• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.209-215
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• 2004

Objective: Embryonic stem (ES) cells could be differentiated into the specific cell types by alternation of culture condition and modification of gene expression. This study was performed to evaluate the differentiation protocol for mouse and human ES cells to insulin secreting cells. Methods: Undifferentiated mouse (JH-I) and human (Miz-hESI) ES cells were cultured on STO feeder layer, and embryoid bodies (EBs) were formed by suspension culture. For the differentiation, EBs were cultured by sequential system with three stage protocol. The differentiating ES cells were collected and marker gene expressions were analyzed by seIni-quantitative RT-PCR in each stage. Amount of secreted insulin levels in culture media of human ES cells were measured by human insulin specific RIA kit. Results: During the differentiation process of human ES cells, GATA-4, a-fetoprotein, glucose transporter-2 and Ngn-3 expression were increased whereas OctA was decreased progressively. Insulin and albuInin mRNAs were expressed from stage IT in mouse ES cells and from stage III in human ES cells. We detected 3.0~7.9 IlU/rnl secretion of insulin from differentiated human ES cells by in vitro culture for 36 days. Conclusion: The sequential culture system could induce the differentiation of mouse and human ES cells into insulin secreting cells. This is the fIrst report of differentiation of human ES cells into insulin secreting cells by in vitro culture with serum and insulin free medium.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.22-22
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• 2001

To produce reconstituted rabbit embryos with fetal fibroblasts, the present study was evaluated the efficiencies of the different fusion and activation conditions as assessments of subsequent development and chromosome in the embryos. New Zealand White rabbits were used throughout the study. Fetal fibroblasts collected from 22-d of fetuses were cultured in DMEM ＋ 10% FBS in 5% $CO_2$ in air. The culture was maintained for 10 passages. In every passage half of cell suspension were kept In frozen. From rabbits treated with FSH in 30% PVP solution and hCG, oocytes were surgically collected from oviducts at 14 h post-hCG injection and stripped off their cumulus cells by re-pipetting in a 300 IU hyaluronidase solution. Oocytes with an extruded first polar body and dense cytoplasm were enucleated by micromanipulation in Ham's F-10 medium＋7.5 g/$m\ell$ cytochalasin B. Euncleation was confirmed under a fluorescence microscope after staining with 5 g/$m\ell$ bisbenzimide for 2 min. Each enucleated oocyte was injected with a fetal fibroblast into a perivitelline space. Reconstructed eggs were compared fusion rates either at 2.0 ㎸/cm or 1.6 ㎸/cm(60 sec, double pulses). After fusion, all eggs were activated with the combination of 5 M ionomycin (5 min) and 10 g/$m\ell$ cycloheximide (CHX, 3h), and cultured in CRlaa medium and transferred into TCM199＋10% FBS on day 3. Although there was not significantly differ in fusion rate between treatments (60%, 2.0 ㎸/cm vs. 79.4%, 1.6 ㎸/cm), none of them in the eggs fused with 2.0 ㎸/cm developed to blastocyst. In comparison of development and chromosome status between different activation treatments (Group 1; 5 M ionomycin/10 g/$m\ell$ CHX, Group 2; 5 M ionomycin/5 g/$m\ell$ CHX ＋ 2 mM DMAP after fusion with 1.6 ㎸/cm), there were not differ in cleavage and development rates (67.3% and 28.9% in Group 1; 67% and 33% in Group 2). All out of 8 embryos evaluated in Group 1 appeared a normal diploid chromosome sets and mean number of cells (Mean SEM) on day 4.5 of culture was 141.5 23.15 (n=8). It can be concluded that the use of cycloheximide has not happened in chromosome abnormalities, and fetal fibroblasts can be used for cloning in rabbit.

• Asian Journal of Turfgrass Science
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• v.18 no.3
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• pp.141-148
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• 2004

In this report, several factors such as infection time and concentration of bacterial suspension, influencing on transient gene expression in Agrobacterium-mediated transformation were evaluated. An appropriate concentration (O.D 600nm = 1.0-1.2) of bateria and 30 min of infection time showed a higher level of GUS expression. To improve transformation efficiency (TE), friable embryogenic calli (FEC) were bombarded by tungsten particles without plasmid DNA, and then co-cultivated with A. tumefaciens LBA4404 which contains pTOK233 super binary vector, carrying neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (hpt) and$\beta-glucuronidase$ (GUS) genes. Three days after co-cultivation with A. tumefaciens and particle bombardment, FEC cultures were transferred to the selection medium (SM: MS medium supplemented with BA 1mg/l, hygromycin 100mg/l, cefotaxime 250 mg/l and vancomycin 200mg/l). They were cultured for 2 weeks and then transferred to the second SM containing hygromycin 50mg/l, cefotaxime 200 mg/l and vancomycin 100mg/l. Later, stable GUS expression was detected 4 to 6 weeks after transfer to the SM. Further, TE from Agrobacterium-mediated transformation after particle bombardment increased to about 3-folds compared with Agrobacterium-mediated transformation without particle bombardment. In the present study, we established an efficient transformation protocol of zoysiagrass by using A. tumefaciens in the combination with particle bombardment for the first time.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.33-37
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• 2012

Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of $Tuj1$ increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous $Igf2$ may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.34 no.6
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• pp.384-390
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• 2012

Purpose: The purpose of this study is to examine in vivo osteogenesis of cultured human periosteal-derived cells and polydioxanone/pluronic F127 scaffold. Methods: Two one-year-old miniature pigs were used in this study. $2{\times}10^6$ periosteal-derived cells in 1 mL medium were seeded by dropping the cell suspension into the polydioxanone/pluronic F127 scaffold. These cell-scaffold constructs were cultured in osteogenic Dulbecco's modified Eagle's medium for 7 days. Under general anesthesia with azaperone and tiletamine-zolazepam, the mandibular body and ramus of the pigs were exposed. Three bony defects were created. Polydioxanone/pluronic F127 scaffold with periosteal-derived cells and the scaffold only were implanted into each defect. Another defect was left empty. Twelve weeks after implantation, the animals were sacrificed. Results: New bone formation was clearly observed in the polydioxanone/pluronic F127 scaffold with periosteal-derived cells. Newly generated bone was also observed in the scaffold without periosteal-derived osteoblasts and empty defect, but was mostly limited to the periphery. Conclusion: These results suggest that cultured human periosteal-derived cells have good osteogenic capacity in a polydioxanone/pluronic F127 scaffold, which provides a proper environment for the osteoblastic differentiation of these cells.

• KSBB Journal
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• v.22 no.2
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• pp.109-113
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• 2007

Suspension culture of recombinant Chinese hamster ovary (CHO) cells producing follicle-stimulating hormone was performed to investigate the effect of glycine betaine on cell growth and FSH production at low culture temperature. At 28$^{\circ}C$, cell growth was suppressed, but cell viability remained high for a longer culture period. When the culture temperature was lowered from 37$^{\circ}C$ to 28$^{\circ}C$, more than 14-fold increase in the maximum FSH titer was achieved. In batch culture at 28$^{\circ}C$, the use of 15 mM glycine betaine (GB) to culture medium resulted in the enhancement of maximum cell density and FSH titer by 11% and 17%, respectively, compared to the culture without GB. In pseudo-perfusion culture at 28$^{\circ}C$ with the exchange of fresh medium containing 15 mM GB, a final FSH of $2,058{\mu}g$ which is approximately 1.4-fold higher as compared to the culture without GB was obtained. This enhanced FSH production with 15 mM GB was not just because of enhanced specific FSH productivity (qFSH), but mainly because of the extended culture longevity. Taken together, this result demonstrates that the application of GB at low culture temperature is feasible to enhance the production of recombinant proteins in rCHO cells.

• Korean Journal of Agricultural Science
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• v.14 no.2
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• pp.197-204
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• 1987

In order to know the growth of suspended cells by explant sources, the change of nitrogen contents of cultured cells following the growth periods, capability of micro-callus formation according to cell plating methods, growth of suspended cells on various media, and efficiency of micro-callus formation by using growth regulators and different N strengths were investigated. 1. When suspension culture was tried by using the callus induced from internode and petiole, cell fresh weight and packed cell volume increased with similar way and the growth reached at stationary phase after 12 culture days. 2. N-contents of cultured cells increased upto 3 days and decreased around 6days. But the values increased again upto 9 days, after that they showed gradual decreases. 3. Of cell plating methods, embedding method was the best for micro-callus formation. 4. Growth of suspened cells showed the rest performanoes, when they were cultured on LM medium with 1/2N strengths and BAP 0.01.2.4-D 0.1, and NAA $1.0mg/{\ell}$, after 15 cultured days(upto 76.9 folds). LM medium was better than MS or GD. The combination of auxin and cytokinin was better for cell growing than auxin-treatment only. 5. Micro-callus from single cell and small cell aggregates was formed only on MS and LM media with 2,4-D $1.0mg/{\ell}$.

• KSBB Journal
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• v.19 no.6 s.89
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• pp.471-477
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• 2004

Oldenlandia diffusa is a Chinese medicinal herb with antitumor activity capable of suppressing the growth of some cancer cell lines. Oleanolic acid and ursolic acid are triterpenoid compounds that exist in Oldenlandia diffusa. Recently, these have been noted for anti-inflammatory, anti-cancer, and hepato-protective effects. Application of both plant growth regulators, 2,4-D and kinetin, was found to be essential for the initiation of callus and suspension cells. Leaf blades of Oldenlandia diffusa was transformed into callus on Schenk and Hildebrandt medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L kinetin, while optimum initiation condition for suspension cells of Oldenlandia diffusa was determined to be 0.75 mg/L 2,4-D and 0.1 mg/L kinetin. Chromatographic separation of oleanolic acid from its derivatives was achieved using Rexchrom S5-100-ODS column. Analytical conditions for oleanolic acid were determined as follows: flow rate at 1.0 mL/min, UV length at 200 nm and mobile phase of $80\%$ acetonitrile and $20\%$ water. Production of secondary metabolites was found to be increased by the treatment with elicitors or signal transducers. The maximum production of oleanolic acid was 99.6 mg/L in cultures with 0.5 mM salicylic acid. It is 1.74 times higher than that of control.

• Horticultural Science & Technology
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• v.35 no.2
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• pp.210-219
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• 2017

This study was conducted to establish an efficient screening method for radish (Raphanus sativus) cultivars that are resistant to black spot, which is caused by Alternaria brassicicola. Seven A. brassicicola isolates were selected and investigated for their ability to produce spores and pathogenicity. Of these isolates, A. brassicicola KACC 40036 and 43923 produced abundant spores in V-8 juice agar medium and showed pathogenicity and strong virulence on radish seedlings. We examined the resistance of 61 commercial cultivars of radish to A. brassicicola KACC40036, and found that there are no highly resistant radish cultivars; however, some cultivars, such as 'Geumbong' and 'Searom', showed weak resistance to A. brassicicola. For further study, we selected four radish cultivars that showed different disease responses to A. brassicicola KACC40036. According to the growth stage of the radish seedlings, inoculum concentration, and incubation temperature of radish, development of black spot on four cultivars has been investigated. The results showed that younger seedlings were more sensitive to A. brassicicola than older seedlings, and the disease severity depended on the concentration of the spore suspension. The disease severity of plants incubated in humidity chamber at $25^{\circ}C$ was greater than that of plants grown at $20^{\circ}C$ or $30^{\circ}C$. Taken together, we suggest the following method for screening for radish plants that are resistant to A. brassicicola: 1) inoculate 16-day-old radish seedlings with an A. brassicicola spore suspension ($2.0{\times}10^5spores{\cdot}mL^{-1}$) using the spray method, 2) incubate the inoculated plants in a humidity chamber at $25^{\circ}C$ for 24 h and then transfer the plants to a growth chamber at $25^{\circ}C$ with 80% relative humidity under a 12 h light/dark cycle, and 3) assess the disease severity of the plants two days after inoculation.

• Applied Chemistry for Engineering
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• v.31 no.1
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• pp.13-18
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• 2020

Iron oxide nanoparticles were microencapsulated using fibroin, a protein polymer of silk fiber, for theragnostic applications. The content of iron oxide was determined to be 4.28% by thermogravimetric analysis and 5.11% by magnetometer. A suspension of murine fibroblast 3T3 cells grown in medium supplemented with iron oxide-microcapsules turned clear in response to the magnetic force and the cells aggregated to the magnet direction. Neodymium magnets placed on the top of the culture dish, and attracted cells to the center of the culture surface. The cells collected on the culture surface aggregated to form a rough spheroid of 2 mm in a diameter after 72 h. In the outer layer of the cell aggregate, cells were relatively large and gathered together to form a dense tissue, but the central part was observed to undergo cell death due to the mass transfer restriction. In the outer layer, iron oxide-microcapsules were lined up like chains in the direction of magnetic force. Using microCT, it was demonstrated that the iron oxides inside the cell aggregate were not evenly distributed but biased to the magnetic direction.