• Asian-Australasian Journal of Animal Sciences
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• 제30권7호
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• pp.1029-1036
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• 2017

Objective: In the livestock industry, the regulatory mechanisms of muscle proliferation and differentiation can be applied to improve traits such as growth and meat production. We investigated the regulatory pathway of MyoD and its role in muscle differentiation in quail myoblast cells. Methods: The MyoD gene was mutated by the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology and single cell-derived MyoD mutant sublines were identified to investigate the global regulatory mechanism responsible for muscle differentiation. Results: The mutation efficiency was 73.3% in the mixed population, and from this population we were able to establish two QM7 MyoD knockout subline (MyoD KO QM7#4) through single cell pick-up and expansion. In the undifferentiated condition, paired box 7 expression in MyoD KO QM7#4 cells was not significantly different from regular QM7 (rQM7) cells. During differentiation, however, myotube formation was dramatically repressed in MyoD KO QM7#4 cells. Moreover, myogenic differentiation-specific transcripts and proteins were not expressed in MyoD KO QM7#4 cells even after an extended differentiation period. These results indicate that MyoD is critical for muscle differentiation. Furthermore, we analyzed the global regulatory interactions by RNA sequencing during muscle differentiation. Conclusion: With CRISPR/Cas9-mediated genomic editing, single cell-derived sublines with a specific knockout gene can be adapted to various aspects of basic research as well as in functional genomics studies.

• Journal of Veterinary Science
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• 제22권4호
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• pp.53.1-53.13
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• 2021

Background: Canine adipose-derived stem cells (cADSCs) exhibit various differentiation properties and are isolated from the canine subcutaneous fat. Although cADSCs are valuable as tools for research on adipogenic differentiation, studies focusing on adipogenic differentiation methods and the underlying mechanisms are still lacking. Objectives: In this study, we aimed to establish an optimal method for adipogenic differentiation conditions of cADSCs and evaluate the role of peroxisome proliferator-activated receptor gamma (PPARγ) and estrogen receptor (ER) signaling in the adipogenic differentiation. Methods: To induce adipogenic differentiation of cADSCs, 3 different adipogenic medium conditions, MDI, DRI, and MDRI, using 3-isobutyl-1-methylxanthine (M), dexamethasone (D), insulin (I), and rosiglitazone (R) were tested. Results: MDRI, addition of PPARγ agonist rosiglitazone to MDI, was the most significantly facilitated cADSC into adipocyte. GW9662, an antagonist of PPARγ, significantly reduced adipogenic differentiation induced by rosiglitazone. Adipogenic differentiation was also stimulated when 17β-estradiol was added to MDI and DRI, and this stimulation was inhibited by the ER antagonist ICI182,780. Conclusions: Taken together, our results suggest that PPARγ and ER signaling are related to the adipogenic differentiation of cADSCs. This study could provide basic information for future research on obesity or anti-obesity mechanisms in dogs.

• Asian-Australasian Journal of Animal Sciences
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• 제31권9호
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• pp.1507-1515
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• 2018

Objective: In the poultry industry, the most important economic traits are meat quality and carcass yield. Thus, many studies were conducted to investigate the regulatory pathways during muscle differentiation. To gain insight of muscle differentiation mechanism during growth period, we identified and validated calcium-related genes which were highly expressed during muscle differentiation through mRNA sequencing analysis. Methods: We conducted next-generation-sequencing (NGS) analysis of mRNA from undifferentiated QM7 cells and differentiated QM7 cells (day 1 to day 3 of differentiation periods). Subsequently, we obtained calcium related genes related to muscle differentiation process and examined the expression patterns by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results: Through RNA sequencing analysis, we found that the transcription levels of six genes (troponin C1, slow skeletal and cardiac type [TNNC1], myosin light chain 1 [MYL1], MYL3, phospholamban [PLN], caveolin 3 [CAV3], and calsequestrin 2 [CASQ2]) particularly related to calcium regulation were gradually increased according to days of myotube differentiation. Subsequently, we validated the expression patterns of calcium-related genes in quail myoblasts. These results indicated that TNNC1, MYL1, MYL3, PLN, CAV3, CASQ2 responded to differentiation and growth performance in quail muscle. Conclusion: These results indicated that calcium regulation might play a critical role in muscle differentiation. Thus, these findings suggest that further studies would be warranted to investigate the role of calcium ion in muscle differentiation and could provide a useful biomarker for muscle differentiation and growth.

• Natural Product Sciences
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• 제14권3호
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• pp.182-186
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• 2008

Scutellaria baicalensis Georgi (SBG) is traditionally used medicinal herb that has anti-oxidant, anticancer and anti-inflammatory effects. In this study, we investigated whether the extracts of SBG have the inhibitory activity in the osteoclast differentiation by using mouse monocytes RAW264.7 cells and primary mouse bone marrow-derived macrophages (BMMs). Methanol extract (ME) from SBG was successively fractionated into methylene chloride (MF), ethylacetate (EF) and n-butanol fraction (BF). The activity assay for tartrateresistant acid phosphatase (TRAP) and Western blot analysis were employed to evaluate the osteoclasts differentiation and the activation of mitogen-activated protein (MAP) kinases, respectively. ME, MF, EF and BF significantly and dose-dependently inhibited osteoclast differentiation without the decrease of cell viability at the concentrations used in this study. In addition, ME significantly inhibited the activation of c-jun-N-terminal kinase (JNK). In conclusion, this study firstly demonstrated that ME of SBG has the potential to inhibit the osteoclast differentiation through the suppression of JNK activation partially.

• International Journal of Stem Cells
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• 제7권2호
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• pp.108-117
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• 2014

Background and Objectives: Genomic imprinting is an inheritance phenomenon by which a subset of genes are expressed from one allele of two homologous chromosomes in a parent of origin-specific manner. Even though fine-tuned regulation of genomic imprinting process is essential for normal development, no other means are available to study genomic imprinting in human during embryonic development. In relation with this bottleneck, differentiation of human embryonic stem cells (hESCs) into specialized lineages may be considered as an alternative to mimic human development. Methods and Results: In this study, hESCs were differentiated into three lineage cell types to analyze temporal and spatial expression of imprinted genes. Of 19 imprinted genes examined, 15 imprinted genes showed similar transcriptional level among two hESC lines and two human induced pluripotent stem cell (hiPSC) lines. Expressional patterns of most imprinted genes were varied in progenitors and fully differentiated cells which were derived from hESCs. Also, no consistence was observed in the expression pattern of imprinted genes within an imprinting domain during in vitro differentiation of hESCs into three lineage cell types. Conclusions: Transcriptional expression of imprinted genes is regulated in a cell type- specific manner in hESCs during in vitro differentiation.

• 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).

• 대한수학회보
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• 제46권6호
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• pp.1135-1140
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• 2009

We will consider the questions of when the products of composition and differentiation are bounded and compact on Bloch and little Bloch spaces.

• Animal Bioscience
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• 제36권12호
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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.

• Journal of Animal Science and Technology
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• 제53권3호
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• pp.223-226
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• 2011

The current study was undertaken to determine the effect of retinoic acid (RA) on proliferation and differentiation of preadipocytes from male and female pigs. The preadipocytes were isolated from new-born male and female pigs by collagenase digestion and washed three times one day after seeding (designated as day 0 of culture). RA was included in the media at various concentratives from day 0 to 2. The cell number was measured on day 2 with hematocytometer after trypsin digestion. Cell differentiation was determined on day 6 by measuring glycerol-3-phosphate dehydrogenase activity. RA (0.1, 1 and 10 uM) showed no effect on proliferation of preadipocytes from both male and female pigs. However, RA significantly decreased differentiation of pig preadipocytes. Degree of differentiation with 0.1 uM, 1 uM and 10 uM of RA treatment was 80%, 41% and 29% respectively, compared with control. Similar inhibitory effect was found in the female pigs; 77%, 28% and 16% respectively. It is interesting that RA treated on cell proliferation stage had no effect on proliferation but had a strong inhibitory effect on differentiation which is happening in the late stage of cell culture.

• Molecules and Cells
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• 제38권1호
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• pp.89-94
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• 2015

The shape and activity of mitochondria are tightly regulated by fusion and fission processes that are essential for maintaining normal cellular function. However, little is known about the involvement of mitochondrial dynamics in the development of the immune system. In this study, we demonstrate that mitochondrial dynamics play a role in the differentiation and migration of immature dendritic cells (imDCs). We show that mitochondrial elongation is induced during GM-CSF-stimulated differentiation of bone marrow progenitors to imDCs accompanied by upregulation of mitochondrial fusion proteins. These processes precede the changes in mitochondrial morphology and connectivity that occur during differentiation. Mfn2 and OPA1, but not Mfn1, are transcriptionally upregulated during differentiation; however, knockdown of Mfn2 and OPA1 does not induce any change in expression of CD11c, CDC80, or CD86. Notably, knockdown of Mfn2 or OPA1 by siRNA in imDCs significantly reduces CCR7 expression and CCL19-mediated migration. These results suggest that the mitochondrial fusion-related proteins Mfn2 and OPA1 are upregulated during bone marrow progenitor differentiation and promote the migration of imDCs by regulating the expression of CCR7.