• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.101-101
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• 2003

Main strategy for a treatment of Parkinson's disease (PD), due to a progressive degeneration of dopaminergic neurons, is a pharmaceutical supplement of dopamine derivatives or ceil replacement therapy. Both of these protocols have pros and cons; former exhibiting a dramatic relief but causing a severe side effects on long-term prescription and latter also having a proven effectiveness but having availability and ethical problems Embryonic stem (ES) cells have several characteristics suitable for this purpose. To investigate a possibility of using ES cells as a carrier of therapeutic gene(s), human ES (hES, MB03) cells were transfected with cDNAs coding for tyrosine hydroxylase (TH) in pcDNA3.1 (+) and the transfectants were selected using neomycin (250 $\mu /ml$). Expression of TH being confirmed, two of the positive clone (MBTH2 & 8) were second transfected with GTP cyclohydrolase 1 (GTPCH 1) in pcDNA3.1 (+)-hyg followed by selection with hygromycin-B (150 $\mu /ml$) and RT-PCR confirmation. By immune-cytochemistry, these genetically modified but undifferentiated dual drug-resistant cells were found to express few of the neuronal markers, such as NF200, $\beta$-tubulin, and MAP2 as well as astroglial marker GFAP. This results suggest that over-production of BH4 by ectopically expressed GTPCH I may be involved in the induction of those markers. Transplantation of the cells into striatum of 6-OHDA- denervated PD animal model relieved symptomatic rotational behaviors of the animals. Immunohistochemical analyses showed the presence of human cells within the striatum of the recipients. These results suggest a possibility of using hES cells as a carrier of therapeutic gene(s).

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.25-33
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• 2006

Objective: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. Methods: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific genes expressions with immunocytochemistry and RT-PCR. Results: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cell specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. Conclusion: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

• Journal of Microbiology and Biotechnology
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• v.22 no.12
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• pp.1790-1794
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• 2012

Recently, it has been suggested that $p27^{Kip1}$, the cell cycle regulatory protein, plays a pivotal role in the progression of normal differentiation in murine embryonic stem (mES) cells. In the current study, we investigated the role of $p27^{Kip1}$ in the regulation of differentiation and apoptotic induction using Western blotting, quantitative real-time RT-PCR, and small interfering RNA (siRNA) assays and confocal laser scanning microscopic analysis of H9 human ES (hES) cells and H9-derived embryoid bodies (EBs) grown for 10 ($EB_{10}$) and 20 days ($EB_{20}$). Our results demonstrate that the proteins $p27^{Kip1}$ and cyclin D3 are strongly associated with cellular differentiation, and, for the first time, show that up-regulation of $p27^{Kip1}$ protects hES cells from inducing differentiation-associated and caspase 3-dependent apoptosis.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.235-244
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• 1995

The present study was designed to demonstrate that ES cell lines efficiently could be isolated from explanted blastocysts of hybrid BCF1 mouse when grown on STO feeder layer derived from mouse fibroblasts in culture medium supplemented with leukemia inhibitory factor (LIF). The expanded blastocysts were attached to mitomycin C-inactivated STO feeder layer and were cultured for 4 days. Four days later the ICM was disaggregated by a short term trypsin treatment (0.25% trypsin / 0.04% EDT A for 2-3 min). The resulting cell suspension was seeded on a new STO feeder layer and covered with DMEM supplemented with 10% FCS, 0.1 mM nonessential amino acid, 0.1 mM sodium pyruvate, 0.1 mM mercaptoethanol and 1,000 U/ml LIF. Colonies of ES-like cells were observed after the second passage. These colonies were repeatedly passaged at approximately 5 day intervals. In this study, five ES-like celllines were isolated by directly explanting blastocysts, but three lines were lost after the 5th passage, possibly due to toxic effects of a new FCS batch. The characterization of developmental potential of isolated cell lines was performed with respect to in vitro differentiation and specific activity of alkaline phosphatase (AP). When cells were cultured in suspension, the aggregates of cell lines were capable of forming simple embryoid bodies (EB), and showed the capacity for forming cystic multilayer EBs. In addition, the cell lines were positive for AP staining, a biochemical marker characteristic of mouse ES cells.

• Development and Reproduction
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• v.12 no.1
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• pp.1-8
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• 2008

Specific protocols to increase the differentiation of neuronal cells from embryonic stem (ES) cells have been well established, such as retinoic acid induction and lineage selection of neuronal cells. For the neuropathological studies, ES-derived neurons (ES neurons) must show normal physiological characteristics related to cell death and survival and should be maintained in vitro for a sufficient time to show insults-specific cell death without spontaneous death. When mouse ES cells were plated onto astrocytes monolayer after retinoic acid induction, most ES cells differentiated into neuronal cells, which were confirmed by the presence of specific neuronal markers, and the cultures were viable for at least four weeks. When these cultures were examined for vulnerability to glutamate excitotoxicity, ES neurons were vulnerable to excitotoxic insults mediated by agonist-specific receptors. The vulnerability to excitotoxic death increased with developmental age of ES neurons in vitro. Specific receptors for Neurotrophin and GDNF family ligands were present in ES neurons. GDNF and NT-3 could modulate the survival and excitotoxic vulnerability of ES neurons. The vulnerability and resistance to toxic insults, which are essential requirements of model culture systems for neuropathological studies, make ES neurons to a useful model culture system. Especially ES cell are highly amenable to genetic modification unlikely to primary neuronal cells, which will give us a chance to answer more complicated neurophysiological questions. Recently there was an outstanding attempt to explore the cellular toxicity using human ES cells (Schrattenholz & Klemm, 2007) and it suggested that ES cells could be a new model system for neurophysiological studies soon and go further a large-scale screening system for pharmacological compounds in the future.

• Molecules and Cells
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• v.37 no.10
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• pp.742-746
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• 2014

The epithelial cell adhesion molecule (EpCAM, also known as CD326) is a transmembrane glycoprotein that is specifically detected in most adenocarcinomas and cancer stem cells. In this study, we performed a Cell systematic evolution of ligands by exponential enrichment (SELEX) experiment to isolate the aptamers against EpCAM. After seven round of Cell SELEX, we identified several aptamer candidates. Among the selected aptamers, EP166 specifically binds to cells expressing EpCAM with an equilibrium dissociation constant (Kd) in a micromolar range. On the other hand, it did not bind to negative control cells. Moreover, EP166 binds to J1ES cells, a mouse embryonic stem cell line. Therefore, the isolated aptamers against EpCAM could be used as a stem cell marker or in other applications in both stem cell and cancer studies.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.5
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• pp.587-594
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• 2000

At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.449-455
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• 2011

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by etopic expression of transcription factors. iPS cells are indistinguishable from ES cells in terms of morphology and stem cell marker expression. Moreover, mouse iPS cells give rise to chimeric mice that are competent for germline transmission. However, mice derived from iPS cells often develop tumors. Furthermore, the low efficiency of iPS cell generation is a big disadvantage for mechanistic studies. Nonviral plasmid.based vectors are free of many of the drawbacks that constrain viral vectors. The histone deacetylase inhibitor valproic acid (VPA) has been shown to improve the efficiency of mouse and human iPS cell generation, and vitamin C (Vc) accelerates gene expression changes and establishment of the fully reprogrammed state. The MEK inhibitor PD0325901 (Stemgent) has been shown to increase the efficiency of the reprogramming of human primary fibroblasts into iPS cells. In this report, we described the generation of mouse iPS cells devoid of exogenous DNA by the simple transient transfection of a nonviral vector carrying 2A-peptide-linked reprogramming factors. We used VPA, Vc, and the MEK inhibitor PD0325901 to increase the reprogramming efficiency. The reprogrammed somatic cells expressed pluripotency markers and formed EBs.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.