• Molecules and Cells
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• v.41 no.7
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• pp.631-638
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• 2018

Spermatogonial stem cells (SSCs) derived from mouse testis are unipotent in regard of spermatogenesis. Our previous study demonstrated that SSCs can be fully reprogrammed into pluripotent stem cells, so called germline-derived pluripotent stem cells (gPS cells), on feeder cells (mouse embryonic fibroblasts), which supports SSC proliferation and induction of pluripotency. Because of an uncontrollable microenvironment caused by interactions with feeder cells, feeder-based SSC reprogramming is not suitable for elucidation of the self-reprogramming mechanism by which SSCs are converted into pluripotent stem cells. Recently, we have established a Matrigel-based SSC expansion culture system that allows longterm SSC proliferation without mouse embryonic fibroblast support. In this study, we developed a new feeder-free SSC self-reprogramming protocol based on the Matrigel-based culture system. The gPS cells generated using a feeder-free reprogramming system showed pluripotency at the molecular and cellular levels. The differentiation potential of gPS cells was confirmed in vitro and in vivo. Our study shows for the first time that the induction of SSC pluripotency can be achieved without feeder cells. The newly developed feeder-free self-reprogramming system could be a useful tool to reveal the mechanism by which unipotent cells are self-reprogrammed into pluripotent stem cells.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 1997.06a
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• pp.51-72
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• 1997

Diapause hormone (DH) is a neuropeptide hormone which is secreted from the suboesophageal ganglion (SG) and is responsible for induction of embryonic diapause of the silkworm, Bombyx mori. DH is isolated from SGs and determined to be a 24 amino acid peptide amide. The cDNA encodes the polyprotein precursor from which DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides are released and become matured. The C-terminal FXPRL-NH2 sequence of DH is essential but not sufficient for expression of full activity. Recently, we have isolated a unique hydrohobic peptide (VAP peptide) with a slight diapause egg induceing activity from organic solvent extracts of the male adult heads of the silkworm. The VAP peptide itself has no diapause inducing activity, but enhances DH activity through reducing ED50 value and the threshold concentration of DH. The DH-PBAN gene is composed of 6 exons interrupted by 5 introns and is expressed in 12 neurosecretory cells of the SG. The incubation of eggs at 25$^{\circ}C$, which induces embryonic diapause in the progeny, caused DH-PBAN mRNA content to increase at 5 different stages in the life cycle. By contrast, a 15$^{\circ}C$ incubation only induced expression of the gene at the late phrase adult stage. The temperature-controlled expression of DH-PBAN gene is closely correlated to the incidence of diapause, indicating that DH-PBAN gene expression is the initial event leading to diapause induction. DH acts to stimulate trehalase activity in developing ovary to bring about hyprglycogenism in mature eggs, a prerequisite metabolism for diapause initiation. Using in vivo and in vitro systems, DH is clearly shown to induce trehalase gene expression in developing ovaries. New protein synthesis is not needed for this process, but a Ca2+-dependent proteinkinase seems to be involved. Quite recently, we have sucessfully applied a new and potent trehalase inhibitor (Trehazoline) to reudce glycogen content in developing ovaries. The eggs deficient in glycogen were also able to enter diapause as the natural eggs do, so that we could provide the new egg system to reconsider the diapause associated metabolism other than the glycogen-sorbitol metabolic system.

• BMB Reports
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• v.45 no.9
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• pp.496-508
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• 2012

Fas-associated protein with death domain (FADD), an adaptor that bridges death receptor signaling to the caspase cascade, is indispensible for the induction of extrinsic apoptotic cell death. Interest in the non-apoptotic function of FADD has greatly increased due to evidence that FADD-deficient mice or dominant-negative FADD transgenic mice result in embryonic lethality and an immune defect without showing apoptotic features. Numerous studies have suggested that FADD regulates cell cycle progression, proliferation, and autophagy, affecting these phenomena. Recently, programmed necrosis, also called necroptosis, was shown to be a key mechanism that induces embryonic lethality and an immune defect. Supporting these findings, FADD was shown to be involved in various necroptosis models. In this review, we summarize the mechanism of extrinsic apoptosis and necroptosis, and discuss the in vivo and in vitro roles of FADD in necroptosis induced by various stimuli.

• International Journal of Oral Biology
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• v.37 no.4
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• pp.175-180
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• 2012

Skin-derived precursor cells (SKPs) are multipotent, sphere-forming and embryonic neural crest-related precursor cells that can be isolated from dermis. It is known that the properties of porcine SKPs can be enhanced by leukemia inhibitory factor (LIF) which is an essential factor for the generation of embryonic stem cells in mice. In our present study, to enhance or maintain the properties of murine SKPs, LIF was added to the culture medium. SKPs were treated with 1,000 IU LIF for 72 hours after passage 3. Quantitative real time RT-PCR was then performed to quantify the expression of the pluripotent stem cell specific genes Oct4, Nanog, Klf4 and c-Myc, and the neural crest specific genes Snai2 and Ngfr. The results show that the expression of Oct4 is increased in murine SKPs by LIF treatment whereas the level of Ngfr is decreased under these conditions. Interestingly, LIF treatment reduced Nanog expression which is also important for cell proliferation in adult stem cells and for osteogenic induction in mesenchymal stem cells. These findings implicate LIF in the maintenance of stemness in SKPs through the suppression of lineage differentiation and in part through the control of cell proliferation.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.102-102
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• 2003

Embryonic stem (ES) cells proliferate extensively in the undifferentiated state and have the potential to differentiate into a variety of cell types in response to various environmental cues. The generation of functional dopaminergic neurons from ES cells is promising for cell replacement therapy to treat Parkinson's disease. We compared the in vitro differentiation potential of pluripotent human embryonic stem (hES, MB03) cells induced with basic fibroblast growth factor (bFGF) or retinoic acid (RA). Both types of treatment resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)- $\alpha$ during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression, compared to control (P<0.05). In contrast, no effect was observed on the rate of mature or glutamic acid decarboxylase-positive neurons. Immunostaining and HPLC analyses revealed the higher levels of TH (20.3%) and dopamine in bFGF and TGF-$\alpha$ treated hES cells than in RA or BDNF treated hES cells. The results indicate that TGF-$\alpha$ may be successfully used in the bFGF induction protocol to yield higher numbers of functional dopaminergic neurons from hES cells.

• Journal of Industrial Convergence
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• v.17 no.4
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• pp.39-43
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• 2019

Mouse embryonic stem cell could show an substitutional materials of cells of neuron differentiation, positively increasing their effectiveness in the treatment of nervous symptom. We examined that mouse embryonic stem cells (mESCs) can be induced to undergo neuronal differentiation. After neuronal induction, the phenotype of mESCs changed towards neuronal morphology and mESCs were injected into the lateral ventricle of the experimental animal brain. Transplanted cells migrated to various parts of the brain and ischemic brain injury by middle cerebral artery occlusion (MCAO) increased their migration to the injured cortex. Intracerebral grafting of mESCs mostly improve sensory and motor nervous system of neurological injury in focal cerebral rats.

• Development and Reproduction
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• v.6 no.1
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• pp.55-66
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• 2002

Embryonic stem cells(ES cells) are derived from the inner cell mass(ICM) of blastocysts, which have the potentials to remain undifferentiated, to proliferate indefinitely in vitro, to differentiate into the derivates of three embryonic germ layers. ES cells are an attractive model system for studying the initial developmental decisions and their molecular mechanisms during embryogenesis. Additionally, ES cells of significant interest to those characterizing the various gene functions utilizing transgenic and gene targeting techniques. We investigated the effects of reproductive hormones, gonadotropins(GTH) and steroids on the induction of differentiation and expressions of their receptor genes using the newly established mouse ES cells. We collected the matured blastocysts of inbred mice C57BL/6J after superovulation and co-cultured with mitotically inactivated STO feeder cells. After 5 passages, we confirmed the expression alkaline phosphatase(Alk P) activity and SSEA-1, 3, 4 expressions. The protocol devised for inducing ES differentiation consisted of an aggregation steps, after 5 days as EBs in hormone treatments(FSH, LH, E$_2$, P$_4$, T) that allows complex signaling to occur between the cells and a dissociation step, induced differentiation through attachment culture during 7 days in hormone treatments. Hormone receptors were not increased in dose-dependent manner. All hormone receptors in ES cells treated reproductive hormones were expressed lower than those of undifferentiated ES cell except for LHR expression in E$_2$-treated ES cells group. After hormone induced differentiation, at least some of the cells are not terminally differentiated, as is evident from the expression of Oct-4, a marker of undifferentiated. To assess their differentiation by gene expression, we analyzed the expression of 7 tissue-specific markers from all three germ layers. Most of hormone-treated group increased in the expression of gata-4 and $\alpha$ -fetoprotein, suggesting reproductive hormone allowed or induced differentiation of endoderm.

• Korean Journal of Ichthyology
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• v.28 no.4
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• pp.260-266
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• 2016

We determined the morphologic characteristics (body weight and abdomen inflation degree) of the mother fish of sevenband grouper, Epinephelus septemfasciatus producing the healthy eggs. Experimental fish were chosen from the reared fish in the sea cage for 6 years. The fish were divided into four size groups by body weight: 4.0~5.0, 5.0~6.0, and 6.0~7.0 kg and four stages (I~IV) by the abdomen inflation degree. After hormone treatment, we observed the ovulation amount of induced eggs, rate of buoyant, fertilization, embryonic survival, and hatching. Egg and oil globule diameter was measured. In order to observe gonadal development, we calculated gonadosomatic index (GSI) and conducted its historical analysis. The ovulation occurred from all experimental fishes over 5.0 kg. The rate of buoyant, fertilization, and embryonic survival was the highest in 6.0~7.0 kg. Hatching rate was the highest in 5.0~6.0 kg. Stage I and II did not induce ovulation. GSI was $0.31{\pm}0.10%$ in stage I, $0.74{\pm}0.25%$ in stage II, $4.68{\pm}0.40%$ in stage III and $6.86{\pm}0.12%$ in stage IV. The rate of buoyant, fertilization, embryonic survival and hatching was the highest in stage III.

• Molecules and Cells
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• v.37 no.10
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• pp.705-712
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• 2014

The early steps of neural development in the vertebrate embryo are regulated by sets of transcription factors that control the induction of proliferative, pluripotent neural precursors, the expansion of neural plate stem cells, and their transition to differentiating neural progenitors. These early events are critical for producing a pool of multipotent cells capable of giving rise to the multitude of neurons and glia that form the central nervous system. In this review we summarize findings from gain- and loss-of-function studies in embryos that detail the gene regulatory network responsible for these early events. We discuss whether this information is likely to be similar in mammalian embryonic and induced pluripotent stem cells that are cultured according to protocols designed to produce neurons. The similarities and differences between the embryo and stem cells may provide important guidance to stem cell protocols designed to create immature neural cells for therapeutic uses.

• BMB Reports
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• v.49 no.7
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• pp.370-375
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• 2016

Ras oncoproteins are small molecular weight GTPases known for their involvement in oncogenesis, which operate in a complex signaling network with multiple effectors. Approximately 25% of human tumors possess mutations in a member of this family. The Raf1/MEK/Erk1/2 pathway is one of the most intensively studied signaling mechanisms. Different levels of regulation account for the inactivation of MAP kinases by MAPK phosphatases in a cell type- and stimuli-dependent manner. In the present study, using three inducible Ras-expressing NIH/3T3 cell lines, we demonstrated that MKP3 upregulation requires the activation of the Erk1/2 pathway, which correlates with the shutdown of this pathway. We also demonstrated, by applying pharmacological inhibitors and effector mutants of Ras, that induction of MKP3 at the protein level is positively regulated by the oncogenic Ras/Raf/MEK/Erk1/2 signaling pathway.