• 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.

• 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.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.219-224
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• 2013

Muscular satellite cell (SC), which is stem cell of postnatal pig, is an important for study of differentiation into adipogenesis, myogenesis, and osteoblastogenesis. In this study, we isolated and examined from pig muscle tissue to determine capacity in proliferate, differentiate, and expression of various genes. Porcine satellite cells (PSC) were isolated from semimembranosus (SM) muscles of 90~100 days old pigs according to standard conditions. The cell proliferation increased in multi-potent cell by Masson's, oil red O, and Alizarin red staining respectively. We performed the expression levels of differentiation related genes using real-time PCR. We found that the differentiation into adipocyte increased expression levels of both fatty acid binding protein 4 (FABP4) and peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) genes (p<0.01). Myocyte increased the expression levels of the myosin heavy chain (MHC), myogenic factor 5 (Myf5), myogenic regulatory factor (MyoD), and Myogenic factor 4 (myogenin) (p<0.01). Osteoblast increased the expression levels of alkaline phosphatase (ALP) (p<0.01). Finally, porcine satellite cells were induced to differentiate towards adipogenic, myogenic, and osteoblastogenic lineages. Our results suggest that muscle satellite cell in porcine may influence cell fate. Understanding the progression of PSC may lead to improved strategies for augmenting meat quality.

• Journal of Animal Science and Technology
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• v.64 no.6
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• pp.1132-1143
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• 2022

Insects are a valuable natural source that can produce a variety of bioactive compounds due to their increasing species diversity. CopA3 is an antimicrobial peptide derived from Copris tripartitus (i.e., the dung beetle). It is known to increase the proliferation of colonic epithelial and neuronal stem cells by regulating cell cycle. This research hypothesized that CopA3 can promote the proliferation of porcine muscle satellite cells (MSCs). The effects of CopA3 on porcine MSCs, which are important for muscle growth and regeneration, remain unclear. Here, we investigated the effects of CopA3 on porcine MSCs. According to viability results, we designed four groups: control (without CopA3) and three treatment groups (treated with 5,10, and 25 ㎍/mL of CopA3). At a CopA3 concentration of 5 ㎍/mL and 10 ㎍/mL, the proliferation of MSCs increased more than that observed in the control group. Furthermore, compared to that in the control, CopA3 treatment increased the S phase but decreased the G0/G1 phase ratio. Additionally, early and late apoptotic cells were found to be decreased in the 5 ㎍/mL group. The expressions of the myogenesis-related transcription factor PAX7 and MYOD proteins were significantly upregulated in the 5 ㎍/mL and 10 ㎍/mL groups, whereas the MYOG protein remained undetected in all group. This study suggested that CopA3 promotes muscle cell proliferation by regulating the cell cycle of MSCs and can regulate the activity of MSCs by increasing the expressions of PAX7 and MYOD.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.225-232
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• 2013

Satellite cells were derived from muscular tissue in postnatal pig. Satellite cell is an important to growth and development in animal tissues or organs. However, the progress underlying induced differentiation is not clear. The aim of this study was to evaluate the morphologic and the transcriptome changes in porcine satellite cell (PSC) treated with insulin, rosiglitazone, or dexamethasone respectively. PSC was obtained from postnatal muscle tissue. In study 1, for study the effect of insulin and FBS on the differentiated satellite cells, cells were cultured at absence or presence of insulin treated with FBS. Total RNA was extracted for determining the expression levels of myogenic PAX3, PAX7, Myf5, MyoD, and myogenin genes by real-time PCR. Myogenic genes decreased expression levels of mRNA in treated with insulin. In study 2, in order to clarify the relationship between rosiglitazone and lipid in differentiated satellite cells, we further examined the effect of FBS on lipid accumulation in the presence or absence of the rosiglitazone and lipid. Significant differences were observed between rosiglitazone and lipid by FBS. The mRNA of FABP4 and $PPAR{\gamma}$ increased in rosiglitazone treatment. In study 3, we examined the effect of dexamethasone on osteogenic differentiation in PSC. The mRNA was increased osteoblasotgenic ALP and ON genes treated with dexamethasone in 2% FBS. Dexamethasone induces osteoblastogenesis in differentiated PSC. Taken together, in differentiated PSCs, FABP4 and $PPAR{\gamma}$ increased to rosiglitazone. Whereas, no differences to FBS and lipid. These results were not comparable with previous reports. Our results suggest that adipogenic, myogenic, and osteoblastogenic could be isolated from porcine skeletal muscle, and identify culture conditions which optimize proliferation and differentiation formation of PSC.

• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.233-245
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• 2013

Muscle satellite cell (SC) is responsible for postnatal muscle growth, repair, and regeneration. Satellite cell is an important source of multi-potent stem cell process and differentiation into adipogenic, myogenic, and osteoblastogenic. The objective of this study was to identify alter of transcriptome during differentiation in porcine satellite cell and to elevated transcriptome at different stages of postnatal development to gain insight into the differences in differentiated PSC. We used RNA-seq technique to investigate the transcriptomes during differentiation in pig muscle. Sequence reads were obtained from Illumina HiSeq2000. Differentially expressed genes (DEG) were detected by EdgeR. Gene ontology (GO) terms are powerful tool for unification among representation genes or products. In study of GO biological terms, functional annotation clustering involved in cell cycle, apoptosis, extracellular matrix, phosphorylation, proteolysis, and cell signaling in differences stage. Taken together, these results would be contributed to a better understanding of muscle biology and processes underlying differentiation. Our results suggest that the source of DEGs could be better understanding of the mechanism of muscle differentiation and transdifferentiation.

• Journal of Embryo Transfer
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• v.27 no.2
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• pp.113-119
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• 2012

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.11
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• pp.1620-1632
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• 2017

Objective: This study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula's extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes. Methods: In a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction. Results: The final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber Ι, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type Ι fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC Ι, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC Ι and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor ${\gamma}$ $coactivator-1{\alpha}$ and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by $4{\mu}g/mL$ and $20{\mu}g/mL$ TCMF water extraction (p<0.05). Both $1{\mu}g/mL$ and $5{\mu}g/mL$ of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC Ι mRNA expression in porcine myocytes was decreased by $5{\mu}g/mL$ TCMF water extraction (p<0.05). Porcine myocyte MyHC Ι and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by $5{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). MyHC Ι and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by $1{\mu}g/mL$ TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by $5{\mu}g/mL$ TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by $1{\mu}g/mL$ in a TCMF petroleum ether extraction (p<0.05). Conclusion: These results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes.

• Journal of Animal Science and Technology
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• v.49 no.1
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• pp.25-32
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• 2007

The current study was undertaken to determine the effect of conjugated linoleic acid(CLA) isomers, cis-9, cis-11(c9c11), cis-9, trans-11(c9t11), trans-9, trans-11(t9t11), trans-10, cis-12(t10c12) on differentiation of pig preadipocytes and myogenic satellite cells during culture. Cells were isolated from new born pigs. The t10c12 isomer decreased differentiation of pig preadipocytes(92%), but not that of myogenic cells. The t9t11 isomer decreased differentiation of preadipocytes(14%) and increased that of myogenic cells (26%). No other CLA isomers affected differentiation of preadipocytes or myogenic cells. The effects of CLA on proliferation of preadipocytes and myogenic cells were small, compared to the effects on differentiation. These results suggest that CLA isomers have different effects on differentiaton of pig preadipocytes and myogenic cells.

• Food Science of Animal Resources
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• v.30 no.5
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• pp.842-850
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• 2010

Sex steroids are known to be involved in skeletal muscle development (anabolic effect) and are frequently used in medicines. It has been known that pork contains a variety of steroids that are mainly synthesized in the gonads (testis and ovary). Thus, the present study was conducted to evaluate the effects of anabolic steroids of pork on the proliferation and differentiation of myogenic satellite cells (MSC). Three different methods (M1, M2, and M3) were developed for the isolation and purification of steroids from porcine tissues. Among three extraction methods that we developed, M3 was the best method with respect to the quantities of steroids and the induction of MSC proliferation. Hormonal analysis showed that the steroid hormone levels were the highest in muscle and fat of intact male than those of castrated males and females. In addition, the highest serum levels of nandrolone and testosterone were detected in intact males, whereas estrone and $17{\beta}$-estradiol levels were similar in the entire experimental serum samples. Expression of androgen receptor (AR), myoD, desmin, and myogenin in bovine muscle cells were significantly up-regulated by the treatment of steroid extracts. The highest increas of myogenin and AR mRNA abundance were observed in the MSCs treated with M3 extract (p<0.001). Altogether, the present research showed the positive effect of steroids on MSC proliferation and differentiation in vitro. These results would certainly imply a beneficial effect of pork consumption on human muscle development.