• 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.65 no.3
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• pp.664-678
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• 2023

To improve culture efficiency of Hanwoo myosatellite cells, these cells were cultured at different temperatures. Hanwoo myosatellite cells were compared with C2C12 cells to observe proliferation and differentiation at culture temperatures of 37℃ and 39℃ and determine the possibility of using them as cultured meat. Immunofluorescence staining using Pax7 and Hoechst, both cells cultured at 37℃ proliferated better than cultured at 39℃ (p < 0.05). When differentiated cells were stained with myosin and Hoechst, there was no significant difference in myotube thickness and Fusion index (p > 0.05). In Western blotting analysis, Hanwoo myosatellite cells were no significant difference in the expression of myosin between cells differentiated at the two temperatures (p > 0.05). C2C12 cells were no significant difference in the expression of myosin between cells differentiated at the two temperatures (p > 0.05). In reverse transcription and quantitative polymerase chain reaction (RT-qPCR) analysis, Hanwoo myosatellite cells cultured at 39℃ had significantly (p < 0.05) higher expression levels of MyHC, MYF6, and MB than those cultured at 37℃. C2C12 cells cultured at 39℃ showed significantly (p < 0.05) higher expression levels of MYOG and MB than those cultured at 37℃. To increase culture efficiency of Hanwoo myosatellite cells, proliferating at 37℃ and differentiating at 39℃ are appropriate. Since results of temperature differences of Hanwoo myosatellite cells were similar to those of C2C12 cells, they could be used as a reference for producing cultured meat using Hanwoo satellite cells.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.4
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• pp.289-296
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• 2020

Spermatogonial stem cells are self-renewal and differentiate into sperm in post-pubertal mammals. There exists a balance between the self-renewal and differentiation in the testes. Spermatogonial stem cells make up only 0.03% of testicular cells in adult mice. These cells maintain sperm production by differentiating after puberty. Therefore, analyzing the expression of genes associated with spermatogenesis is critical for understanding differentiation. The present study aimed to establish the postnatal period of cells in relation to spermatogenesis. To study the expression of differentiated and undifferentiated marker genes in enriched spermatogonial stem cells, in vitro culture was performed and cells from pup (6-8-day-old) and adult (4-months-old) testicular tissues were isolated. As a result, undifferentiated genes, Pax7, Plzf, GFRa1, Etv5 and Bcl6b, were highly increased in cultured spermaotogonial stem cells compared with pup and adult testicular cells. On the other hands, differentiated gene, c-kit was highly increased in adult testicular cells, Also Stra8 gene was highly increased in pup and adult testicular cells. This study provides a better understanding of spermatogenesis-associated gene expression during postnatal periods.

• 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 Reproduction and Biotechnology
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• v.38 no.1
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• pp.26-31
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• 2023

Chicken embryonic stem (ES) cells have great potential and provide a powerful tool to investigate embryonic development and to manipulate genetic modification in a genome. However, very limited studies are available on the functional characterization and robust expansion of chicken ES cells compared to other species. Here, we have developed a method to generate chicken embryonic stem cell-like cells under pluripotent culture conditions. The chicken embryonic stem cell-like cells were cultivated long-term over several passages of culture without loss of pluripotency in vitro and had the specific expression of key stem cell markers. Furthermore, they showed severe changes in morphology and a significant reduction in pluripotent genes after siRNA-mediated NANOG knockdown. Collectively, these results demonstrate the efficient generation of chicken embryonic stem cell-like cells from EGK stage X blastoderm-derived singularized cells and will facilitate their potential use for various purposes, such as biobanking genetic materials and understanding stemness in the fields of animal biotechnology.

• Animal Bioscience
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• v.34 no.8
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• pp.1321-1330
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• 2021

Objective: Transgenic hens hold a great promise to produce various valuable proteins. Through virus transduction into stage X embryo, the transgene expression under the control of constructed chicken ovalbumin promoters has been successfully achieved. However, a validation system that can evaluate differently developed ovalbumin promoters in in vitro, remains to be developed. Methods: In the present study, chicken oviduct epithelial cells (cOECs) were isolated from oviduct tissue and shortly cultured with keratinocyte complete medium supplemented with chicken serum. The isolated cells were characterized with immunofluorescence, western blot, and flow cytometry using oviduct-specific marker. Chicken mutated ovalbumin promoter (Mut-4.4-kb-pOV) was validated in these cells using luciferase reporter analysis. Results: The isolated cOECs revealed that the oviduct-specific marker, ovalbumin protein, was clearly detected by immunofluorescence, western blot, and flow cytometry analysis revealed that approximately 79.40% of the cells contained this protein. Also, luciferase reporter analysis showed that the constructed Mut-4.4-kb-pOV exhibited 7.1-fold (p<0.001) higher activity in the cOECs. Conclusion: Collectively, these results demonstrate the efficient isolation and characterization of cOECs and validate the activity of the constructed ovalbumin promoter in the cultured cOECs. The in vitro validation of the recombinant promoter activity in cOECs can facilitate the production of efficient transgenic chickens for potential use as bioreactors.

• Asian-Australasian Journal of Animal Sciences
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• v.2 no.4
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• pp.595-598
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• 1989

Bovine embryos/ova obtained from in-vitro fertilization were either co-cultured on a monolayer of bovine cumulus cells or cultured in medium alone. Embryos/ova co-cultured with cumulus cells developed to 8-cell (30.9%), morula (29.8%) and blastocyst stages (26.6%) after 3-4, 5-6, and 7-8 days of culture, respectively, while embryos/ova cultured in medium alone failed to develop beyond 8-cell (0-13.3%), morula (0-1.5%) and blastocyst stages (0%). The results of this study demonstrated the beneficial effect of cumulus cells on the development of bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.9
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• pp.1242-1248
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• 2005

This study was designed to investigate the in vitro developmental ability and apoptosis of bovine embryos nucleartransferred (NT) with frozen-thawed or cooled donor cells. Cultured adult bovine ear cells were used as donor cells after sub-culturing to confluence (CC), cooling to 4$^{\circ}C$ for 48 h, or freezing-thawing (FT). Apoptotic cells in blastocysts were evaluated for apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Fusion, cleavage and blastocyst rates were 69.0 (167/242), 68.8 (115/167), and 29.9 (50/167) with CC cells, 70.4 (88/125), 69.3 (61/88), and 29.6 (26/88) with cooled cells and 66.1 (117/177), 70.1 (82/117), and 13.7 (16/117) with FT cells, respectively. Blastocyst rates of NT embryos derived from FT cells were significantly lower than those from CC or cooled cells (p<0.05). In addition, NT blastocysts produced by using FT cells showed significantly higher apoptosis rates (6.4${\pm}$4.0%) than those produced by CC (2.8${\pm}$1.7%) or cooled (2.3${\pm}$1.3%) cells. However, cooling of donor cells had no significant adverse effect on blastocyst rate as well as apoptosis rate. Therefore, our results suggest that cooled cells may be used as an alternative to freshly cultured confluent culture cells, as donor cells, for the production of Somatic nuclear cloned cattle.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.8
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• pp.1220-1225
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• 2008

This study was conducted to investigate the protective effect of genistein on the antioxidative defence system and membrane fluidity in chick skeletal muscle cells after supplementation with 0, 20, 40, and $80{\mu}mol/L$ genistein in $50{\mu}mol/L$ $FeSO_4/H_2O_2$ treated cells for 24 h. Genistein supplementation recovered the decreased activity of total superoxide dismutase induced by $FeSO_4/H_2O_2$, significantly increased glutathione peroxidase activity (p<0.05) and decreased malondialdehyde production (p<0.05). The treatment of 80 mol/L genistein in $FeSO_4/H_2O_2$ treated cells decreased the secretion of creatine kinase (p<0.05). Fluorescence polarization values and microviscosities observed with $FeSO_4/H_2O_2$ treated cells were significantly higher than those observed with no $FeSO_4/H_2O_2$ treated cells. The addition of $80{\mu}mol/L$ genistein improved the increased fluorescence polarization value (p<0.05) caused by $FeSO_4/H_2O_2$ treatment. The microviscosity value was significantly decreased by adding genistein (p<0.05). In conclusion, genistein protected skeletal muscle cells from oxidative damage by improving antioxidative status and membrane fluidity.

• 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.