• Food Science of Animal Resources
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• v.40 no.5
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• pp.852-859
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• 2020

The muscle stem cells of domestic animals are of interest to researchers in the food and biotechnology industries for the production of cultured meat. For producing cultured meat, it is crucial for muscle stem cells to be efficiently isolated and stably maintained in vitro on a large scale. In the present study, we aimed to optimize the method for the enrichment of pig muscle stem cells using a magnetic-activated cell sorting (MACS) system. Pig muscle stem cells were collected from the biceps femoris muscles of 14 d-old pigs of three breeds [Landrace×Yorkshire×Duroc (LYD), Berkshire, and Korean native pigs] and cultured in skeletal muscle growth medium-2 (SkGM-2) supplemented with epidermal growth factor (EGF), dexamethasone, and a p38 inhibitor (SB203580). Approximately 30% of total cultured cells were nonmyogenic cells in the absence of purification in our system, as determined by immunostaining for cluster of differentiation 56 (CD56) and CD29, which are known markers of muscle stem cells. Interestingly, following MACS isolation using the CD29 antibody, the proportion of CD56⁺/CD29⁺ muscle stem cells was significantly increased (91.5±2.40%), and the proportion of CD56 single-positive nonmyogenic cells was dramatically decreased. Furthermore, we verified that this method worked well for purifying muscle stem cells in the three pig breeds. Accordingly, we found that CD29 is a valuable candidate among the various marker genes for the isolation of pig muscle stem cells and developed a simple sorting method based on a single antibody to this protein.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.3
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• pp.203-208
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• 2013

As the abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis and vascular restenosis, a candidate drug with antiproliferative properties is needed. We investigated the antiproliferative action and underlying mechanism of a newly synthesized naphthoquinone derivative, 5,8-dimethoxy-2-nonylamino-naphthalene-1,4-dione (2-nonylamino-DMNQ), using VSMCs treated with platelet-derived growth factor (PDGF). 2-Nonylamino-DMNQ inhibited proliferation and cell number of VSMCs induced by PDGF, but not epidermal growth factor (EGF), in a concentration-dependent manner without any cytotoxicity. This derivative suppressed PDGF-induced $[^3H]$-thymidine incorporation, cell cycle progression from $G_0/G_1$ to S phase, and the phosphorylation of phosphor-retinoblastoma protein (pRb) as well as the expression of cyclin E/D, cyclin-dependent kinase (CDK) 2/4, and proliferating cell nuclear antigen (PCNA). Importantly, 2-nonylamino-DMNQ inhibited the phosphorylation of PDGF receptor${\beta}$(PDGF-$R{\beta}$) enhanced by PDGF at $Tyr^{579}$, $Tyr^{716}$, $Tyr^{751}$, and $Tyr^{1021}$ residues. Subsequently, 2-nonylamino-DMNQ inhibited PDGF-induced phosphorylation of STAT3, ERK1/2, Akt, and $PLC{\gamma}1$. Therefore, our results indicate that 2-nonylamino-DMNQ inhibits PDGF-induced VSMC proliferation by blocking PDGF-$R{\beta}$ autophosphorylation, and subsequently PDGF-$R{\beta}$-mediated downstream signaling pathways.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.4
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• pp.333-340
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• 2003

Objective: This study was performed to examine the maturation and the development to the blastocyst stage of immature oocytes collected from patients with high risk of ovarian hyperstimulation syndrome (OHSS). Materials and Methods: Cumulus-oocyte complexes (COCs) were collected following only HCGpriming for non stimulated IVF-ET cycles of the patients. At the time of oocyte collection, COCs were classified into three groups in accordance with their appearance (Group I: oocytes with dispersed cumulus cells; Group II: oocytes with compacted cumulus cells; Group III: oocytes with sparse cumulus cells). The in vitro maturation and blastocyst development rates of the COCs were compared among these groups. From August 2001 to June 2002, 48 IVM/IVF-ET cycles from 42 patients (mean age: $32.4{\pm}3.8$ years) were performed. To prevent the occurrence of OHSS, the patients were primed with 10, 000 IU HCG alone 36 h before oocyte collection without gonadotropin stimulation. Oocytes were aspirated on cycle days from 7 to 13. The normal COCs were classified into three groups according to their appearance. The aspirated immature oocytes were cultured in YS maturation medium containing 30% (v/v) human follicular fluid (HFF), 1 IU/ml FSH, 10 IU/ml HCG and 10 ng/ml rhEGF. Fertilization was induced by intracytoplasmic sperm injection (ICSI). All zygotes were co-cultured with cumulus cells in $10{\mu}l$ YS medium containing 10% HFF until day 7 after oocyte collection. Blastocyst transfer was performed on day 5 after ICSI. Results: Th e mean number of oocytes cultured in the IVM/IVF cycles was $24.7{\pm}10.6$. Of 1185 COCs, those assigned to Group I, II and III were 470 (39.7%), 414 (35.0%) and 301 (25.4%), respectively. The maturation rate (94.5%, 444/470, p<0.05) in Group I was significantly higher than those of Group II (62.8%, 260/414) and Group III (73.1%, 220/301). Especially, 30.9% of COCs in Group I (145/470) was matured on the day of oocyte aspiration. There were no differences in the rates of fertilization and cleavage among the three groups. The development rate to the blastocyst stage in Group I (54.6%, 206/377, p<0.05) was also significantly higher than those in Group II (33.0%, 68/206) and Group III (30.1%, 52/173). Twenty-four clinical pregnancies (50.0%) was obtained and 22 pregnancies (45.8%) are ongoing. Implantation rate in the present study was 24.6%. Conclusion: These results suggest that there is a positive correlation between the appearance of COCs and the developmental competence of the immature oocytes in non stimulated IVM/IVF cycles.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.355-359
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• 1997

Leiomyomas, which are the commonest pelvic tumors in women, are originated from myometrial cells. Although the exact initial pathophysiologic event of the leiomyoma is not known, recent evidences suggested that the effects of sex steroid hormones in the process of tumor growth are mediated by local production of growth factors including epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II). If we look at the effects of other cytokines, it was suggested that basic fibroblast growth factor (bFGF) may stimulate the proliferation of myometrial and leiomyomas cells. And it was reported that interferon-${\alpha}$ inhibit the action of bFGF. Therefore, we examined the effect of bFGF and interferon-${\alpha}$ on the proliferation of leiomyoma and myometrial cells. bFGF stimulated the myometrial and leiomyoma cells significantly at the concentration of 1ng/ml (p<0.05) and 5ng/ml (p<0.05). However, Interferon-${\alpha}$ inhibited the cell proliferation of myometrial and leiomyoma cells significantly at the concentration of 100U/ml (p<0.05) and 1000U/ml (p<0.05). And the stimulated effects of bFGF with the various concentration on the myometrial and leiomyoma cells ware inhibited by interferon-${\alpha}$ with 100U/ml. Therefore, we concluded that bFGF may stimulate the myometrial and leiomyoma cell proliferation and interferon-${\alpha}$ may inhibit the myometrial and leiomyoma cell proliferation through blocking the effect of basic fibroblast growth factor.

• Journal of Embryo Transfer
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• v.22 no.4
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• pp.245-249
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• 2007

The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), $10{\mu}g/ml$ follicle stimulating hormone (FSH), $35{\mu}g/ml$ luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$ and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within $39.1{\sim}70%$ in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.

• Proceedings of the Korean Vacuum Society Conference
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• 2010.02a
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• pp.44-45
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• 2010

We are currently conducting studies on culturing and biocompatibility assessment of various cells such as neural stem cells and induced pluripotent stem cells(IPS cells) on carbon nanotube (CNT), on nerve regeneration electrodes, and on silicon wafers with a focus on developing nerve integrated CNT based bio devices for interfacing with living organisms, in order to develop brain-machine interfaces (BMI). In addition, we are carried out the chemical modification of carbon nanotube (mainly SWCNTs)-based bio-nanosensors by the plasma ion irradiation (plasma activation) method, and provide a characteristic evaluation of a bio-nanosensor using bovine serum albumin (BSA)/anti-BSA binding and oligonucleotide hybridization. On the other hand, the researches in the case of "novel plasma" have been widely conducted in the fields of chemistry, solid physics, and nanomaterial science. From the above-mentioned background, we are conducting basic experiments on direct irradiation of body tissues and cells using a micro-spot atmospheric pressure plasma source. The device is a coaxial structure having a tungsten wire installed inside a glass capillary, and a grounded ring electrode wrapped on the outside. The conditions of plasma generation are as follows: applied voltage: 5-9 kV, frequency: 1-3 kHz, helium (He) gas flow: 1-1.5 L/min, and plasma irradiation time: 1-300 sec. The experiment was conducted by preparing a culture medium containing mouse fibroblasts (NIH3T3) on a culture dish. A culture dish irradiated with plasma was introduced into a $CO_2$-incubator. The small animals used in the experiment involving plasma irradiation into living tissue were rat, rabbit, and pick and are deeply anesthetized with the gas anesthesia. According to the dependency of cell numbers against the plasma irradiation time, when only He gas was flowed, the growth of cells was inhibited as the floatation of cells caused by gas agitation inside the culture was promoted. On the other hand, there was no floatation of cells and healthy growth was observed when plasma was irradiated. Furthermore, in an experiment testing the effects of plasma irradiation on rats that were artificially given burn wounds, no evidence of electric shock injuries was found in the irradiated areas. In fact, the observed evidence of healing and improvements of the burn wounds suggested the presence of healing effects due to the growth factors in the tissues. Therefore, it appears that the interaction due to ion/radicalcollisions causes a substantial effect on the proliferation of growth factors such as epidermal growth factor (EGF), nerve growth factor (NGF), and transforming growth factor (TGF) that are present in the cells.

• The Journal of Pediatrics of Korean Medicine
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• v.21 no.3
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• pp.33-55
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• 2007

Objectives In the present study, the author intended to investigate whether bojung-ikgitang-gamibang(BJGB) and seonbang-paedoktang(SBPT) significantly affect in vivo and in vitro mucin secretion from airway epithelial cells. Methods In vivo experiment, mice's mucin which is on a hypersecretion of airway mucin, mice's tracheal goblet cells in hyperplasia and mice's intraepithelial mucosubstances were exposed with SO2for3weeks. Effects of orally-administered BJGB and SBPT during 1 week on vivo mucin secretion and hyperplasia of tracheal goblet cells were assessed by using both enzyme-linked immunosorbent assay(ELISA) and staining goblet cells with alcian blue. In vitro experiment, confluent hamster tracheal surface epithelial(HTSE) cells were metabolically radiolabeled with 3H-glucosamine for 24hrs and chased for 30 min in the presence of each agent to figure out the effectiveness of 3H-mucin secretion. Total elution profiles of control spent media and treatment sample through Sepharose CL-4B column were analyzed. The effects of each agent on contractility of isolated tracheal smooth muscle and effects of each agent on MUC5AC gene expression in cultured HTSE cells were investigated. Also, possible cytotoxicities of each agent were assessed by measuring lactate dehydrogenase(LDH) release. Additionally, effects of BJGB and SBPT on both MUC5AC gene expression in cultured HTSE cells and TNF- or EGF-induced MUC5AC gene expression in human airway epithelial cells (NCI-H292) were investigated. Results (1) BJGB and SBPT inhibited hypersecretion of in vivo mucin. SBPT also inhibited the increase the number of goblet cells. However, BJGB did not affect the increase of number of goblet cells; (2) BJGB significantly increased mucin secretion from cultured HTSE cells, without significant cytotoxicity, and chiefly affected the 'mucin' secretion; (3) SBPT did not affect mucin secretion from cultured HTSE cells without significant cytotoxicity, and also did not affect the secretion of the other releseable glycoproteins; (4) BJGB and SBPT did not affect Ach-induced contraction of isolated tracheal smooth muscle; (5) SBPT significantly inhibit the expression levels of MUC5AC gene and BJGB significantly increased the expression levels of MUC5AC gene in both HTSE cells and NCI-H292 cells. Conclusions BJGB and SBPT can not only affect the secretion of mucin but also affect the expression of mucin gene. The author suggests that the effects BJGB and SBPT with their components should be further investigated and it is highly desirable to find from oriental medical prescriptions, novel agents which might regulate hypersecretion of mucin from airway epithelial cells.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.124-124
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• 2003

The successful development of embryos cloned by nuclear transfer (NT)have been dependent on a wide range of known factors including cell cycle of donor and recipient ooplast, oocyte quality, NT procedure and oocyte activation. The present study compared the development of cloned porcine embryos following different activation treatments. Cumulus-oocyte complexes (COCs) were aspirated from 26 mm follicles of slaughterhouse ovaries and cultured for 22 h in NCSU #23 medium supplemented with 10% porcine follicular fluid, 0.57 mM cysteine, 0.5 g/mL LH, 0.5 g/mL FSH and 10 ng/mL EGF. The COCs were further cultured for an additional 22 h in the same medium at $39{\cird}C$ in an atmosphere of 5% $CO_2$ in air, without hormonal supplements. Primary cultures of fibroblasts isolated from a female fetus on day 40 of gestation were established in DMEM + 15% FCS. For nuclear donation, cells at the 5th-6th passage were cultured in DMEM +0.5% FCS for 5 days in order to arrest the cells in G0/Gl. After enucleation, oocytes were reconstructed by transfer of donor cells and fusion with three DC pulses (1.4 KV/cm, 30 sec) in 0.28 M mannitol containing 0.01 mM $CaCl_2$ and 0.01 mM $MgCl_2$. Eggs were then divided into three treatment groups, control (without further treatment, Group 1), eggs cultured in 10 g/ml cycloheximide (CHX) for 5 h (Group 2), and eggs cultured in 1.9 mM 6-dimethylaminopurine (6-DMAP) for 5 h (Group 3). The eggs were then cultured in sets of 30 in 60 I drops of NCSU#23 supplemented with 4mg/ml BSA (essentially fatty acid free) until day 7 at $39{\circ}C$ in a humidified atmosphere of 5% $CO_2$. On day 4 the culture were fed by adding 20 I NCSU #23 supplemented with 10% FBS. Development rates into blastocysts were significantly higher (P<0.05) in Group 3 embryos compared to Group 1 controls ($27.6 \mu 2.7% vs. 20.1 \mu 4.1%$, respectively), but rates did not differ in Group 2 compared to control ($23.8 \mu 5.7%$). Total cell number in Group 3 blastocysts was however significantly higher (P<0.05) than in Groups 1 and 2 ($44.6 \mu 2.4 vs. 19.9 \mu 1.9 and 21.9 \mu 2.1$, respectively). These results suggest that 6-DMAP is more efficient than cycloheximide in the activation of electrically fused NT oocytes during in vitro production of cloned porcine embryos.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.62-62
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• 2003

This study was carried out to investigate the effects of activation agents on parthenogenetic activation of pig oocytes matured in vitro. The medium used for oocyte maturation was tissue culture medium (TCM) 199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin $B_{l2}$, 25 mM Hepes, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Cumulus-free oocytes involving first polar body were activated by exposure to various concentrations of ethanol and exposure time of ethanol in Hepes-buffered NCSU23 medium. Also, oocytes were activated by electric pulse alone or combination with ethanol. For electrical activation, oocytes were rinsed twice in 0.3 M mannitol solution supplemented with 0.1 mM CaC1$_2$, 0.2 mM MgC1$_2$, 0.5 mM Hopes and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1 mm apart which was overlaid with the same activation solution. Oocytes were activated with a single DC pulse of 1.3 ㎸/cm for 30 $\mu$sec. After activation treatments, oocytes were washed three times with Hepes-buffered NCSU23 medium and were washed twice with NCSU23 culture medium containing 0.4% BSA, and then cultured in 500 ${mu}ell$ of the same medium for 20 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. The activation rates of oocytes were higher in 6, 7 and 8% ethanol concentrations compared with 0, 5, 9 and 10% ethanol concentrations. Significantly more oocytes (29.3~33.7%) were activated in the exposure for 8, 10, 12 and 15 min than those in the exposure for 0 and 5 min, but there was no difference due to exposure to 8% ethanol for 8 to 15 min. Electric pulse treatment followed by exposure to ethanol significantly improved the rate of oocyte activation (61.9%) compared with that of other 3 treatments. In conclusion, the optimal activation treatment of ethanol exposure alone for the in-vitro matured pig oocytes was 8% ethanol for 8 to 15 min. Electric pulse treatment followed by ethanol exposure significantly improved the rate of activation.n.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.265-274
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• 2009

Objective: MicroRNAs (miR) are known to repress target genes at post-transcriptional level and play important roles in development and maturation of cell. However, the expression profiles of miR during ovarian follicle maturation have not been fully elucidated. Here, we designed this study to investigate the expression profiles of miR in oocytes and granulose cells (G-cells) after in vitro culture according to gonadotropins and adding hCG. Methods: Ovaries from 12-day-old mice (C57BL6) were removed and preantral follicles were isolated and cultured in $20\;{\mu}L$-drop of culture media with supplementation of either rFSH, rLH, or rFSH+rLH. After their full maturation, follicles were incubated with rhCG and rEGF. RNA was isolated from oocytes and G-cells, and real-time PCR were performed with primers of miR known to be expressed in the mouse ovary (mmu-miR-16, -miR-27a, -miR-126, -miR-721). Results: FSH+LH group showed the highest ovulation and MII rates among gonadotropin groups. The profiles of miRs in oocytes and G-cells differed according to gonadotropin groups and adding hCG. The profiles of miRs showed divergent changes between oocytes and G-cells. Conclusion: miR expression profiles are altered by gonadotropins and supplementation of hCG during in vitro maturation of murine follicles. Target gene study must be necessary to validate these findings.