scholarly journals 3D skeletal muscle fascicle engineering is improved with TGF-β1 treatment of myogenic cells and their co-culture with myofibroblasts

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4939 ◽  
Author(s):  
Jessica Krieger ◽  
Byung-Wook Park ◽  
Christopher R. Lambert ◽  
Christopher Malcuit
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Muscle Fiber ◽  
Muscle Regeneration ◽  
Muscle Cells ◽  
Culture Conditions ◽  
Myogenic Cells ◽  
Culture Model ◽  
Tgf Β1

Background Skeletal muscle wound healing is dependent on complex interactions between fibroblasts, myofibroblasts, myogenic cells, and cytokines, such as TGF-β1. This study sought to clarify the impact of TGF-β1 signaling on skeletal muscle cells and discern between the individual contributions of fibroblasts and myofibroblasts to myogenesis when in co-culture with myogenic cells. 3D tissue-engineered models were compared to equivalent 2D culture conditions to assess the efficacy of each culture model to predictively recapitulate the in vivo muscle environment. Methods TGF-β1 treatment and mono-/co-cultures containing human dermal fibroblasts or myofibroblasts and C2C12 mouse myoblasts were assessed in 2D and 3D environments. Three culture systems were compared: cell monolayers grown on 2D dishes and 3D tissues prepared via a self-assembly method or collagen 1-based hydrogel biofabrication. qPCR identified gene expression changes during fibroblast to myofibroblast and myoblast differentiation between culture conditions. Changes to cell phenotype and tissue morphology were characterized via immunostaining for myosin heavy chain, procollagen, and α-smooth muscle actin. Tissue elastic moduli were measured with parallel plate compression and atomic force microscopy systems, and a slack test was employed to quantify differences in tissue architecture and integrity. Results TGF-β1 treatment improved myogenesis in 3D mono- and co-cultures containing muscle cells, but not in 2D. The 3D TGF-β1-treated co-culture containing myoblasts and myofibroblasts expressed the highest levels of myogenin and collagen 1, demonstrating a greater capacity to drive myogenesis than fibroblasts or TGF-β1-treatment in monocultures containing only myoblasts. These constructs possessed the greatest tissue stability, integrity, and muscle fiber organization, as demonstrated by their rapid and sustained shortening velocity during slack tests, and the highest Young’s modulus of 6.55 kPA, approximate half the stiffness of in situ muscle. Both self-assembled and hydrogel-based tissues yielded the most multinucleated, elongated, and aligned muscle fiber histology. In contrast, the equivalent 2D co-culture model treated with TGF-β1 completely lacked myotube formation through suppression of myogenin gene expression. Discussion These results show skeletal muscle regeneration can be promoted by treating myogenic cells with TGF-β1, and myofibroblasts are superior enhancers of myogenesis than fibroblasts. Critically, both TGF-β1 treatment and co-culturing skeletal muscle cells with myofibroblasts can serve as myogenesis accelerators across multiple tissue engineering platforms. Equivalent 2D culture systems cannot replicate these affects, however, highlighting a need to continually improve in vitro models for skeletal muscle development, discovery of therapeutics for muscle regeneration, and research and development of in vitro meat products.

Studies of the dynamics of skeletal muscle regeneration: the mouse came back!

1998 ◽  
Vol 76 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Judy E Anderson
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Magnetic Resonance ◽  
Magnetic Resonance Spectroscopy ◽  
Muscle Fiber ◽  
Muscle Regeneration ◽  
Skeletal Muscle Regeneration ◽  
Resonance Spectroscopy ◽  
Dystrophic Mouse

Regeneration of skeletal muscle tissue includes sequential processes of muscle cell proliferation and commitment, cell fusion, muscle fiber differentiation, and communication between cells of various tissues of origin. Central to the process is the myosatellite cell, a quiescent precursor cell located between the mature muscle fiber and its sheath of external lamina. To form new fibers in a muscle damaged by disease or direct injury, satellite cells must be activated, proliferate, and subsequently fuse into an elongated multinucleated cell. Current investigations in the field concern modulation of the effectiveness of skeletal muscle regeneration, the regeneration-specific role of myogenic regulatory gene expression distinct from expression during development, the impact of growth and scatter factors and their respective receptors in amplifying precursor numbers, and promoting fusion and maturation of new fibers and the ultimate clinical therapeutic applications of such information to alleviate disease. One approach to muscle regeneration integrates observations of muscle gene expression, proliferation, myoblast fusion, and fiber growth in vivo with parallel studies of cell cycling behaviour, endocrine perturbation, and potential biochemical markers of steps in the disease-repair process detected by magnetic resonance spectroscopy techniques. Experiments on muscles from limb, diaphragm, and heart of the mdx dystrophic mouse, made to parallel clinical trials on human Duchenne muscular dystrophy, help to elucidate mechanisms underlying the positive treatment effects of the glucocorticoid drug deflazacort. This review illustrates an effective combination of in vivo and in vitro experiments to integrate the distinctive complexities of post-natal myogenesis in regeneration of skeletal muscle tissue.Key words: satellite cell, cell cycling, HGF/SF, c-met receptor, MyoD, myogenin, magnetic resonance spectroscopy, mdx dystrophic mouse, deflazacort.

Calf Blood Compound (CFC) and Homeopathic Drug Induce Differentiation of Primary Human Skeletal Muscle Cells

2019 ◽  
Vol 40 (12) ◽  
pp. 803-809 ◽  
Author(s):  
Eva K. Langendorf ◽  
Anja Klein ◽  
Pol M. Rommens ◽  
Philipp Drees ◽  
Ulrike Ritz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Cell Viability ◽  
Human Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
In Vivo Studies ◽  
Human Skeletal Muscle Cells ◽  
Regulated Gene Expression

AbstractThe use of injections to treat structural muscle injuries is controversially discussed. In our controlled in vitro study, we investigated the biological impact of Actovegin and Traumeel alone and in combination on primary human skeletal muscle cells. Cells were characterized by immunofluorescence staining for myogenic factor 5 (Myf5) and MyoD, and cultured with or without Actovegin and / or Traumeel. The effects of these agents were assayed by cell viability and gene expression of the specific markers MyoD, Myf5, neural adhesion molecule (NCAM), and CD31. Myotube formation was determined by myosin staining. Neither Actovegin nor Traumeel showed toxic effects or influenced cell viability significantly. High volumes of Actovegin down-regulated gene expression of NCAM after 3 days but had no effect on MyoD, Myf5, and CD31 gene expression. High volumes of Traumeel inhibited MyoD gene expression after 3 days, whereas after 7 days MyoD expression was significantly up-regulated. The combination of both agents did not significantly influence cell viability or gene expression. This is the first study demonstrating that Actovegin and Traumeel potentially modulate human skeletal muscle cells. The relevance of these in vitro findings has to be highlighted in further in vivo studies.

scholarly journals Modeling the transport of nuclear proteins along single skeletal muscle cells

2020 ◽  
Vol 117 (6) ◽  
pp. 2978-2986 ◽  
Author(s):  
Hermes Taylor-Weiner ◽  
Christopher L. Grigsby ◽  
Duarte M. S. Ferreira ◽  
José M. Dias ◽  
Molly M. Stevens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Molecular Weight ◽  
Transcription Factors ◽  
Protein Transport ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Nuclear Proteins ◽  
Transcriptional Responses

Skeletal muscle cells contain hundreds of myonuclei within a shared cytoplasm, presenting unique challenges for regulating gene expression. Certain transcriptional programs (e.g., postsynaptic machinery) are segregated to specialized domains, while others (e.g., contractile proteins) do not show spatial confinement. Furthermore, local stimuli, such as denervation, can induce transcriptional responses that are propagated along the muscle cells. Regulated transport of nuclear proteins (e.g., transcription factors) between myonuclei represents a potential mechanism for coordinating gene expression. However, the principles underlying the transport of nuclear proteins within multinucleated cells remain poorly defined. Here we used a mosaic transfection model to create myotubes that contained exactly one myonucleus expressing a fluorescent nuclear reporter and monitored its distribution among all myonuclei. We found that the transport properties of these model nuclear proteins in myotubes depended on molecular weight and nuclear import rate, as well as on myotube width. Interestingly, muscle hypertrophy increased the transport of high molecular weight nuclear proteins, while atrophy restricted the transport of smaller nuclear proteins. We have developed a mathematical model of nuclear protein transport within a myotube that recapitulates the results of our in vitro experiments. To test the relevance to nuclear proteins expressed in skeletal muscle, we studied the transport of two transcription factors—aryl hydrocarbon receptor nuclear translocator and sine oculis homeobox 1—and found that their distributions were similar to the reporter proteins with corresponding molecular weights. Together, these results define a set of variables that can be used to predict the spatial distributions of nuclear proteins within a myotube.

Retinoic Acid Receptor Agonists Suppress Muscle Fatty Infiltration in Mice

2021 ◽  
Vol 49 (2) ◽  
pp. 332-339
Author(s):  
Hideyuki Shirasawa ◽  
Noboru Matsumura ◽  
Masaki Yoda ◽  
Kazumasa Okubo ◽  
Masayuki Shimoda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Rotator Cuff ◽  
Rotator Cuff Tear ◽  
Gene Expression Analysis ◽  
Retinoic Acid Receptor ◽  
Adipogenic Differentiation ◽  
Fatty Infiltration ◽  
Cuff Tear

Background: The infiltration of fat tissue into skeletal muscle, a condition referred to as muscle fatty infiltration or fatty degeneration, is regarded as an irreversible event that significantly compromises the motor function of skeletal muscle. Purpose: To investigate the effect of retinoic acid receptor (RAR) agonists in suppressing the adipogenic differentiation of fibroadipogenic progenitors (FAPs) in vitro and fatty infiltration after rotator cuff tear in mice. Study Design: Controlled laboratory study. Methods: FAPs isolated from mouse skeletal muscle were cultured in adipogenic differentiation medium in the presence or absence of an RAR agonist. At the end of cell culture, adipogenic differentiation was evaluated by gene expression analysis and oil red O staining. A mouse model of fatty infiltration—which includes the resection of the rotator cuff, removal of the humeral head, and denervation the supraspinatus muscle—was used to induce fatty infiltration in the supraspinatus muscle. The mice were orally or intramuscularly administered with an RAR agonist after the surgery. Muscle fatty infiltration was evaluated by histology and gene expression analysis. Results: RAR agonists effectively inhibited the adipogenic differentiation of FAPs in vitro. Oral and intramuscular administration of RAR agonists suppressed the development of muscle fatty infiltration in the mice after rotator cuff tear. In accordance, we found a significant decrease in the number of intramuscular fat cells and suppressed expression in adipogenic markers. RAR agonists also increased the expression of the transcripts for collagens; however, an accumulation of collagenous tissues was not histologically evident in the present model. Conclusion: Muscle fatty infiltration can be alleviated by RAR agonists through suppressing the adipogenic differentiation of FAPs. The results also suggest that RAR agonists are potential therapeutic agents for treating patients who are at risk of developing muscle fatty infiltration. The consequence of the increased expression of collagen transcripts by RAR agonists needs to be clarified. Clinical Relevance: RAR agonists can be used to prevent the development of muscle fatty infiltration after rotator cuff tear. Nevertheless, further studies are mandatory in a large animal model to examine the safety and efficacy of intramuscular injection of RAR agonists.

scholarly journals Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kornphimol Kulthong ◽  
Guido J. E. J. Hooiveld ◽  
Loes Duivenvoorde ◽  
Ignacio Miro Estruch ◽  
Victor Marin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Culture Conditions ◽  
Gene Set Enrichment Analysis ◽  
Shear Stresses ◽  
Static Culture ◽  
Dynamic Culture ◽  
Intestinal Segments ◽  
On Chip

AbstractGut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

scholarly journals Impact of Adenosine A2 Receptor Ligands on BCL2 Expression in Skeletal Muscle Cells

2021 ◽  
Vol 11 (5) ◽  
pp. 2272
Author(s):  
Mansour Haddad
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Adenosine Receptors ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Qrt Pcr ◽  
A2a Receptor ◽  
Adenosine A2a ◽  
Bcl2 Expression ◽  
Bcl2 Gene

Background: Adenosine plays the role of regulating cell differentiation, proliferation, and apoptosis in various kinds of cells through the B-cell lymphoma 2 (BCL2) pathway. Objectives: Since anti-apoptotic (BCL2) expression plays a role in controlling apoptosis in some cell lines, this study was designed to investigate whether adenosine analogue, NECA (non-selective adenosine receptors agonist), selective adenosine A2B receptor antagonist, PSB 603, and a selective adenosine A2A receptor agonist, CG21680, affect BCL2-gene expression in the skeletal muscle cells of rats. The purpose of this investigation was to test the hypothesis that CG21680 treatment would significantly intensify BCL2 gene expression in rat skeletal muscle. Methods: Flasks measuring 25 cm2 were employed in culturing the rat L6 skeletal muscle cells. After treating these differential cells, the relative mRNA expression level for the BCL2 gene, at varying conditions of treatment, was measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: From the qRT-PCR analysis results, it was concluded that BCL2 expression was markedly amplified after selective adenosine A2A receptor agonist, CGS21680 (p < 0.01) treatment. More prospective validation for the adenosine receptors’ contribution in modulating apoptosis by NECA was delivered by the outcomes from the combined pre-treatment of the cells with NECA and PSB 603. These outcomes show that when starved skeletal muscle cells are treated with a combination of NECA and 100 nM PSB 603, there was a substantial decrease in comparison to either treatment used on its own. Conclusions: This study’s results showed, for the first time, an increase in BCL2 gene expression within skeletal muscle after CGS21680 treatment. Hence, the prospective escalation in BCL2 protein expression might have a protective role to play against apoptosis and avert damage to the skeletal muscle.

In Vitro Effects of Soy Phytoestrogens on Rat L6 Skeletal Muscle Cells

2005 ◽  
Vol 8 (3) ◽  
pp. 327-331 ◽  
Author(s):  
K.L. Jones ◽  
J. Harty ◽  
M.J. Roeder ◽  
T.A. Winters ◽  
W.J. Banz
Keyword(s):  
Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
In Vitro Effects ◽  
L6 Skeletal Muscle Cells

Characterization of β-adrenoceptors on rat skeletal muscle cells grown in vitro

1990 ◽  
Vol 40 (5) ◽  
pp. 1043-1048 ◽  
Author(s):  
Marie-Helene Disatnik ◽  
Sanford R. Sampson ◽  
Asher Shainberg
Keyword(s):  
Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Rat Skeletal Muscle
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