine P

2018-01-01

Multiple pathways may exist for age-related tongue muscle degeneration. Cell death is one mechanism contributing to muscle atrophy and decreased function. We hypothesized with aging, apoptosis, and apoptotic regulators would be increased, and muscle fiber size and number would be reduced in extrinsic tongue muscles. Cell death indices, expression of caspase-3 and Bcl-2, and measures of muscle morphology and number were determined in extrinsic tongue muscles of young and old rats. Significant increases in cell death, caspase-3, and Bcl-2 were observed in all extrinsic tongue muscles along with reductions in muscle fiber number in old rats. We demonstrated that apoptosis indices increase with age in lingual muscles and that alterations in apoptotic regulators may be associated with age-related degeneration in muscle fiber size and number. These observed apoptotic processes may be detrimental to muscle function, and may contribute to degradation of cranial functions with age. Muscle Nerve 57: E29-E37, 2018. © 2017 Wiley Periodicals, Inc.

Human induced pluripotent stem cell-derived vascular smooth muscle cells

DEFF Research Database (Denmark)

Ayoubi, Sohrab; Sheikh, Søren P; Eskildsen, Tilde V

2017-01-01

. To this end, human induced pluripotent stem cells (hiPSCs) have generated great enthusiasm, and have been a driving force for development of novel strategies in drug discovery and regenerative cell-therapy for the last decade. Hence, investigating the mechanisms underlying the differentiation of hi......PSCs into specialized cell types such as cardiomyocytes, endothelial cells, and vascular smooth muscle cells (VSMCs) may lead to a better understanding of developmental cardiovascular processes and potentiate progress of safe autologous regenerative therapies in pathological conditions. In this review, we summarize...

Myostatin in relation to physical activity and dysglycaemia and its effect on energy metabolism in human skeletal muscle cells.

Science.gov (United States)

Hjorth, M; Pourteymour, S; Görgens, S W; Langleite, T M; Lee, S; Holen, T; Gulseth, H L; Birkeland, K I; Jensen, J; Drevon, C A; Norheim, F

2016-05-01

Some health benefits of exercise may be explained by an altered secretion of myokines. Because previous focus has been on upregulated myokines, we screened for downregulated myokines and identified myostatin. We studied the expression of myostatin in relation to exercise and dysglycaemia in skeletal muscle, adipose tissue and plasma. We further examined some effects of myostatin on energy metabolism in primary human muscle cells and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. Sedentary men with or without dysglycaemia underwent a 45-min acute bicycle test before and after 12 weeks of combined endurance and strength training. Blood samples and biopsies from m. vastus lateralis and adipose tissue were collected. Myostatin mRNA expression was reduced in skeletal muscle after acute as well as long-term exercise and was even further downregulated by acute exercise on top of 12-week training. Furthermore, the expression of myostatin at baseline correlated negatively with insulin sensitivity. Myostatin expression in the adipose tissue increased after 12 weeks of training and correlated positively with insulin sensitivity markers. In cultured muscle cells but not in SGBS cells, myostatin promoted an insulin-independent increase in glucose uptake. Furthermore, muscle cells incubated with myostatin had an enhanced rate of glucose oxidation and lactate production. Myostatin was differentially expressed in the muscle and adipose tissue in relation to physical activity and dysglycaemia. Recombinant myostatin increased the consumption of glucose in human skeletal muscle cells, suggesting a complex regulatory role of myostatin in skeletal muscle homeostasis. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Impaired macrophage and satellite cell infiltration occurs in a muscle-specific fashion following injury in diabetic skeletal muscle.

Directory of Open Access Journals (Sweden)

Matthew P Krause

Full Text Available Systemic elevations in PAI-1 suppress the fibrinolytic pathway leading to poor collagen remodelling and delayed regeneration of tibialis anterior (TA muscles in type-1 diabetic Akita mice. However, how impaired collagen remodelling was specifically attenuating regeneration in Akita mice remained unknown. Furthermore, given intrinsic differences between muscle groups, it was unclear if the reparative responses between muscle groups were different.Here we reveal that diabetic Akita muscles display differential regenerative responses with the TA and gastrocnemius muscles exhibiting reduced regenerating myofiber area compared to wild-type mice, while soleus muscles displayed no difference between animal groups following injury. Collagen levels in TA and gastrocnemius, but not soleus, were significantly increased post-injury versus controls. At 5 days post-injury, when degenerating/necrotic regions were present in both animal groups, Akita TA and gastrocnemius muscles displayed reduced macrophage and satellite cell infiltration and poor myofiber formation. By 10 days post-injury, necrotic regions were absent in wild-type TA but persisted in Akita TA. In contrast, Akita soleus exhibited no impairment in any of these measures compared to wild-type soleus. In an effort to define how impaired collagen turnover was attenuating regeneration in Akita TA, a PAI-1 inhibitor (PAI-039 was orally administered to Akita mice following cardiotoxin injury. PAI-039 administration promoted macrophage and satellite cell infiltration into necrotic areas of the TA and gastrocnemius. Importantly, soleus muscles exhibit the highest inducible expression of MMP-9 following injury, providing a mechanism for normative collagen degradation and injury recovery in this muscle despite systemically elevated PAI-1.Our findings suggest the mechanism underlying how impaired collagen remodelling in type-1 diabetes results in delayed regeneration is an impairment in macrophage

Cell death induced by gamma irradiation of developing skeletal muscle

International Nuclear Information System (INIS)

Olive, M.; Blanco, R.; Rivera, R.; Cinos, C.; Ferrer, I.

1995-01-01

Newborn Sprague-Dawley rats were exposed to a single dose of 2 Gy gamma rays and killed from 6 h to 5 d later. Increased numbers of dying cells, characterised by their extreme chromatin condensation and often nuclear fragmentation were seen in skeletal muscle 6 h after irradiation. Dying cells decreased to nearly normal values 48 h later. In situ labelling of nuclear DNA fragmentation identified individual cells bearing fragmented DNA. The effects of gamma rays were suppressed following cycloheximide i.p. at a dose of 1 μg/g body weight given at the time of irradiation. Taken together, the present morphological and pharmacological results suggest that gamma ray induced cell death in skeletal muscle is apoptotic, and that the process is associated with protein synthesis. Finally, proliferating cell nuclear antigen-immunoreactive cells, which were abundant in control rats, decreased in number 48 h after irradiation. However, a marked increase significantly above normal age values was observed at the 5th day, thus suggesting that regeneration occurs following irradiation-induced cell death in developing muscle. (author)

Exercise-induced muscle-derived cytokines inhibit mammary cancer cell growth.

Science.gov (United States)

Hojman, Pernille; Dethlefsen, Christine; Brandt, Claus; Hansen, Jakob; Pedersen, Line; Pedersen, Bente Klarlund

2011-09-01

Regular physical activity protects against the development of breast and colon cancer, since it reduces the risk of developing these by 25-30%. During exercise, humoral factors are released from the working muscles for endocrinal signaling to other organs. We hypothesized that these myokines mediate some of the inhibitory effects of exercise on mammary cancer cell proliferation. Serum and muscles were collected from mice after an exercise bout. Incubation with exercise-conditioned serum inhibited MCF-7 cell proliferation by 52% and increased caspase activity by 54%. A similar increase in caspase activity was found after incubation of MCF-7 cells with conditioned media from electrically stimulated myotubes. PCR array analysis (CAPM-0838E; SABiosciences) revealed that seven genes were upregulated in the muscles after exercise, and of these oncostatin M (OSM) proved to inhibit MCF-7 proliferation by 42%, increase caspase activity by 46%, and induce apoptosis. Blocking OSM signaling with anti-OSM antibodies reduced the induction of caspase activity by 51%. To verify that OSM was a myokine, we showed that it was significantly upregulated in serum and in three muscles, tibialis cranialis, gastronemius, and soleus, after an exercise bout. In contrast, OSM expression remained unchanged in subcutaneous and visceral adipose tissue, liver, and spleen (mononuclear cells). We conclude that postexercise serum inhibits mammary cancer cell proliferation and induces apoptosis of these cells. We suggest that one or more myokines secreted from working muscles may be mediating this effect and that OSM is a possible candidate. These findings emphasize that role of physical activity in cancer treatment, showing a direct link between exercise-induced humoral factors and decreased tumor cell growth.

Evaluation of hematopoietic potential generated by transplantation of muscle-derived stem cells in mice.

Science.gov (United States)

Farace, Francoise; Prestoz, Laetitita; Badaoui, Sabrina; Guillier, Martine; Haond, Celine; Opolon, Paule; Thomas, Jean-Leon; Zalc, Bernard; Vainchenker, William; Turhan, Ali G

2004-02-01

Muscle tissue of adult mice has been shown to contain stem cells with hematopoietic repopulation ability in vivo. To determine the functional characteristics of stem cells giving rise to this hematopoietic activity, we have performed hematopoietic reconstitution experiments by the use of muscle versus marrow transplantation in lethally irradiated mice and followed the fate of transplanted cells by Y-chimerism using PCR and fluorescence in situ hybridization (FISH) analysis. We report here that transplantation of murine muscle generate a major hematopoietic chimerism at the level of CFU-C, CFU-S, and terminally-differentiated cells in three generations of lethally irradiated mice followed up to 1 year after transplantation. This potential is totally abolished when muscle grafts were performed by the use of muscle from previously irradiated mice. As compared to marrow transplantation, muscle transplants were able to generate similar potencies to give rise to myeloid, T, B, and natural killer (NK) cells. Interestingly, marrow stem cells that have been generated in primary and then in secondary recipients were able to contribute efficiently to myofibers in the muscle tissue of tertiary recipients. Altogether, our data demonstrate that muscle-derived stem cells present a major hematopoietic repopulating ability with evidence of self-replication in vivo. They are radiation-sensitive and similar to marrow-derived stem cells in terms of their ability to generate multilineage hematopoiesis. Finally, our data demonstrate that muscle-derived hematopoietic stem cells do not lose their ability to contribute to myofiber generation after at least two rounds of serial transplantation, suggesting a potential that is probably equivalent to that generated by marrow transplantation.

Regulation of collagen biosynthesis in cultured bovine aortic smooth muscle cells

International Nuclear Information System (INIS)

Stepp, M.A.

1986-01-01

Aortic smooth muscles cells have been implicated in the etiology of lesions which occur in atherosclerosis and hypertension. Both diseases involve proliferation of smooth muscle cells and accumulation of excessive amounts of extracellular matrix proteins, including collagen type I and type III produced by the smooth muscle cells. To better understand the sites of regulation of collagen biosynthesis and to correlate these with the growth rate of the cells, cultured bovine aortic smooth muscle cells were studied as a function of the number of days (3 to 14) in second passage. Cells grew rapidly up to day 6 when confluence was reached. The total incorporation of [ 3 H]-proline into proteins was highest at day 3 and decreased to a constant level after the cultures reached confluence. In contrast, collagen protein production was lowest before confluence and continued to increase over the entire time course of the experiments. cDNA clones for the α1 and α2 chains of type I and the α1 chain of type III collagen were used to quantitate the steady state level of collagen mRNAs. RNA was tested in a cell-free translation system. Changes in the translational activity of collagen mRNAs parallelled the observed increases in collagen protein production. Thus, at later time points, collagen mRNAs are more active in directing synthesis of preprocollagens, even though less collagen mRNA is present. The conclusion is that the site of regulation of the expression of collagen genes is a function of the growth rate of cultured smooth muscle cells

Muscle Progenitor Cell Regenerative Capacity in the Torn Rotator Cuff

Science.gov (United States)

Meyer, Gretchen A.; Farris, Ashley L.; Sato, Eugene; Gibbons, Michael; Lane, John G.; Ward, Samuel R.; Engler, Adam J.

2014-01-01

Chronic rotator cuff (RC) tears affect a large portion of the population and result in substantial upper extremity impairment, shoulder weakness, pain and limited range of motion. Regardless of surgical or conservative treatment, persistent atrophic muscle changes limit functional restoration and may contribute to surgical failure. We hypothesized that deficits in the skeletal muscle progenitor (SMP) cell pool could contribute to poor muscle recovery following tendon repair. Biopsies were obtained from patients undergoing arthroscopic RC surgery. The SMP population was quantified, isolated and assayed in culture for its ability to proliferate and fuse in-vitro and in-vivo. The SMP population was larger in muscles from cuffs with partial tears compared with no tears or full thickness tears. However, SMPs from muscles in the partial tear group also exhibited reduced proliferative ability. Cells from all cuff states were able to fuse robustly in culture and engraft when injected into injured mouse muscle, suggesting that when given the correct signals, SMPs are capable of contributing to muscle hypertrophy and regeneration regardless of tear severity. The fact that this does not appear to happen in-vivo helps focus future therapeutic targets for promoting muscle recovery following rotator cuff repairs and may help improve clinical outcomes. PMID:25410765

Flow rate calibration to determine cell-derived microparticles and homogeneity of blood components.

Science.gov (United States)

Noulsri, Egarit; Lerdwana, Surada; Kittisares, Kulvara; Palasuwan, Attakorn; Palasuwan, Duangdao

2017-08-01

Cell-derived microparticles (MPs) are currently of great interest to screening transfusion donors and blood components. However, the current approach to counting MPs is not affordable for routine laboratory use due to its high cost. The current study aimed to investigate the potential use of flow-rate calibration for counting MPs in whole blood, packed red blood cells (PRBCs), and platelet concentrates (PCs). The accuracy of flow-rate calibration was investigated by comparing the platelet counts of an automated counter and a flow-rate calibrator. The concentration of MPs and their origins in whole blood (n=100), PRBCs (n=100), and PCs (n=92) were determined using a FACSCalibur. The MPs' fold-changes were calculated to assess the homogeneity of the blood components. Comparing the platelet counts conducted by automated counting and flow-rate calibration showed an r 2 of 0.6 (y=0.69x+97,620). The CVs of the within-run and between-run variations of flow-rate calibration were 8.2% and 12.1%, respectively. The Bland-Altman plot showed a mean bias of -31,142platelets/μl. MP enumeration revealed both the difference in MP levels and their origins in whole blood, PRBCs, and PCs. Screening the blood components demonstrated high heterogeneity of the MP levels in PCs when compared to whole blood and PRBCs. The results of the present study suggest the accuracy and precision of flow-rate calibration for enumerating MPs. This flow-rate approach is affordable for assessing the homogeneity of MPs in blood components in routine laboratory practice. Copyright © 2017 Elsevier Ltd. All rights reserved.

Influence of exercise and aging on extracellular matrix composition in the skeletal muscle stem cell niche.

Science.gov (United States)

Garg, Koyal; Boppart, Marni D

2016-11-01

Skeletal muscle is endowed with a remarkable capacity for regeneration, primarily due to the reserve pool of muscle resident satellite cells. The satellite cell is the physiologically quiescent muscle stem cell that resides beneath the basal lamina and adjacent to the sarcolemma. The anatomic location of satellite cells is in close proximity to vasculature where they interact with other muscle resident stem/stromal cells (e.g., mesenchymal stem cells and pericytes) through paracrine mechanisms. This mini-review describes the components of the muscle stem cell niche, as well as the influence of exercise and aging on the muscle stem cell niche. Although exercise promotes ECM reorganization and stem cell accumulation, aging is associated with dense ECM deposition and loss of stem cell function resulting in reduced regenerative capacity and strength. An improved understanding of the niche elements will be valuable to inform the development of therapeutic interventions aimed at improving skeletal muscle regeneration and adaptation over the life span. Copyright © 2016 the American Physiological Society.

A novel paradigm links mitochondrial dysfunction with muscle stem cell impairment in sepsis.

Science.gov (United States)

Chatre, Laurent; Verdonk, Franck; Rocheteau, Pierre; Crochemore, Clément; Chrétien, Fabrice; Ricchetti, Miria

2017-10-01

Sepsis is an acute systemic inflammatory response of the body to microbial infection and a life threatening condition associated with multiple organ failure. Survivors may display long-term disability with muscle weakness that remains poorly understood. Recent data suggest that long-term myopathy in sepsis survivors is due to failure of skeletal muscle stem cells (satellite cells) to regenerate the muscle. Satellite cells impairment in the acute phase of sepsis is linked to unusual mitochondrial dysfunctions, characterized by a dramatic reduction of the mitochondrial mass and hyperactivity of residual organelles. Survivors maintain the impairment of satellite cells, including alterations of the mitochondrial DNA (mtDNA), in the long-term. This condition can be rescued by treatment with mesenchymal stem cells (MSCs) that restore mtDNA alterations and mitochondrial function in satellite cells, and in fine their regenerative potential. Injection of MSCs in turn increases the force of isolated muscle fibers and of the whole animal, and improves the survival rate. These effects occur in the context of reduced inflammation markers that also raised during sepsis. Targeting muscle stem cells mitochondria, in a context of reduced inflammation, may represent a valuable strategy to reduce morbidity and long-term impairment of the muscle upon sepsis. Copyright © 2017 Elsevier B.V. All rights reserved.

PGC-1α regulates alanine metabolism in muscle cells.

Science.gov (United States)

Hatazawa, Yukino; Qian, Kun; Gong, Da-Wei; Kamei, Yasutomi

2018-01-01

The skeletal muscle is the largest organ in the human body, depositing energy as protein/amino acids, which are degraded in catabolic conditions such as fasting. Alanine is synthesized and secreted from the skeletal muscle that is used as substrates of gluconeogenesis in the liver. During fasting, the expression of PGC-1α, a transcriptional coactivator of nuclear receptors, is increased in the liver and regulates gluconeogenesis. In the present study, we observed increased mRNA expression of PGC-1α and alanine aminotransferase 2 (ALT2) in the skeletal muscle during fasting. In C2C12 myoblast cells overexpressing PGC-1α, ALT2 expression was increased concomitant with an increased alanine level in the cells and medium. In addition, PGC-1α, along with nuclear receptor ERR, dose-dependently enhanced the ALT2 promoter activity in reporter assay using C2C12 cells. In the absence of glucose in the culture medium, mRNA levels of PGC-1α and ALT2 increased. Endogenous PGC-1α knockdown in C2C12 cells reduced ALT2 gene expression level, induced by the no-glucose medium. Taken together, in the skeletal muscle, PGC-1α activates ALT2 gene expression, and alanine production may play roles in adaptation to fasting.

Muscle Atrophy Reversed by Growth Factor Activation of Satellite Cells in a Mouse Muscle Atrophy Model

DEFF Research Database (Denmark)

Hauerslev, Simon; Vissing, John; Krag, Thomas O

2014-01-01

mechanism that may contribute to the progressive muscle wasting seen in severely affected patients with muscular dystrophy and significant on-going regeneration. This treatment could potentially be applied to many conditions that feature muscle wasting to increase muscle bulk and strength.......Muscular dystrophies comprise a large group of inherited disorders that lead to progressive muscle wasting. We wanted to investigate if targeting satellite cells can enhance muscle regeneration and thus increase muscle mass. We treated mice with hepatocyte growth factor and leukemia inhibitory...... factor under three conditions: normoxia, hypoxia and during myostatin deficiency. We found that hepatocyte growth factor treatment led to activation of the Akt/mTOR/p70S6K protein synthesis pathway, up-regulation of the myognic transcription factors MyoD and myogenin, and subsequently the negative growth...

Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

Science.gov (United States)

Yu, Fang-Jian; Zeng, Chang-Jun; Zhang, Yan; Wang, Cheng-Dong; Xiong, Tie-Yi; Fang, Sheng-Guo; Zhang, He-Min

2015-06-01

The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

The Application of Homogenate and Filtrate from Baltic Seaweeds in Seedling Growth Tests

Directory of Open Access Journals (Sweden)

Izabela Michalak

2017-02-01

Full Text Available Algal filtrate and homogenate, obtained from Baltic seaweeds, were applied in seedling growth tests. Radish seeds were used in order to assess algal products phytotoxicity and their biostimulant effect on growth and nutrient uptake. Algal filtrate, at concentrations ranging from 5.0% to 100% was used for seed soaking and as a liquid biostimulant (soil and foliar application. Algal homogenate was developed for seed coating. Algal filtrate and homogenate were also enriched with Zn(II ions in order to examine the influence on metal ion complexation. The optimal doses of algal filtrate and homogenate, as well as soaking time were established. Multi-elemental analyses of the raw biomass, filtrate, homogenate, and radish were also performed using ICP-OES (Inductively Coupled Plasma—Optical Emission Spectrometry. The best results in terms of seedlings’ length and weight were obtained using clear filtrate at a concentration of 50% applied to the soil and for homogenate applied at a dose of 50 mg/g of seeds. Clear filtrate at a concentration of 50% used for seed soaking for one hour showed the best results. The applied algal products increased the content of elements in seedlings. Among the tested products, a concentration of 50% algal filtrate is recommended for future pot and field experiments.

File list: InP.Emb.10.AllAg.Muscle_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: InP.Emb.20.AllAg.Muscle_cells [Chip-atlas[Archive

Lifescience Database Archive (English)

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The pathway to muscle fibrosis depends on myostatin stimulating the differentiation of fibro/adipogenic progenitor cells in chronic kidney disease.

Science.gov (United States)

Dong, Jiangling; Dong, Yanjun; Chen, Zihong; Mitch, William E; Zhang, Liping

2017-01-01

Fibrosis in skeletal muscle develops after injury or in response to chronic kidney disease (CKD), but the origin of cells becoming fibrous tissue and the initiating and sustaining mechanisms causing muscle fibrosis are unclear. We identified muscle fibro/adipogenic progenitor cells (FAPs) that potentially differentiate into adipose tissues or fibrosis. We also demonstrated that CKD stimulates myostatin production in muscle. Therefore, we tested whether CKD induces myostatin, which stimulates fibrotic differentiation of FAPs leading to fibrosis in skeletal muscles. We isolated FAPs from mouse muscles and found that myostatin stimulates their proliferation and conversion into fibrocytes. In vivo, FAPs isolated from EGFP-transgenic mice (FAPs-EGFP) were transplanted into muscles of mice with CKD or into mouse muscles that were treated with myostatin. CKD or myostatin stimulated FAPs-EGFP proliferation in muscle and increased α-smooth muscle actin expression in FAP-EGFP cells. When myostatin was inhibited with a neutralizing peptibody (a chimeric peptide-Fc fusion protein), the FAP proliferation and muscle fibrosis induced by CKD were both suppressed. Knocking down Smad3 in cultured FAPs interrupted their conversion into fibrocytes, indicating that myostatin directly converts FAPs into fibrocytes. Thus, counteracting myostatin may be a strategy for preventing the development of fibrosis in skeletal muscles of patients with CKD. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro

DEFF Research Database (Denmark)

Wewer, U M; Iba, K; Durkin, M E

1998-01-01

differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining...... cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced...... that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis....

Muscle glycogen and cell function--Location, location, location.

Science.gov (United States)

Ørtenblad, N; Nielsen, J

2015-12-01

The importance of glycogen, as a fuel during exercise, is a fundamental concept in exercise physiology. The use of electron microscopy has revealed that glycogen is not evenly distributed in skeletal muscle fibers, but rather localized in distinct pools. In this review, we present the available evidence regarding the subcellular localization of glycogen in skeletal muscle and discuss this from the perspective of skeletal muscle fiber function. The distribution of glycogen in the defined pools within the skeletal muscle varies depending on exercise intensity, fiber phenotype, training status, and immobilization. Furthermore, these defined pools may serve specific functions in the cell. Specifically, reduced levels of these pools of glycogen are associated with reduced SR Ca(2+) release, muscle relaxation rate, and membrane excitability. Collectively, the available literature strongly demonstrates that the subcellular localization of glycogen has to be considered to fully understand the role of glycogen metabolism and signaling in skeletal muscle function. Here, we propose that the effect of low muscle glycogen on excitation-contraction coupling may serve as a built-in mechanism, which links the energetic state of the muscle fiber to energy utilization. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

Energy Technology Data Exchange (ETDEWEB)

Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.; Daniel, Mark L.; Cu, Sharon R.; McLoon, Linda K., E-mail: mcloo001@tc.umn.edu

2011-04-01

The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin{sup -/-} (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a

Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

International Nuclear Information System (INIS)

Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.; Daniel, Mark L.; Cu, Sharon R.; McLoon, Linda K.

2011-01-01

The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin -/- (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation of

Conditional Loss of Pten in Myogenic Progenitors Leads to Postnatal Skeletal Muscle Hypertrophy but Age-Dependent Exhaustion of Satellite Cells.

Science.gov (United States)

Yue, Feng; Bi, Pengpeng; Wang, Chao; Li, Jie; Liu, Xiaoqi; Kuang, Shihuan

2016-11-22

Skeletal muscle stem cells (satellite cells [SCs]) are normally maintained in a quiescent (G 0 ) state. Muscle injury not only activates SCs locally, but also alerts SCs in distant uninjured muscles via circulating factors. The resulting G Alert SCs are adapted to regenerative cues and regenerate injured muscles more efficiently, but whether they provide any long-term benefits to SCs is unknown. Here, we report that embryonic myogenic progenitors lacking the phosphatase and tensin homolog (Pten) exhibit enhanced proliferation and differentiation, resulting in muscle hypertrophy but fewer SCs in adult muscles. Interestingly, Pten null SCs are predominantly in the G Alert state, even in the absence of an injury. The G Alert SCs are deficient in self-renewal and subjected to accelerated depletion during regeneration and aging and fail to repair muscle injury in old mice. Our findings demonstrate a key requirement of Pten in G 0 entry of SCs and provide functional evidence that prolonged G Alert leads to stem cell depletion and regenerative failure. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Macrophages and mast cells in dystrophic masseter muscle: a light and electron microscopic study

DEFF Research Database (Denmark)

Kirkeby, S; Mikkelsen, H

1988-01-01

Macrophages and mast cells in masseter muscle from normal and dystrophic mice were studied by light and electron microscopy. Acid phosphatase activity and FITC-dextran were used to identify and describe macrophages. Toluidine blue was used as a marker for mast cells. In dystrophic muscle, the num......Macrophages and mast cells in masseter muscle from normal and dystrophic mice were studied by light and electron microscopy. Acid phosphatase activity and FITC-dextran were used to identify and describe macrophages. Toluidine blue was used as a marker for mast cells. In dystrophic muscle...

Diseased muscles that lack dystrophin or laminin-α2 have altered compositions and proliferation of mononuclear cell populations

Directory of Open Access Journals (Sweden)

Miller Jeffrey

2005-04-01

Full Text Available Abstract Background Multiple types of mononucleate cells reside among the multinucleate myofibers in skeletal muscles and these mononucleate cells function in muscle maintenance and repair. How neuromuscular disease might affect different types of muscle mononucleate cells had not been determined. In this study, therefore, we examined how two neuromuscular diseases, dystrophin-deficiency and laminin-α2-deficiency, altered the proliferation and composition of different subsets of muscle-derived mononucleate cells. Methods We used fluorescence-activated cell sorting combined with bromodeoxyuridine labeling to examine proliferation rates and compositions of mononuclear cells in diseased and healthy mouse skeletal muscle. We prepared mononucleate cells from muscles of mdx (dystrophin-deficient or Lama2-/- (laminin-α2-deficient mice and compared them to cells from healthy control muscles. We enumerated subsets of resident muscle cells based on Sca-1 and CD45 expression patterns and determined the proliferation of each cell subset in vivo by BrdU incorporation. Results We found that the proliferation and composition of the mononucleate cells in dystrophin-deficient and laminin-α2-deficient diseased muscles are different than in healthy muscle. The mdx and Lama2-/- muscles showed similar significant increases in CD45+ cells compared to healthy muscle. Changes in proliferation, however, differed between the two diseases with proliferation increased in mdx and decreased in Lama2-/- muscles compared to healthy muscles. In particular, the most abundant Sca-1-/CD45- subset, which contains muscle precursor cells, had increased proliferation in mdx muscle but decreased proliferation in Lama2-/- muscles. Conclusion The similar increases in CD45+ cells, but opposite changes in proliferation of muscle precursor cells, may underlie aspects of the distinct pathologies in the two diseases.

Modulation of Stem Cells Differentiation and Myostatin as an approach to Counteract fibrosis in Muscle Dystrophy and Regeneration after Injury

Science.gov (United States)

2010-03-01

Duchenne muscular dystrophy (DMD), hampers cell therapy in the muscle , and is a feasible therapeutic target. Myostatin (Mst), a...17 Figure 18 Figure 19 Figure 20 Figure 21 • Muscle lipofibrotic degeneration characterizes Duchenne muscular dystrophy (DMD), hampers cell therapy...SUBJECT TERMS Myostatin, muscle dystrophy , stem cells, myogenesis, Oct-4; Duchenne 16. SECURITY CLASSIFICATION OF: U 17. LIMITATION OF ABSTRACT

Adhesion of axolemmal fragments to Schwann cells: a signal- and target-specific process closely linked to axolemmal induction of Schwann cell mitosis

International Nuclear Information System (INIS)

Sobue, G.; Pleasure, D.

1985-01-01

Radioiodinated rat CNS axolemmal fragments adhered to cultured rat Schwann cells by a time-, temperature-, and concentration-dependent process independent of extracellular ionized calcium. Adhesion showed target and signal specificity; axolemmal fragments adhered to endoneurial or dermal fibroblasts to a much lesser extent than to Schwann cells, and plasma membrane fragments from skeletal muscle, erythrocytes, or PNS myelin adhered to Schwann cells to a lesser extent than did axolemmal fragments. Brief trypsinization removed 94 to 97% of bound radioactivity from Schwann cells previously incubated with 125 I-axolemmal fragments for up to 24 hr, indicating that adhesion was largely a surface phenomenon rather than the result of rapid internalization of axolemmal fragments by the Schwann cells. When adhesion was compared to the axolemmal mitogenic response of Schwann cells, the concentration of axolemmal fragments yielding half-maximal adhesion was the same as the concentration producing half-maximal stimulation of Schwann cell mitosis. Trypsin digestion, homogenization, or heating of axolemmal fragments before application to cultured Schwann cells diminished adhesion and axolemmal fragment-induced stimulation of Schwann cell mitosis in a parallel fashion. Whereas adhesion of axolemmal fragments to the surfaces of the cultured Schwann cells reached completion within 4 hr in this assay system, induction of Schwann cell mitosis by the fragments required contact with Schwann cells for a minimum of 6 to 8 hr and reached a maximum when the axolemmal fragments had adhered to the Schwann cells for 24 hr or more

Cell Identity Switching Regulated by Retinoic Acid Signaling Maintains Homogeneous Segments in the Hindbrain.

Science.gov (United States)

Addison, Megan; Xu, Qiling; Cayuso, Jordi; Wilkinson, David G

2018-06-04

The patterning of tissues to form subdivisions with distinct and homogeneous regional identity is potentially disrupted by cell intermingling. Transplantation studies suggest that homogeneous segmental identity in the hindbrain is maintained by identity switching of cells that intermingle into another segment. We show that switching occurs during normal development and is mediated by feedback between segment identity and the retinoic acid degrading enzymes, cyp26b1 and cyp26c1. egr2, which specifies the segmental identity of rhombomeres r3 and r5, underlies the lower expression level of cyp26b1 and cyp26c1 in r3 and r5 compared with r2, r4, and r6. Consequently, r3 or r5 cells that intermingle into adjacent segments encounter cells with higher cyp26b1/c1 expression, which we find is required for downregulation of egr2b expression. Furthermore, egr2b expression is regulated in r2, r4, and r6 by non-autonomous mechanisms that depend upon the number of neighbors that express egr2b. These findings reveal that a community regulation of retinoid signaling maintains homogeneous segmental identity. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

PITX2 Enhances the Regenerative Potential of Dystrophic Skeletal Muscle Stem Cells.

Science.gov (United States)

Vallejo, Daniel; Hernández-Torres, Francisco; Lozano-Velasco, Estefanía; Rodriguez-Outeiriño, Lara; Carvajal, Alejandra; Creus, Carlota; Franco, Diego; Aránega, Amelia Eva

2018-04-10

Duchenne muscular dystrophy (DMD), one of the most lethal genetic disorders, involves progressive muscle degeneration resulting from the absence of DYSTROPHIN. Lack of DYSTROPHIN expression in DMD has critical consequences in muscle satellite stem cells including a reduced capacity to generate myogenic precursors. Here, we demonstrate that the c-isoform of PITX2 transcription factor modifies the myogenic potential of dystrophic-deficient satellite cells. We further show that PITX2c enhances the regenerative capability of mouse DYSTROPHIN-deficient satellite cells by increasing cell proliferation and the number of myogenic committed cells, but importantly also increasing dystrophin-positive (revertant) myofibers by regulating miR-31. These PITX2-mediated effects finally lead to improved muscle function in dystrophic (DMD/mdx) mice. Our studies reveal a critical role for PITX2 in skeletal muscle repair and may help to develop therapeutic strategies for muscular disorders. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Characterization of DLK1+ cells emerging during skeletal muscle remodeling in response to myositis, myopathies, and acute injury

DEFF Research Database (Denmark)

Andersen, Ditte C; Petersson, Stine J; Jørgensen, Louise H

2009-01-01

, DLK1 was upregulated in all human myopathies analyzed, including Duchenne- and Becker muscular dystrophies. Substantial numbers of DLK1(+) satellite cells were observed in normal neonatal and Duchenne muscle, and furthermore, myogenic DLK1(+) cells were identified during muscle regeneration in animal...

Differential Gene Expression Profiling of Dystrophic Dog Muscle after MuStem Cell Transplantation.

Science.gov (United States)

Robriquet, Florence; Lardenois, Aurélie; Babarit, Candice; Larcher, Thibaut; Dubreil, Laurence; Leroux, Isabelle; Zuber, Céline; Ledevin, Mireille; Deschamps, Jack-Yves; Fromes, Yves; Cherel, Yan; Guevel, Laetitia; Rouger, Karl

2015-01-01

Several adult stem cell populations exhibit myogenic regenerative potential, thus representing attractive candidates for therapeutic approaches of neuromuscular diseases such as Duchenne Muscular Dystrophy (DMD). We have recently shown that systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generates the remodelling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. This global effect, which is observed in the clinically relevant DMD animal model, leads us to question here the molecular pathways that are impacted by MuStem cell transplantation. To address this issue, we compare the global gene expression profile between healthy, GRMD and MuStem cell treated GRMD dog muscle, four months after allogenic MuStem cell transplantation. In the dystrophic context of the GRMD dog, disease-related deregulation is observed in the case of 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. Importantly, we reveal the impact of MuStem cell transplantation on several molecular and cellular pathways based on a selection of 31 genes displaying signals specifically modulated by the treatment. Concomitant with a diffuse dystrophin expression, a histological remodelling and a stabilization of GRMD dog clinical status, we show that cell delivery is associated with an up-regulation of genes reflecting a sustained enhancement of muscle regeneration. We also identify a decreased mRNA expression of a set of genes having metabolic functions associated with lipid homeostasis and energy. Interestingly, ubiquitin-mediated protein degradation is highly enhanced in GRMD dog muscle after systemic delivery of MuStem cells. Overall, our results provide the first high-throughput characterization of GRMD dog muscle and throw new light on the complex molecular

Differential Gene Expression Profiling of Dystrophic Dog Muscle after MuStem Cell Transplantation.

Directory of Open Access Journals (Sweden)

Florence Robriquet

Full Text Available Several adult stem cell populations exhibit myogenic regenerative potential, thus representing attractive candidates for therapeutic approaches of neuromuscular diseases such as Duchenne Muscular Dystrophy (DMD. We have recently shown that systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generates the remodelling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD dog. This global effect, which is observed in the clinically relevant DMD animal model, leads us to question here the molecular pathways that are impacted by MuStem cell transplantation. To address this issue, we compare the global gene expression profile between healthy, GRMD and MuStem cell treated GRMD dog muscle, four months after allogenic MuStem cell transplantation.In the dystrophic context of the GRMD dog, disease-related deregulation is observed in the case of 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. Importantly, we reveal the impact of MuStem cell transplantation on several molecular and cellular pathways based on a selection of 31 genes displaying signals specifically modulated by the treatment. Concomitant with a diffuse dystrophin expression, a histological remodelling and a stabilization of GRMD dog clinical status, we show that cell delivery is associated with an up-regulation of genes reflecting a sustained enhancement of muscle regeneration. We also identify a decreased mRNA expression of a set of genes having metabolic functions associated with lipid homeostasis and energy. Interestingly, ubiquitin-mediated protein degradation is highly enhanced in GRMD dog muscle after systemic delivery of MuStem cells.Overall, our results provide the first high-throughput characterization of GRMD dog muscle and throw new light on the complex

Airway smooth muscle cells : regulators of airway inflammation

NARCIS (Netherlands)

Zuyderduyn, Suzanne

2007-01-01

Airways from asthmatic subjects are more responsive to bronchoconstrictive stimuli than airways from healthy subjects. Airway smooth muscle (ASM) cells mediate contraction of the airways by responding to the bronchoconstrictive stimuli, which was thought to be the primary role of ASM cells. In this

Action of Obestatin in Skeletal Muscle Repair: Stem Cell Expansion, Muscle Growth, and Microenvironment Remodeling

Science.gov (United States)

Gurriarán-Rodríguez, Uxía; Santos-Zas, Icía; González-Sánchez, Jessica; Beiroa, Daniel; Moresi, Viviana; Mosteiro, Carlos S; Lin, Wei; Viñuela, Juan E; Señarís, José; García-Caballero, Tomás; Casanueva, Felipe F; Nogueiras, Rubén; Gallego, Rosalía; Renaud, Jean-Marc; Adamo, Sergio; Pazos, Yolanda; Camiña, Jesús P

2015-01-01

The development of therapeutic strategies for skeletal muscle diseases, such as physical injuries and myopathies, depends on the knowledge of regulatory signals that control the myogenic process. The obestatin/GPR39 system operates as an autocrine signal in the regulation of skeletal myogenesis. Using a mouse model of skeletal muscle regeneration after injury and several cellular strategies, we explored the potential use of obestatin as a therapeutic agent for the treatment of trauma-induced muscle injuries. Our results evidenced that the overexpression of the preproghrelin, and thus obestatin, and GPR39 in skeletal muscle increased regeneration after muscle injury. More importantly, the intramuscular injection of obestatin significantly enhanced muscle regeneration by simulating satellite stem cell expansion as well as myofiber hypertrophy through a kinase hierarchy. Added to the myogenic action, the obestatin administration resulted in an increased expression of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) and the consequent microvascularization, with no effect on collagen deposition in skeletal muscle. Furthermore, the potential inhibition of myostatin during obestatin treatment might contribute to its myogenic action improving muscle growth and regeneration. Overall, our data demonstrate successful improvement of muscle regeneration, indicating obestatin is a potential therapeutic agent for skeletal muscle injury and would benefit other myopathies related to muscle regeneration. PMID:25762009

Adropin Contributes to Anti-Atherosclerosis by Suppressing Monocyte-Endothelial Cell Adhesion and Smooth Muscle Cell Proliferation

Directory of Open Access Journals (Sweden)

Kengo Sato

2018-04-01

Full Text Available Adropin, a peptide hormone expressed in liver and brain, is known to improve insulin resistance and endothelial dysfunction. Serum levels of adropin are negatively associated with the severity of coronary artery disease. However, it remains unknown whether adropin could modulate atherogenesis. We assessed the effects of adropin on inflammatory molecule expression and human THP1 monocyte adhesion in human umbilical vein endothelial cells (HUVECs, foam cell formation in THP1 monocyte-derived macrophages, and the migration and proliferation of human aortic smooth muscle cells (HASMCs in vitro and atherogenesis in Apoe−/− mice in vivo. Adropin was expressed in THP1 monocytes, their derived macrophages, HASMCs, and HUVECs. Adropin suppressed tumor necrosis factor α-induced THP1 monocyte adhesion to HUVECs, which was associated with vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 downregulation in HUVECs. Adropin shifted the phenotype to anti-inflammatory M2 rather than pro-inflammatory M1 via peroxisome proliferator-activated receptor γ upregulation during monocyte differentiation into macrophages. Adropin had no significant effects on oxidized low-density lipoprotein-induced foam cell formation in macrophages. In HASMCs, adropin suppressed the migration and proliferation without inducing apoptosis via ERK1/2 and Bax downregulation and phosphoinositide 3-kinase/Akt/Bcl2 upregulation. Chronic administration of adropin to Apoe−/− mice attenuated the development of atherosclerotic lesions in the aorta, with reduced the intra-plaque monocyte/macrophage infiltration and smooth muscle cell content. Thus, adropin could serve as a novel therapeutic target in atherosclerosis and related diseases.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

KAUST Repository

Castagnetti, Francesco; Fiacco, Elisabetta; Imbriano, Carol; Latella, Lucia

2017-01-01

with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor

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

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.