Formation of skeletal muscle in vivo from the mouse C2 cell line

1992 ◽  
Vol 102 (4) ◽  
pp. 779-787 ◽  
Author(s):  
J.E. Morgan ◽  
S.E. Moore ◽  
F.S. Walsh ◽  
T.A. Partridge
Keyword(s):  
Skeletal Muscle ◽  
Cell Line ◽  
Muscle Cell ◽  
Muscle Fibres ◽  
Neural Cell Adhesion ◽  
Anterior Tibial ◽  
Undifferentiated Cells ◽  
Vicryl Suture

The C2 muscle cell line is myogenic in vitro and has been extensively used in studies of muscle cell differentiation. Here, we have investigated the myogenicity in vivo of C2 cells implanted into suitable sites in the mouse. Large amounts of new muscle were formed when C2 cells were implanted into sites in nude mice which were undergoing regeneration following whole muscle grafting and in scaffolding of freeze-killed muscle or vicryl suture in the anterior tibial compartment. When implanted into regenerating muscle, C2 cells fused with the host muscle to form mosaic fibres; when implanted into inert sites, they formed muscle of largely donor origin. C2-derived muscle fibres appeared to become innervated, but the progression of N-CAM (neural cell adhesion molecule) isoform changes in such regenerates indicated that they did not become fully mature. Proliferating, undifferentiated cells of C2 origin form tumours in older grafts; however, this was more pronounced in the absence of competition from host muscle cells. In the short term, C2 cells can form large amounts of muscle in vivo for biochemical analysis. In addition, C2 cells are easily manipulable in vitro; genes of interest may be transfected into them prior to implantation of the cells into skeletal muscle and the effects of these genes in vivo may thus be examined.

An In Vitro and In Vivo Evidence That Downregulation of Leukemia Inhibitory Factor (LIF) Receptor (LIF-R) Decreases the Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2561-2561
Author(s):  
Marcin Wysoczynski ◽  
Katarzyna Miekus ◽  
Anna Marcinkowska ◽  
Anna Janowska-Wieczorek ◽  
Mariusz Z. Ratajczak
Keyword(s):  
Skeletal Muscle ◽  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Leukemia Inhibitory Factor ◽  
Biological Significance ◽  
Human Cells ◽  
Inhibitory Factor

Abstract Rhabdomyosarcoma (RMS) and skeletal muscle-derived tumors frequently infiltrate bone marrow (BM). We have demonstrated that the stromal-derived factor (SDF)-1-CXCR4 receptor (Blood2002;100:2597) and hepatocyte growth factor (HGF)-c-Met receptor (Cancer Res. 2003;63:7926) play an important role in RMS metastasis to BM. Leukemia inhibitory factor (LIF) is a well known factor that plays an important role in skeletal muscle development/regeneration and similarly as SDF-1 and HGF is secreted by BM stroma. This prompted us to examine whether the LIF-LIF receptor (LIF-R) axis affects the biology/metastasis of RMS cells. We employed in our studies, human established RMS cell lines, as well as RMS samples isolated from patients and noticed that LIF-R was expressed not only on established human RMS cell lines (7/7) but more importantly, it was also detectable in patient samples (23/23). We also found that in RMS cells LIF stimulatesphosphorylation of MAPKp42/44, AKT and STAT3,chemotaxis and adhesion andincreases resistance to cytostatics (e.g., etoposide). These LIF-mediated effects were inhibited after downregulating the LIF-R by siRNA. To learn more on the biological significance of the LIF-LIF-R axis in vivo we employed two models. First, human RMS cells (RH-30) were exposed or not exposed to LIF-R siRNA and subsequently injected into SCID™-Beige immunodeficient mice. To estimate the number of RMS cells that seed to BM and liver in these animals, we isolated DNA and using real- time RT-PCR, amplified human a-satellite sequences and murine b-actin. The number of human cells present in murine organs was subsequently calculated from a standard curve derived from mixing varying numbers of human cells with a constant number of murine cells. We noticed that downregulation of LIF-R by siRNA significantly decreased the number of human RMS cells in murine BM and liver (x4 and x2 respectively). In a second model, the RH30 cell line was selected by repetitive chemotaxis for cells that are highly responsive to LIF (RH-30 L) and subsequently the cells from parental RH-30 cell line and RH-30 L cells were injected intramuscularly. Six weeks after tumour inoculation, we detected more metastasis in bone marrow and lungs in mice injected with RH-30L cells as compared to parental RH-30 clone (x6 and x3 respectively). In conclusion, we present evidence for the first time that the inhibition of LIF-LIF-R axis may decrease the invasive potential of human RMS both in vitro and in vivo. Hence, molecular targeting of LIF-LIF-R axis could possibly become a more effective new strategy to control the progression and metastasis of RMS.

scholarly journals Prion Infection of Muscle Cells In Vitro

2007 ◽  
Vol 81 (9) ◽  
pp. 4615-4624 ◽  
Author(s):  
Wendy M. Dlakic ◽  
Eric Grigg ◽  
Richard A. Bessen
Keyword(s):  
Skeletal Muscle ◽  
Cell Line ◽  
Muscle Cells ◽  
C2c12 Cells ◽  
C2c12 Myotubes ◽  
C2c12 Myoblasts ◽  
Peripheral Cell ◽  
Prion Infection

ABSTRACT The prion agent has been detected in skeletal muscle of humans and animals with prion diseases. Here we report scrapie infection of murine C2C12 myoblasts and myotubes in vitro following coculture with a scrapie-infected murine neuroblastoma (N2A) cell line but not following incubation with a scrapie-infected nonneuronal cell line or a scrapie brain homogenate. Terminal differentiation of scrapie-infected C2C12 myoblasts into myotubes resulted in an increase in the expression of the disease-specific prion protein, PrPSc. The amount of scrapie infectivity or PrPSc in C2C12 myotubes was comparable to the levels found in scrapie-infected N2A cells, indicating that a high level of infection was established in muscle cells. Subclones of scrapie-infected C2C12 cells produced high levels of PrPSc in myotubes, and the C-terminal C2 polypeptide fragment of PrPSc was found based on deglycosylation and PrPSc-specific immunoprecipitation of cell lysates. This is the first report of a stable prion infection in muscle cells in vitro and of a long-term prion infection in a nondividing, differentiated peripheral cell type in culture. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals.

scholarly journals ID: 1022 Acellular muscle scaffolds: Histological and biochemical evaluation

2017 ◽  
Vol 4 (S) ◽  
pp. 97
Author(s):  
Jaroslav Mokry ◽  
Hana Hrebíková ◽  
Jana Chvátalová ◽  
Rishikaysh Pisal ◽  
Stanislav Filip ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Chemical Components ◽  
Hypotonic Solution ◽  
Myogenic Cells ◽  
Promising Alternative ◽  
Anterior Tibial Muscle ◽  
Extracellular Matrix Components ◽  
Anterior Tibial

Anterior tibial muscle of C57Bl6/J mice was subjected to decellularization with hypotonic solution, detergents and DNase. Resulting acellular scaffolds were examined to characterize the content of chromatin, cell cytoplasm and extracellular matrix components incl. basal laminas, fibres and glycoproteins. Although the sarcoplasm and cell nuclei were removed, the general skeletal muscle microarchitecture with ECM of stromal components remained well preserved at light and electron microscopic levels. Moreover, basal laminas contouring honeycomb-like structures left after removal of myofibres and vascular endothelium remained intact. Immunostaining of scaffolds for collagen IV and laminin confirmed positivity of basal laminas. Histochemical staining of deparaffinised scaffold sections identified well organized fibres after staining with green trichrome, Sirius red, Weigert’s resorcin fuchsin and Gomori impregnation. Chemical analysis gave evidence of reduced dsDNA and well-preserved collagen according to high hydroxyproline content and laminin as documented by Western blotting. We cultured scaffolds seeded with murine myogenic cells in vitro and confirmed their cytocompatibility as the cells were able to adhere, grow and migrate through the ECM without affecting the scaffold structure. Myogenic cells were able to migrate in the endomysium and start to fuse. Implantation of decellularized scaffolds into an artificial cavity inside of anterior tibial muscle of mice in vivo confirmed the scaffolds were colonized soon by recipient inflammatory cells without formation of foreign body giant cells. Scaffolds were well integrated with recipient skeletal muscle and gradually resorbed within 3 weeks. Our results confirm decellularized muscle scaffold is a promising alternative for rebuilding the skeletal muscle organ as it can preserve basic chemical components and the tissue microstructure and show biocompatibility for myogenic cells as demonstrated in vitro and in vivo.

scholarly journals Neuregulin 1 Drives Morphological and Phenotypical Changes in C2C12 Myotubes: Towards De Novo Formation of Intrafusal Fibres In Vitro

2022 ◽  
Vol 9 ◽  
Author(s):  
Philip Barrett ◽  
Tom J. Quick ◽  
Vivek Mudera ◽  
Darren J. Player
Keyword(s):  
Skeletal Muscle ◽  
Muscle Spindle ◽  
De Novo ◽  
C2c12 Myotubes ◽  
Muscle Fibres ◽  
Neuregulin 1 ◽  
Gene And Protein Expression ◽  
Intrafusal Fibres

Muscle spindles are sensory organs that detect and mediate both static and dynamic muscle stretch and monitor muscle position, through a specialised cell population, termed intrafusal fibres. It is these fibres that provide a key contribution to proprioception and muscle spindle dysfunction is associated with multiple neuromuscular diseases, aging and nerve injuries. To date, there are few publications focussed on de novo generation and characterisation of intrafusal muscle fibres in vitro. To this end, current models of skeletal muscle focus on extrafusal fibres and lack an appreciation for the afferent functions of the muscle spindle. The goal of this study was to produce and define intrafusal bag and chain myotubes from differentiated C2C12 myoblasts, utilising the addition of the developmentally associated protein, Neuregulin 1 (Nrg-1). Intrafusal bag myotubes have a fusiform shape and were assigned using statistical morphological parameters. The model was further validated using immunofluorescent microscopy and western blot analysis, directed against an extensive list of putative intrafusal specific markers, as identified in vivo. The addition of Nrg-1 treatment resulted in a 5-fold increase in intrafusal bag myotubes (as assessed by morphology) and increased protein and gene expression of the intrafusal specific transcription factor, Egr3. Surprisingly, Nrg-1 treated myotubes had significantly reduced gene and protein expression of many intrafusal specific markers and showed no specificity towards intrafusal bag morphology. Another novel finding highlights a proliferative effect for Nrg-1 during the serum starvation-initiated differentiation phase, leading to increased nuclei counts, paired with less myotube area per myonuclei. Therefore, despite no clear collective evidence for specific intrafusal development, Nrg-1 treated myotubes share two inherent characteristics of intrafusal fibres, which contain increased satellite cell numbers and smaller myonuclear domains compared with their extrafusal neighbours. This research represents a minimalistic, monocellular C2C12 model for progression towards de novo intrafusal skeletal muscle generation, with the most extensive characterisation to date. Integration of intrafusal myotubes, characteristic of native, in vivo intrafusal skeletal muscle into future biomimetic tissue engineered models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications.

scholarly journals Bioengineered Fascicle-Like Skeletal Muscle Tissue Constructs

2012 ◽  
Author(s):  
Devin Neal ◽  
Mahmut Selman Sakar ◽  
H. Harry Asada
Keyword(s):  
Skeletal Muscle ◽  
Cell Density ◽  
Muscle Cell ◽  
Muscle Tissue ◽  
Skeletal Muscle Tissue ◽  
Muscle Injuries ◽  
Tissue Constructs

Tissue engineered skeletal muscle constructs have and will continue to be valuable in treating, and testing various muscle injuries and diseases. However a significant drawback to numerous methods of producing 3D skeletal muscle constructs grown in vitro is that muscle cell density as a fraction of total volume or mass, is often significantly lower than muscle found in vivo. Therefore a method to increase muscle cell density within a construct is needed.

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