• Asian-Australasian Journal of Animal Sciences
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• 제20권4호
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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 Animal Science and Technology
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• 제63권4호
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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).

• 현장농수산연구지
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• 제23권1호
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• pp.117-128
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• 2021

한우의 성장단계별 부위 근육발달을 이해하는 것은 도체율 개선에 따른 소득증대와 증체율 증가에 따른 생산효율 향상에 긍정적인 영향을 미친다. 본 연구에서는 한우의 등심과 사태 유래 근육위성세포를 분리 후 세포단위의 발달 및 분화를 비교하여 transcriptome 단위의 작용기전을 제시하였다. 한우의 부위별 근육 유래 근육위성세포의 근섬유의 양은 4일에 가장 높게 나타났고 이후 감소하였다. 한우의 근육위성세포의 발달 단계에 따라 발현되는 총 전사체 유전자의 종류는 사태근육 위성세포에서 높게 나타났다. 등심과 사태 근육 유래 위성세포의 발달단계에 따라 유의적인 차등 유전자 453개를 찾아냈고 이를 이용한 기능유 전체 분석이 필요하다. 등심과 사태유래 근육위성세포를 이용한 동일조건 분화 비교에서 사태유래 근육 위성세포의 분화 시 myosin complex, skeletal muscle contraction, troponin complex, skeletal muscle tissue development 와 같은 근섬유 형성관련 유전자의 발현이 높게 나타나는 것으로 보아 같은 개체의 근육조직에서도 부위별로 차등 발달이 되고 있다는 것을 알 수 있다. 기존 연구에서는 근육의 성장에 대한 이해를 위해 사양과 영양관련 시험이 많이 이루어졌다. 향후 세포단위의 연구들이 많이 이루어져 작용기작에 대한 생물정보 자료를 추가로 적용한다면 한우의 정밀사양을 적용할수 있는 바탕이 마련될 것이다. 또한 근육위성세포의 연구는 추후 동물실험 윤리제도 강화에 따른 비동물 전임상 screening 시험 활용과 대체단백질 산업의 주요 이슈인 배양육 소재 개발 연구와 같이 축산시험연구의 지속적인 확장성에 많은 영향을 미칠 것으로 생각된다.

• Journal of Animal Science and Technology
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• 제54권3호
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• pp.219-226
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• 2012

Gender specificity in muscle growth and development is well known. Genesis of muscle is dependent on proliferation and differentiation potential of resident myogenic satellite cells (MSCs) present in muscle fibers. Multipotential capacity of forming myocyte, osteocyte, and adipocyte like cell makes MSCs a unique stem cell. To understand the molecular mechanism involved in determination of muscle quality due to difference in hormone concentration of different gender of animals, MSCs were isolated from bovine skeletal muscle and cultured in male, female, and castrated serum supplemented media. DNA microarray used consisted of 24,000 spots with 70 mer oligo in each spot. A total of 88 genes were up-regulated and 551 genes were down-regulated by more than two fold. Among up-regulated gene, 33, 34, and 21 genes were found up-regulated in cells grown in male, female, and castrated serum, respectively. Interestingly, male serum showed 4, female 11 and castrated male showed 4 genes expressed highly in each gender. Further study on the highly up-regulated gene may unfold the mystery of gender specificity found in muscle development. Also, the identification of differentially expressed genes in gender-specific serum will add information on infrastructure of bovine genome research.

• Journal of Animal Science and Technology
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• 제52권4호
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• pp.329-336
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• 2010

본 연구는 소의 부위별 근육에 특이하게 발현하는 유전자 마커를 발굴하여 소고기의 부위를 과학적으로 판명할 수 있는 기술을 개발하고자 실시하였다. 이러한 연구 목표 아래 먼저 사태(Beef shank), 등심(Longissimus dorsi), 양지(Deep pectoral), 홍두깨(Semitendinosus) 부위의 근육조직에서 MSC (myogenic satellite cell, 근육줄기세포)를 순수 분리하고 이를 MFC (myotube-formed cell; 근관이 형성된 세포)로 분화시키거나 ALC (adipocyte-like cell; 지방세포와 유사한 세포)로 이형분화 시킨 후 3가지의 세포로 부터 각각의 RNA를 추출하였다. 이렇게 추출한 RNA는 24,000개의 bovine oligo-nucelotide (70 mer)가 집적된 microarray를 이용해 4개의 조직 중 1개의 조직에서만 MSC의 분화(MFC) 또는 이형분화 과정에서 mRNA의 발현이 증감을 보이는 유전자 135개를 먼저 발굴하였다. 135개의 유전자에 대해 microarray 분석에 사용한 동일한 RNA를 이용하여 real-time PCR 기술로 검증한 결과 총 29개의 유전자가 microarray 분석 결과와 유사함을 보였다. 29개의 유전자를 다시 4개 부위의 생체 조직에서 추출한 RNA를 이용해 real-time PCR 방법으로 분석한 결과 TS (thymi- dlyate synthase), TE (tropoelastin), RAD52(similar RAD52 motifcontaining protein 1), unknown gene), MLC2 (myosin light 2, regulatory cardiac, slow), TXNIP (thioredoxin-interating protein) 6개의 유전자만이 다른 부위에 비해 사태 부위에서 현저한 발현의 차이를 나타냈다. 결론적으로 본 연구를 통해 소 부위별 근육을 구분할 수 있는 과학적 기술의 토대를 확립하였다.

• 농업과학연구
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• 제45권4호
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• pp.715-723
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• 2018

The present study was done to investigate the effect of co-culturing muscle satellite cells (MSCs) and intramuscular preadipocytes (IPs) on the differentiation of adipocytes and muscle cells isolated from Korean native cattle. MSCs and IPs were single-cultured in 10% fetal bovine serum/Dulbecco's modified Eagles medium (FBS/DMEM) for 48 h followed by culturing in 5% FBS/DMEM as the growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without any additives for the single- or co-culture of muscle cells and intramuscular adipocytes to induce the differentiation of both cell types. Cell differentiation was measured by morphological investigation and cytosolic enzyme analysis of glycerol-3-phosphate dehydrogenase (GPDH) for the adipocytes and creatine kinase (CK) for the muscle cells. In the morphological test, the presence of muscle cells did not stimulate adipocyte differentiation showing more differentiation of the adipocytes in the single-culture compared to the co-culture condition. However, the differentiation of muscle cells was promoted by adipocytes in the co-culture. The results of the enzymatic analysis were highly associated with the morphological results with a statistically higher GPDH activity (p < 0.05) appearing in the single-culture than in the co-culture, whereas the opposite was true for the CK activity of the muscle cells (p < 0.05). By manipulating in vivo the milieu using a co-culture, we could detect the difference in the rate of cell differentiation and suggest that a co-culture system is a more reliable and precise technique compared to a single-culture. Further studies on various co-culture trials including supplementation of differentiating substances, gene expression analysis, etc. should be done to obtain practical and fundamental data.

• 한국축산식품학회지
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• 제43권6호
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• pp.1017-1030
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• 2023

Fetal bovine serum (FBS), which contains various nutrients, comprises 20% of the growth medium for cell-cultivated meat. However, ethical, cost, and scientific issues, necesitates identification of alternatives. In this study, we investigated commercially manufactured serum-free media capable of culturing Hanwoo satellite cells (HWSCs) to identify constituent proliferation enhancing factors. Six different serum-free media were selected, and the HWSC proliferation rates in these serum-free media were compared with that of control medium supplemented with 20% FBS. Among the six media, cell proliferation rates were higher only in StemFlex^TM Medium (SF) and Mesenchymal Stem Cell Growth Medium DXF (MS) than in the control medium. SF and MS contain high fibroblast growth factor 2 (FGF2) concentrations, and we found upregulated FGF2 protein expression in cells cultured in SF or MS. Activation of the fibroblast growth factor receptor 1 (FGFR1)-mediated signaling pathway and stimulation of muscle satellite cell proliferation-related factors were confirmed by the presence of related biomarkers (FGFR1, FRS2, Raf1, ERK, p38, Pax7, and MyoD) as indicated by quantitative polymerase chain reaction, western blotting, and immunocytochemistry. Moreover, PD173074, an FGFR1 inhibitor suppressed cell proliferation in SF and MS and downregulated related biomarkers (FGFR1, FRS2, Raf1, and ERK). The promotion of cell proliferation in SF and MS was therefore attributed to FGF2, which indicates that FGFR1 activation in muscle satellite cells may be a target for improving the efficiency of cell-cultivated meat production.

• Journal of Animal Science and Technology
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• 제51권6호
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• pp.459-470
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• 2009

The aim of this study was to analyze the proteome of proliferating bovine satellite cells from longissimus dorsi, deep pectoral and semitendinosus muscle depots which had been subjected to hormonal deprivation or addition in culture. For hormone deprivation or addition studies, the cells were either grown in 10% charcoal-dextran stripped fetal bovine serum (CD-FBS) or in 10% FBS supplemented medium. Further to analyze the effect of insulin like growth factor (IGF-1) and testosterone (TS), the cells were grown in 10% CD-FBS containing IGF-1 (10 ng/ml) or TS (10 nM). Results have shown that hormone deprivation had a negative impact on proliferation of the cells from each of the muscle depots. In case of IGF-1 and TS addition, the proliferation levels were low compared with that of the cells grown in 10% FBS. Hence, to gain the insights of the proteins that are involved in such divergent levels of proliferation, the proteome of such satellite cells proliferating under the above mentioned conditions were analyzed using 2D-DIGE and MALDI-ToF/ToF. Thirteen proteins during hormone deprivation and nine proteins from hormone addition were found to be differentially expressed in all the cultures of the cells from the three depots. Moreover, the results highlighted in this study offer a role for each differentially expressed protein with respect to its effect on positive or negative regulation of cell proliferation.

• 한국축산식품학회지
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• 제30권5호
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• pp.842-850
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• 2010

성호르몬은 근육의 발달(아나볼릭 효과)에 관여하는 것으로 잘 알려져 왔으며 인체의 치료제로 넓게 사용되고 있다. 돼지고기에는 주로 성선(정소와 난소)에서 생산되어 고기에 축적된 다양한 아나볼릭 효과를 나타내는 성호르몬이 많이 존재하는 것으로 알려져 있다. 따라서 본 연구는 돼지고기에 존재하는 아나볼릭 스테로이드를 추출하여 근육세포의 증식과 분화에 미치는 영향을 파악하고자 실시하였다. 본 연구에서 돼지의 각 조직으로부터 아나볼릭 스테로이드를 추출하는 3가지 방법(M1, M2, M3)을 개발하였다. 3가지 모든 추출방법에서 아나볼릭 스테로이드가 추출되었으며 특히 M3에서 조직 1g당 가장 많은 양을 획득할 수 있었다. M3를 이용하여 아나볼릭 스테로이드를 추출했을 때 돼지고기의 근육과 지방에서는 스테로이드 호르몬 양이 수컷에서 가장 높았고, 혈청에서는 nandrolone 과 testosterone의 양은 수컷에서 가장 높았으며, estrone와 $17{\beta}$-estradiol의 양은 비슷하였다. 근육세포 증식은 돼지 아나볼릭 스테로이드의 농도 의존적으로 촉진되었으며, M3에서 가장 빠르게 증식되었다. 근육세포의 분화는 추출된 아나볼릭 스테로이드 처리구가 대조구보다 더 효과적으로 분화가 잘 되는 것을 세포 모양과 유전자(AR, myoD, desmin, myogenin)를 통하여 확인할 수 있었다. 특히 M3에서 추출된 아나볼릭 스테로이드에서 myogenin과 AR 유전자가 유의적으로 높게 발현되었다(p<0.001). 전체적으로 돼지의 아나볼릭 스테로이드는 근육세포의 성장과 분화를 촉진하였다. 따라서 돼지고기에 있는 아나볼릭 스테로이드의 섭취를 통해 인체 근육의 성장과 발달에 긍정적 효과를 나타낼 수 있을 것으로 사료된다.

• Journal of Animal Science and Technology
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• 제63권4호
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• pp.934-953
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• 2021

Ciglitazone is a member of the thiazolidinedione family, and specifically binds to peroxisome proliferator-activated receptor-γ (PPARγ), thereby promoting adipocyte differentiation. We hypothesized that ciglitazone as a PPARγ ligand in the absence of an adipocyte differentiation cocktail would increase adiponectin and adipogenic gene expression in bovine satellite cells (BSC). Muscle-derived BSCs were isolated from six, 18-month-old Yanbian Yellow Cattle. The BSC were cultured for 96 h in differentiation medium containing 5 µM ciglitazone (CL), 10 µM ciglitazone (CM), or 20 µM ciglitazone (CH). Control (CON) BSC were cultured only in a differentiation medium (containing 2% horse serum). The presence of myogenin, desmin, and paired box 7 (Pax7) proteins was confirmed in the BSC by immunofluorescence staining. The CL, CM, and CH treatments produced higher concentrations of triacylglycerol and lipid droplet accumulation in myotubes than those of the CON treatment. Ciglitazone treatments significantly increased the relative expression of PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα), C/EBPβ, fatty acid synthase, stearoyl-CoA desaturase, and perilipin 2. Ciglitazone treatments increased gene expression of Pax3 and Pax7 and decreased expression of myogenic differentiation-1, myogenin, myogenic regulatory factor-5, and myogenin-4 (p < 0.01). Adiponectin concentration caused by ciglitazone treatments was significantly greater than CON (p < 0.01). RNA sequencing showed that 281 differentially expressed genes (DEGs) were found in the treatments of ciglitazone. DEGs gene ontology (GO) analysis showed that the top 10 GO enrichment significantly changed the biological processes such as protein trimerization, negative regulation of cell proliferation, adipocytes differentiation, and cellular response to external stimulus. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that DEGs were involved in the p53 signaling pathway, PPAR signaling pathway, biosynthesis of amino acids, tumor necrosis factor signaling pathway, non-alcoholic fatty liver disease, PI3K-Akt signaling pathway, and Wnt signaling pathway. These results indicate that ciglitazone acts as PPARγ agonist, effectively increases the adiponectin concentration and adipogenic gene expression, and stimulates the conversion of BSC to adipocyte-like cells in the absence of adipocyte differentiation cocktail.