• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.177-187
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• 2023

Background: The grading of Hanwoo (Korean native cattle) is based on four economic traits, and efforts have been continuously made to improve the genetic traits associated with these traits. There is a technology to predict the expected grade based on the 4 economic genetic SNP characteristics of Korean cattle calves using single nucleotide polymorphism (SNP) technology. Selection of highly proliferative, self-renewing, and differentiating satellite cells from cattle is a key technology in the cultured meat industry. Methods: We selected the Hanwoo with high and low-scored of genomic estimated breeding value (GEBV) by using the Hanwoo 50K SNP bead chip. We then isolated the bovine satellite cells from the chuck mass. We then conducted comparative analyses of cell proliferation, immunocytochemistry, qRT-PCR at short- and long-term culture. We also analyzed the differentiation capability at short term culture. Results: Our result showed that the proliferation was significantly high at High scored GEBV (Hs-GEBV) compared to Low scored GEBV (Ls-GEBV) at short- and long-term culture. The expression levels of Pax3 were significantly higher in Hs-GEBV bovine satellite cells at long-term culture. However, there were no significant differences in the expression levels of Pax7 between Hs- and Ls-GEBV bovine satellite cells at short- and long- term culture. The expression levels of MyoG and MyHC were significantly high at Ls-GEBV bovine satellite cells. Conclusions: Our results indicated that selection of bovine satellite cells by Hanwoo 50K SNP bead chip could be effective selection methods for massive producing of satellite cells.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.11
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• pp.1519-1526
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• 2010

Serum lipid (SL) is a commercially available cholesterol-rich, proteinaceous compound extracted from bovine serum. Here we investigated the adipogenic transdifferentiation potential of SL on bovine myogenic satellite cells. Exposure of satellite cells to SL could generate lipid droplets on day 2, and further exposure to SL increased cytoplasmic lipid accumulation giving adipocyte morphology. The expression analysis of PPAR gamma and GPDH adipocyte markers along with Oil-red-O staining results confirmed the transdifferentiation potential of SL. When cells were treated at different concentrations (5, 10, 20, $40{\mu}l$/ml) of SL, the results indicated that even levels as low as $5{\mu}l$ SL /ml could induce transdifferentiation, and maximum induction was obtained at $20{\mu}l$ SL/ml. After treatment with SL at different concentrations the expression levels of PPAR gamma varied significantly (p<0.05), whereas the expression of other adipogenic transcription factors showed no difference, indicating that SL acts through PPAR gamma. The combined effect of SL and troglitazone proved to be the best combination for induction of transdifferentiation compared to the individual effect of SL or troglitazone. Thus, overall results clearly show that SL induces transdifferentiation of bovine myogenic satellite cells to adipocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.9
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• pp.1288-1302
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• 2011

The aim of this study was to analyze the proteome expression of bovine satellite cells from longissimus dorsi (LD), deep pectoral (DP) and semitendinosus (ST) muscle depots during in vitro myogenic differentiation. Proteomic profiling by twodimensional gel electrophoresis and mass spectrometry of differentiating satellite cells revealed a total of 38 proteins that were differentially regulated among the three depots. Among differentially regulated proteins, metabolic proteins like lactate dehydrogenase (LDH), malate dehydrogenase (MDH) were found to be up regulated in ST, while alpha-enolase (NNE) in LD and DP depot satellite cells were down regulated. Also, our analysis found that there was a prominent up regulation of cytoskeletal proteins like actin, actincapping protein and transgelin along with chaperone proteins like heat shock protein beta 1 (HSPB 1) and T-complex protein 1 (TCP-1). Among other up regulated proteins, LIM domain containing protein, annexin 2 and Rho GDP-dissociation inhibitor 1 (Rho GDI) are observed, which were already proven to be involved in the myogeneis. More interestingly, satellite cells from ST depot were found to have a higher myotube formation rate than the cells from the other two depots. Taken together, our results demonstrated that, proteins involved in glucose metabolism, cytoskeletal modeling and protein folding plays a key role in the myogenic differentiation of bovine satellite cells.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.5
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• pp.649-658
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• 2010

Myogenic satellite cells (MSCs) are adult stem cells that activate and differentiate into myotubes. These stem cells are multipotent as they transdifferentiate into adipocyte-like cells, nerve cells and osteocytes. The effects of steroid hormones ($E_2$ and testosterone) were studied as a further step toward understanding the mechanism of MSCs proliferation and differentiation. In this study, MSCs were grown continuously for 87 days, implying that there may be a group of MSCs that continue to proliferate rather than undergoing differentiation. Isolated MSCs were cultured in Dulbecco's Modified Eagle's Medium supplemented with adult male, female or castrated bovine serum to observe the effect of steroid hormones on MSC proliferation. Cell proliferation was the highest in cultures supplemented with male serum followed by female and castrated serum. The positive effect of male hormone on MSC proliferation was confirmed by the observation of testosterone-mediated increased proliferation of cells cultured in medium supplemented with castrated serum. Furthermore, steroid hormone treatment of MSCs increased lipid accumulation in myotubes. Oil-Red-O staining showed that 17${\beta}$-estradiol ($E_2$) treatment avidly increased lipid accumulation, followed by $E_2$+testosterone and testosterone alone. To our knowledge, this is the first report of lipid accumulation in myotubes due to steroids in the absence of an adipogenic environment, and the effect of steroid hormones on cell proliferation using different types of adult bovine serum, a natural hormonal system. In conclusion, we found that sex steroids affect MSCs proliferation and differentiation, and lipid accumulation in myotubes.

• 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 Animal Science and Technology
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• v.63 no.4
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• pp.919-933
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• 2021

We hypothesized that the unsaturated fatty acid palmitoleic acid (POA) could promote the expression of adipogenic/lipogenic genes in bovine skeletal muscle satellite cells (BSCs). The BSCs were cultured in a growth medium containing 10% fetal bovine serum. When the cells reached 80%-90% confluence, we used the differentiation medium with 5% horse serum for differentiation for 96 h. The differentiation medium contained 50 µM, 100 µM and 200 µM POA. Control BSC were cultured only in differentiation media. Compared with the control BSC, the POA BSC significantly up-regulated the expression of paired box 3 (Pax3) and paired box 7 (Pax7) and down-regulated myogenin gene expression (p < 0.01), which indicates a depression in muscle fiber development. However, all POA treatments up-regulated the expression of the adipocyte transcription factors peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein alpha and beta (C/EBP α and C/EBP β), and other genes (p < 0.01) and increased the expression of PAT-family proteins and the concentration of adiponectin in the media. These results indicate that POA can convert part of BSCs into adipocytes.

• Journal of Life Science
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• v.22 no.10
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• pp.1316-1323
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• 2012

The present study was conducted to investigate whether myoblasts can be transdifferentiated into adipocytes by ectopic expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor-${\gamma}$ ($PPAR{\gamma}$), CCAAT/enhancer-binding protein-${\alpha}$ (C/$EBP{\alpha}$), sterol regulatory element binding protein-1c (SREBP1c), and Krueppel-like factor 5 (KLF5), in primary bovine satellite cells. Transcription factors were transiently transfected into primary bovine myoblasts, and the cells were cultured with adipogenic differentiation medium for 2 days and then cultured on growth medium for an additional 8 days. Ectopic expression of $PPAR{\gamma}$ or C/$EBP{\alpha}$ alone was insufficient to induce adipogenesis in myoblasts. However, overexpression of both $PPAR{\gamma}$ and C/$EBP{\alpha}$ in myoblasts was able to induce adipogenic transdifferentiation as indicated by the appearance of mature adipocytes, the induction of adipogenic gene expressions, and the suppression of myogenic gene expressions. In addition, KLF5 and $PPAR{\gamma}$ co-transfected bovine myoblasts were converted to adipocytes but not in cells transfected with only KLF5 expression vector. Overexpression of SREBP1c alone was sufficient to induce transdifferentiation from myoblasts into adipocytes. These results demonstrate that primary bovine satellite cells can be transdifferentiated into adipocytes either by single ectopic expression or combined expression of adipogenic transcription factors in a culture system.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.3
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• pp.411-419
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• 2015

We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC) and intramuscular preadipocytes (IPA) were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco's Modified Eagle Medium (DMEM) and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC) or 5% FBS/DMEM (IPA) with antibiotics. Media also contained $10{\mu}g/mL$ insulin and $10{\mu}g/mL$ pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with $40{\mu}M$ palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001) carnitine palmitoyltransferase-1 beta ($CPT1{\beta}$) gene expression, but the increase in $CPT1{\beta}$ gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17-fold increases). Oleic and linoleic acid decreased (p = 0.001) stearoyl-CoA desaturase (SCD) gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased $AMPK{\alpha}$ gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism.

• Molecules and Cells
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• v.22 no.2
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• pp.239-245
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• 2006

This study examined whether adult bovine muscle satellite cells from 30-month-old Hanwoo cattle are multipotential. The satellite cells were found to have the potential to proliferate and differentiate into myoblasts with the formation of multinucleated cells. In addition, treatment with the peroxisome proliferator activating receptor-${\gamma}$ ($PPAR{\gamma}$) agonist, rosiglitazone, promoted their trans-differentiation into adipocytes with significant increases in glycerol accumulation and glycerol-3-phosphate dehydrogenase activity. Western blot analysis revealed that increased levels of the adipocyte fatty acid-binding protein, $PPAR{\gamma}$ and of CCAAT/enhancerbinding protein were closely related to rosiglitazoneinduced differentiation of the cells. These findings demonstrate that satellite cells from adult Hanwoo cattle are multipotent, and that their trans-differentiation into adipocytes can be induced by rosiglitazone.