• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.104-104
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• 2003

As an effort to direct differentiation of human embryonic stem (hES, MB03) cells to dopamine-producing neuronal cells, Nurr1 was transfected using conventional transfection protocol into MB03 and examined the expression of tyrosine hydroylase (TH) after differentiation induced by retinoic acid (RA) and ascorbic acid (AA). Experimentally, cells were transfected with linearized Nurr1 cDNA in pcDNA3.1 (+)-hygovernight followed by selection in medium containing hygromycin-B (150 $\mu$/ml). Expression of Nurr1 mRNA was confirmed by RT-PCR and protein by immunocytochemistry in the drug resistant clones. In order to study the effect of Nurr1 protein on the differentiation pattern of ES cells, one of the positive clones (MBNr24) was allowed to form embryoid body (EB) for 2 days and were induced to differentiate for another 4 days using RA (1 $\mu M$) and AA (50 mM) (2-/4+ protocol) followed by selection in N2 medium for 10 or 20 days. After 10 days in N2 medium, cells immunoreactive to anti-GFAP, anti-TH, or anti-NF200 antibodies were 38.8%, 11%, and 20.5%, respectively. After 20 days in N2 medium, cells expressing GFAP, TH, or NF200 were 28%, 15% and 44.8%, respectively but approximately 9% of MB03 expressed TH protein when the cells were induced to differentiate using a similar prorocol, These results suggest that ectopic expression of Nurr1 enhances generation of TH+ cells as well as neuronal cells when hES cells were differentiated by 2-/4+ protocol.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.12
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• pp.1721-1728
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• 2015

Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (-420/+181) bovine NANOG 5'-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (-420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.189-192
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• 2012

Although embryonic stem cells (ESCs) or ES-like cells are reported from many mammalian species other than the mouse, the culture system for murine ESCs may not be suitable to the other species. Previously many other research groups have modified either human or mouse ESC culture systems for bovine ESC culture. In this study, we compared three different culture mediums consisting of DMEM, ${\alpha}$-MEM or KnockOut$^{TM}$-DMEM (KO), which are modified from human or mouse ESC culture system, for the generation of bovine ESCs. In this study, some pre-requisite events which are important for establishment and long-term propagation of ESCs such as inner cell mass (ICM) attachment on feeder cells, primary colony formation and sustainability after passaging. Once the ICM clumps attached on feeder cells, this was designated as passage 0. In regards to the rate of ICM attachment, ${\alpha}$-MEM was superior to the other systems. For primary colony formation, there was no difference between DMEM and ${\alpha}$-MEM whereas KO showed lower formation rate than the other groups. For passaging, the colonies were split into 2~4 pieces and passed every 5~6 days. From passage 1 to passage 3, DMEM system seemed to be appropriate for maintaining putative bovine ESCs. On the other hand, ${\alpha}$-MEM tended to be more suitable after passage 6. Although ${\alpha}$-MEM support to maintain a ES-like cell progenies to passage 15, all three culture systems which are modified from human or mouse ESC culture media failed to retain the propagation and long-term culture of putative bovine ESCs. Our findings imply that more optimized alternative culture system is required for establishing bovine ESC lines.

• Development and Reproduction
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• v.20 no.1
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• pp.63-71
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• 2016

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KO-SR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xeno-free conditions for clinical grade hESCs culture will be useful data in future clinical studies.

• BMB Reports
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• v.48 no.5
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• pp.256-265
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• 2015

Cardiovascular and neurodegenerative diseases are major health threats in many developed countries. Recently, target tissues derived from human embryonic stem (hES) cells and induced pluripotent stem cells (iPSCs), such as cardiomyocytes (CMs) or neurons, have been actively mobilized for drug screening. Knowledge of drug toxicity and efficacy obtained using stem cell-derived tissues could parallel that obtained from human trials. Furthermore, iPSC disease models could be advantageous in the development of personalized medicine in various parts of disease sectors. To obtain the maximum benefit from iPSCs in disease modeling, researchers are now focusing on aging, maturation, and metabolism to recapitulate the pathological features seen in patients. Compared to pediatric disease modeling, adult-onset disease modeling with iPSCs requires proper maturation for full manifestation of pathological features. Herein, the success of iPSC technology, focusing on patient-specific drug treatment, maturation-based disease modeling, and alternative approaches to compensate for the current limitations of patient iPSC modeling, will be further discussed. [BMB Reports 2015; 48(5): 256-265]

• Anatomy and Cell Biology
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• v.56 no.2
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• pp.179-184
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• 2023

The Functional endoscopic sinus surgery through transnasal approach is a common modality of treatment for disorders of the nasal cavity, paranasal air sinuses as well as cranial cavity. The olfactory fossa (OF) is located along the superior aspect of cribriform plate which varies in shape and depth. This variable measurement of the depth of OF is mostly responsible for greater risk of intracranial infiltration during endoscopic procedures in and around the nasal cavity. The morphology of frontal and ethmoid sinus (ES) vary from simple to complex. This cadaveric study is planned to improve the ability of the otolaryngologist, radiologist to understand the possible morphological variations and plan steps of less invasive "precision surgery" to have a safe and complication free procedures. A total of 37 human head regions were included in the study. For classification of OF, Modified Kero's classification was used. The size, shape and cells of frontal and ES were noted. We found, type II (60.8%) OF was more common followed by type I (29.7%) than type III (9.5%). The shape of frontal sinus was comma shaped (55.4%) followed by oval (18.9%) than irregular (16.2%). Most common two cells type of ES was seen in 50.0% of both anterior and posterior ES. Out of 74 ES, 8.1% of Onodi cells and 14.9% of agger nasi cells were seen.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.1-8
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• 2011

Techniques to evaluate gene expression profiling, such as sufficiently sensitive cDNA microarrays or real-time quantitative PCR, are efficient methods for monitoring human pluripotent stem cell (hESC/iPSC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turn-around time, and the involvement of highly specialized technical expertise. Hence, there is an urgency of rapid, cost-effective, robust, yet sensitive method development for routine screening of hESCs/hiPSCs. A critical requirement in hESC/hiPSC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all three germ layers, including ectoderm, mesoderm, and endoderm. To quantify the modulation of gene expression in hESCs/hiPSC during their propagation, expansion, and differentiation via embryoid body (EB) formation, we developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR platform technology. Among the 9 gene primers tested, 5 were pluripotent markers comprising set 1, and 3 lineage-specific markers were combined as set 2, respectively. We found that these 2 sets were not only effective in determining the relative differentiation in hESCs/hiPSCs, but were easily reproducible. In this study, we used the hES/hiPS cell lines to standardize the technique. This multiplex RT-PCR assay is flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC/hiPSC lines during routine maintenance and directed differentiation.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.95-95
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• 2002

Human embryonic stem (hES) cells can be induced to differentiate into tyrosine hydroxylase expressing (TH+) cells that may serve as an alternative for cell replacement therapy for Parkinson's disease (PD). To examine in vitro differentiation of hES (MB03, registered in NIH) cells into TH+ cells, hES cells were induced to differentiate according to the 4-/4+ protocol using retinoic acid (RA), ascorbic acid (AA), and/or lithium chloride (LiCl) followed by culture in N2 medium for 14 days, during which time the differentiation occurs. Immunocytochemical stainings of the cells revealed that approximately 21.1% of cells treated with RA plus AA expressed TH protein that is higher than the ratio of TH+ cells seen in any other treatment groups (RA, RA+LiCl or RA+AA+LiCl). In order to see the differentiation pattern in vivo and the ability of in vitro differentiation-induced cells in easing symptomatic motor function of PD animal model, cells (2 $\times$ 10$^{5}$ cells/2${mu}ell$) undergone 4-/4+ protocol using RA plus AA without any further treatment were transplanted into unilateral striatum of MPTP-lesioned PD animal model (C57BL/6). Following the surgery, motor behavior of the animals was examined by measuring the retention time on an accelerating rotar-rod far next 10 weeks. No significant differences in retention time of the animals were noticed until 2 weeks post-graft; however, it increased markedly at 6 weeks and 10 weeks time point after the surgery. Immunohistochemical studies confirmed that a reasonable number of TH+ cells were found at the graft site as well as other remote sites, showing the migrating nature of embryonic stem cells. These results suggest that in viかo differentiated hES cells relieve symptomatic motor behavior of PD animal model and should be considered as a promising alternative for the treatment of PD.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.1
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• pp.26-34
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• 2009

Porcine embryonic stem (ES) cells have a great potential as tools for transgenic animal production and studies of regulation of differentiation genes. Although several studies showed successful derivation of porcine ES-like cells, these cells were not maintained long-term in culture. Therefore, this study was conducted to establish porcine pluripotent ES-like cells using in vivo fertilized embryos and to maintain these cells in long term culture. Porcine ES-like cells from in vivo embryos obtained by immunosurgery or whole explant culture were successfully cultured for over 56 passages. Morphology of porcine ES-like cells was flat-shaped with a monolayer type colony. These cells stained for alkaline phosphatase throughout the culture. Furthermore, porcine ES-like cells reacted with antibodies against Oct-4, SSEA-1, SSEA-4, Tra-1-60, and Tra-1-81, which are typical markers of undifferentiated stem cells. To characterize the ability of porcine ES-like cells to differentiate into three germ layers, embryoid body formation was induced. After plating of these cells, porcine ES-like cells were spontaneously differentiated into various cell types of all three germ layers. In addition, porcine ES-like cells were successfully derived from IVF blastocysts in media containing human recombinant basic fibroblast growth factor.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.75-75
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• 2003

Since the establishment of embryonic stem cell, pluripotency of the cells was known to allow differentiation of the cells into various cell types consisting whole body. Several protocols have been developed to induce expression of specific genes.. However, no precise protocol that will generate a single type of the cells from stem cells has been reported. In order to produce cells suitable for transplantion into brain of PD animal model, which arouse due to a progressive degeneration of dopaminergic neurons in midbrain, human embryonic stem cell (hESC, MB03) was transfected with cDNAs cording for tyrosine hydroxylase (TH). Successful transfection was confirmed by western immunoblotting. Newly transfected cell line (TH#2/MB03) was induced to differentiate by the two neurogenic factors retinoic acid (RA) and b-FGF. Exp. I) Upon differentiation using RA/ascorbic acid (AA), embryoid bodies (EB, for 4days) derived from hES cells were exposed to RA (10$^{-6}$ M)/AA (50 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14, 21, or 28 days. Exp. II) When bFGF was used, neuronal precursor cells were selected for 8 days in N2 medium after EB formation. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14, 21 or 28 days. By indirect immunocytochemical studies, proportion of cells expressing NF200 increased rapidly from 20% at 7 days to 70 % at 28 days in RA/AA-treated group, while those cells expressing NF160 decreased from 80% at 7 days to 10% at 28 days upon differentiation in N2 medium. However, in differentiation by RA/AA treatment system, there was a significant increase in proportion of neuron maturity (73%) at day 14 after N2 medium. TH#2/MB03 cells expressing TH are >90% when matured at the absence of either bDNF or TGF-$\alpha$. These results suggested that TH#2/MB03 cells could be differentiated in vitro into mature neurons by RA/AA.