• Animal Bioscience
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• v.36 no.12
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• pp.1775-1784
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• 2023

Objective: The aim of this study was to reveal the role and regulatory mechanism of miR-188-5p in the proliferation and differentiation of goat muscle satellite cells. Methods: Goat skeletal muscle satellite cells isolated in the pre-laboratory were used as the test material. First, the expression of miR-188-5p in goat muscle tissues at different developmental stages was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In addition, miR-188-5p was transfected into goat skeletal muscle satellite cells by constructing mimics and inhibitors of miR-188-5p, respectively. The changes of differentiation marker gene expression were detected by qPCR method. Results: It was highly expressed in adult goat latissimus dorsi and leg muscles, goat fetal skeletal muscle, and at the differentiation stage of muscle satellite cells. Overexpression and interference of miR-188-5p showed that miR-188-5p inhibited the proliferation and promoted the differentiation of goat muscle satellite cells. Target gene prediction and dual luciferase assays showed that miR-188-5p could target the 3'untranslated region of the calcium/calmodulin dependent protein kinase II beta (CAMK2B) gene and inhibit luciferase activity. Further functional studies revealed that CAMK2B promoted the proliferation and inhibited the differentiation of goat muscle satellite cells, whereas si-CAMK2B restored the function of miR-188-5p inhibitor. Conclusion: These results suggest that miR-188-5p inhibits the proliferation and promotes the differentiation of goat muscle satellite cells by targeting CAMK2B. This study will provide a theoretical reference for future studies on the molecular mechanisms of skeletal muscle development in goats.

• Animal Bioscience
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• v.36 no.12
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• pp.1889-1897
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• 2023

Objective: 'Cultured meat' has been suggested as means of solving the problems associated with overpopulation and gas emissions. Satellite cells are a major component in the production of cultured meat; however, these cells cannot be maintained in vitro over long periods. Fibronectin is a glycoprotein that affects biological processes such as cell adhesion, differentiation, and migration. Unfortunately, the characteristics of porcine satellite cells grown in a long-term culture when exposed to fibronectin-coated dishes are unknown. The objective of this study was to investigate the appropriate concentration of fibronectin coated dishes for proliferation and maintenance of porcine satellite cells at long-term culture. Methods: In this study, we isolated the satellite cells and fibroblast cells with pre-plating method. We next analyzed the cell doubling time, cell cycle, and rate of expressed paired box 7 (Pax7) and myogenic differentiation 1 (MyoD1) in porcine satellite cells cultured with 20 ㎍/mL of fibronectin-, gelatin-, and non-coated dishes at early and late passage. We then analyzed the proliferation of porcine satellite cells with various concentrations of mixed gelatin/fibronectin. We next determined the optimal concentration of fibronectin that would encourage proliferation and maintenance of porcine satellite cells in a long-term culture. Results: Doubling time was lowest when 20 ㎍/mL of fibronectin was used (as tested during an early and late passage). Levels of expressed Pax7 and MyoD1, assessed using immunocytochemistry, were highest in cells grown using fibronectin-coated dishes. The proliferation of gelatin/fibronectin mixed coatings had no significant effect on porcine satellite cells. The concentration of 5 ㎍/mL fibronectin coated dishes showed the lowest doubling time and maintained expression of Pax7. Conclusion: Fibronectin with 5㎍/mL effectively maintains porcine satellite cells, a discovery that will be of interest to those developing the next generation of artificial meats.

• Journal of Animal Science and Technology
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• v.63 no.4
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• pp.673-680
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• 2021

The purpose of this study was to establish a basic principal procedure for the processing of cultured meat. The first stage involved isolating satellite cells from the desired muscle of an animal using enzymatic digestion (i.e., by using proteases, collagenases, and pronases). The second stage involved culturing the isolated muscle satellite cells in a growth medium containing fetal bovine serum and penicillin/streptomycin with growth factors for an optimal period of time. The second stage involved a basic method for the isolated muscle cells to proliferate while sub-culturing to further induce differentiation in gelatin-coated culture dishes with the general culture medium. The third stage involved the induction of differentiation of muscle satellite cells or formation of myotubes using myogenic medium. Lastly, the fourth stage involved the identification of cell differentiation or myotube formation (myogenesis) using fluorescent dyes. Moreover, the principle of these protocols can be applied to perform primary culture of animal cells. This study will assist beginners with the technical aspects of culturing meat (isolation, cultivation, and differentiation of muscle satellite cells as well as identification of myotube formation for myogenesis).

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.8
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• pp.1171-1177
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• 2015

The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse) have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells.

• Journal of Animal Science and Technology
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• v.52 no.4
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• pp.329-336
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• 2010

Myogenic satellite cells (MSCs) are mononuclear, multipotent progenitors of adult skeletal muscle possessing a capacity of forming adipocyte-like cells (ALC). To identify the skeletal muscle type-specific myogenic and adipogenic genes during MSCs differentiation, total RNA was extracted from bovine MSCs, myotube-formed cell (MFC), and ALC from each of Beef shank, Longissimus dorsi, Deep pectoral, and Semitendinosus. DNA microarray analysis (24,000 oligo chip) comparing MSCs with MFC and ALC, respectively, revealed 135 differentially expressed genes (> 4 fold) among four cuts. Real-time PCR confirmed expression of 29 genes. Furthermore, the whole tissue sample RNAs analysis showed 6 differentially expressed genes in Beef shank. Among which, 1 gene in MSCs, 4 in MFC, and 1 in ALCs were highly expressed. This study will provide an insight for better understanding the molecular mechanism of differentiation of skeletal muscle type-specific MSCs. The identified genes may be used as marker to distinguish skeletal muscle types.

• Animal Bioscience
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• v.36 no.2
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• pp.295-306
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• 2023

Objective: Inhibiting the p38 mitogen-activated protein kinase (MAPK) signaling pathway delays differentiation and increases proliferation of muscle stem cells in most species. Here, we aimed to investigate the effect of p38 inhibitor (p38i) treatment on the proliferation and differentiation of chicken muscle stem cells. Methods: Chicken muscle stem cells were collected from the muscle tissues of Hy-line Brown chicken embryos at embryonic day 18, then isolated by the preplating method. Cells were cultured for 4 days in growth medium supplemented with dimethyl sulfoxide or 1, 10, 20 μM of p38i, then subcultured for up to 4 passages. Differentiation was induced for 3 days with differentiation medium. Each treatment was replicated 3 times. Results: The proliferation and mRNA expression of paired box 7 gene and myogenic factor 5 gene, as well as the mRNA expression of myogenic differentiation marker gene myogenin were significantly higher in p38i-treated cultures than in control (p<0.05), but immunofluorescence staining and mRNA expression of myosin heavy chain (MHC) were not significantly different between the two groups. Oil red O staining of accumulated lipid droplets in differentiated cell cultures revealed a higher lipid density in p38i-treated cultures than in control; however, the expression of the adipogenic marker gene peroxisome proliferator activated receptor gamma was not significantly different between the two groups. Conclusion: p38 inhibition in chicken muscle stem cells improves cell proliferation, but the effects on myogenic differentiation and lipid accumulation require additional analysis. Further studies are needed on the chicken p38-MAPK pathway to understand the muscle and fat development mechanism.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.5
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• pp.685-695
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• 2011

Satellite cells are skeletal muscle progenitor/stem cells that reside between the basal lamina and plasma membranes of skeletal fibers in vivo. These cells can give rise to both myogenic and adipogenic cells. Given the possible role for differentiation of satellite cells into adipocytes in marbling and in some pathological disorders like sarcopenia, knowledge of the proteins involved in such process remains obscure. Using two-dimensional polyacrylamide gel electrophoresis coupled with mass spectrometry, we investigated the proteins that are differentially expressed during adipogenic differentiation of satellite cells from bovine longissimus muscle. Our proteome mapping strategy to identify the differentially expressed intracellular proteins during adipogenic differentiation revealed a total of 25 different proteins. The proteins up-regulated during adipogenic differentiation of satellite cells like Cathepsin H precursor, Retinal dehydrogenase 1, Enoyl-CoA hydratase, Ubiquinol-cytochrome-c reductase, T-complex protein 1 subunit beta and ATP synthase D chain were found to be associated with lipid metabolism. The down-regulated proteins like LIM protein, annexin proteins, cofilin-1, Rho GDP-dissociation inhibitor 1 and septin-2, identified in the present study were found to be associated with myogenesis. These results clearly demonstrate that the adipogenic conversion of muscle satellite cells is associated with the up-regulated and down-regulated proteins involved in adipogenesis and myogenesis respectively.

• Journal of Oral Medicine and Pain
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• v.26 no.1
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• pp.39-50
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• 2001

In order to investigate the effect of Transforming growth factor ${\beta}1$(below TGF-${\beta}1$) and osteogenic protein-1(below Op-1) onto the myogenic differentiation, C2C12 satellite myoblastic cell line was cultured and treated with both growth factors. At first morphological changes with microscopical examination were examined, and isolated total RNA to analyse mRNA expression of bone marker proteins, muscle regulatory proteins, TGF-${\beta}$ receptor and their ligands by Northern blot analysis. And cellular proliferative inducibility of both growth factors was also tested to C2C12 cells. Incubating the cell with $5ng/m{\ell}$ of TGF-${\beta}1$ until 4 days almost inhibited multinucleated myotube formation expressing muscular regulatory proteins, and induced decreasing Id proteins. However, no osteoblastic phenotypes was induced by TGF-${\beta}1$ in C2C12 cells. The mRNA expression of TGF-${\beta}$ receptors with TGF-${\beta}1$ was conversed after 48 hours cultured. Type I TGF-${\beta}$ receptor was seemed to play a role in negative signalling for inhibition of myogenic differentiation. OP-1 dose dependently induced ALP activity, osteopontine production and bone sialoprotein production at concentrations above $100ng/m{\ell}$ and osteocalcin production at concentrations above $300ng/m{\ell}$. The concentration of OP-1 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubation with above $100ng/m{\ell}$ OP-1 suppressed the expression of mRNA for muscular egulatory proteins from 2 days after incubation. Expression of Id-1, 2, 3 mRNA were stimulated by OP-1 at concentration above $300ng/m{\ell}$. When C2C12 cells were treated with both growth factors, TGF-${\beta}1$ potentiated the inhibitory effect of OP-1 on myotube formation and expression of mRNA for myogenin at 12 days. And TGF-${\beta}1$ reduced osteocalcin and bone sialoprotein production induced by OP-1 at 12 days in C2C12 cells. Both growth factor had no mitogenic effect. These results indicate that OP-1 converts the differentiation pathway of C2C12 myoblasts into that of osteoblastic lineage cells and it's not heritable, but TGF-${\beta}1$ does not and has reversible inhibitory activity on the myogenic differentiation. TGF-${\beta}1$ and OP-1 play a role in myogenic differentiation via different mechanism between them.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.4
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• pp.501-510
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• 2007

The present study was conducted to establish primary bovine muscle satellite cell (MSC) culture conditions and to investigate the effects of various steroid hormones on transcription of the genes involved in muscle cell proliferation and differentiation. Of three different types of proteases (type II collagenase, pronase and trypsin-EDTA) used to hydrolyze the myogenic satellite cells from muscle tissues, trypsin-EDTA treatment yielded the highest number of cells. The cells separated by hydrolysis with type II collagenase and incubated on gelatin-coated plates showed an enhanced cell attachment onto the culture plate and cell proliferation at an initial stage of cell growth. In this study, the bovine MSCs were maintained in vitro up to passage 16 without revealing any significant morphological change, and even to when the cells died at passage 21 with decreased or almost no cell growth or deformities. When the cells were incubated in a steroid-depleted environment (DMEM(-)/10% CDFBS (charcoal-dextran stripped FBS)), they grew slowly initially, and were widened and deformed. In addition, when the cells were transferred to an incubation medium containing steroid (DMEM(+)/10% FBS), the deformed cells resumed their growth and returned to a normal morphology, suggesting that steroid hormones are crucial in maintaining normal MSC morphology and growth. The results demonstrated that treatments with 19-nortestosterone and testosterone significantly increased AR gene expression (p<0.05), implying that both testosterone and 19-nortestosterone bind with AR and that the hormone bound-AR complex up-regulates the genes of its own receptor (AR) plus other genes involved in satellite cell growth and differentiation in bovine muscle.

• Journal of Biomedical Engineering Research
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• v.31 no.1
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• pp.81-86
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• 2010

Laser irradiation is known to affect various tissues such as skin, bone, nerve, and skeletal muscle. Laser irradiation promotes ATP synthesis, facilitates wound healing, and stimulates cell proliferation and angiogenesis. In skeletal muscle, laser irradiation is related to the proliferation of skeletal muscle satellite cells. Normal skeletal muscle contains remodeling capacity from myogenic cells that are derived from mononuclear satellite cells. Their processes are activated by the expression of genes related with myogenesis such as muscle-specific transcription factors (MyoD and Myf5) and VEGF (vascular endothelial growth factor). In this study, we hypothesized that laser irradiation would enhance and regulate muscle cell proliferation and regeneration through modulation of the gene expressions related with the differentiation of skeletal muscle satellite cells. $C_2C_{12}$ myoblastic cells were exposed to continuous/non-continuous laser irradiation (660nm/808nm) for 10 minutes daily for either 1 day or 5 days. After laser irradiation, cell proliferation and gene expression (MyoD, Myf5, VEGF) were quantified. Continuous 660nm laser irradiation significantly increased cell proliferation and gene expression compared to control, continuous 808nm laser irradiation, and non-continuous 660nm laser irradiation groups. These results indicate that continuous 660nm laser irradiation can be applied to the treatment and regeneration of skeletal muscle tissue.