• 한국발생생물학회지:발생과생식
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• 제13권3호
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• pp.173-181
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• 2009

One of the most extensively studied populations of multipotent adult stem cells are mesenchymal stem cells (MSCs). MSCs derived from the human umbilical cord vein (HUC-MSCs) are morphologically and immunophenotypically similar to MSCs isolated from bone marrow. HUC-MSCs are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. Since neural tissue has limited intrinsic capacity of repair after injury, the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesenchymal-like stem cells from the human umbilical cord vein, and studied transdifferentiation-promoting conditions in neural cells. Dopaminergic neuronal differentiation of HUC-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulfoxide (DMSO) and butylated hydroxyanisole (BHA) in N2 medium and N2 supplement. The immunoreactive cells for $\beta$-tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. HUC-MSCs treated with bFGF, SHH and FGF8 were differentiated into dopaminergic neurons that were immunopositive for tyrosine hydroxylase (TH) antibody. HUC-MSCs treated with DMSO and BHA rapidly showed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including NeuroD1, $\beta$-tubulin III, GFAP and nestin was markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after neural differentiation, we confirmed the differentiation of dopaminergic neurons by TH/$\beta$-tubulin III positive cells. In conclusion, HUC-MSCs can be differentiated into dopaminergic neurons and these findings suggest that HUC-MSCs are alternative cell source of therapeutic treatment for neurodegenerative diseases.

• 대한수의학회지
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• 제60권4호
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• pp.203-207
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• 2020

This study presents neuronal migration pattern of dopamine (DA) neurons generated in separate regions occupying the ventral mesencephalic territory. A single pulse 5-bromodeoxyuridine (BrdU) was administered at embryonic day (E)10-E15. Distribution of tyrosine hydroxylase (TH) positive cells was determined at E13-postnatal day 0 (P0) by immunohistochemistry. BrdU positive cells labeled at E10 were spread out uniformly in the mesencephalon from E13 to E15, migrating through dorsal and ventral routes at E17 and P0. TH expression labeled at E10 was observed at E13 in the ventromedial region and clearly formed in the ventral tegmental area (VTA) at E15. At E17, TH expression in the substantia nigra (SN) was observed in the ventrolateral region, spreading more outward of the mesencephalon at P0. Generation of TH-positive cells labeled at E13 was also observed in VTA and SN of the mesencephalon at E17 and P0. The expression of these cells labeled after E15 was markedly decreased. These results demonstrated that an almost complete primary structure of DA neuron was formed at the early embryonic stage in the ventral mesencephalon, showing the most active neuronal migration was occurred at E13-E17.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.62-63
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• 2005

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.62-63
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• 2005

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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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.

• Journal of Korean Neurosurgical Society
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• 제38권6호
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• pp.456-464
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• 2005

Objective : The authors investigated whether rotenone induces cellular death also in non-dopaminergic neurons and high concentration of potassium ion can show protective effect for non-dopaminergic neuron in case of rotenone-induced cytotoxicity. Methods : Neuro 2A cells was treated with rotenone, and their survival as well as cell death mechanism was estimated using 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium[MTT] assay, Lactate dehydrogenase[LDH] release assay, fluorescence microscopy, and agarose gel electrophoresis. The changes in rotenone-treated cells was also studied after co-treatment of 50mM KCl. And the protective effect of KCl was evaluated by mitochondrial membrane potential assay and compared with the effects of various antioxidants. Results : Neuro 2A cells treated with rotenone underwent apoptotic death showing chromosome condensation and fragmentation as well as DNA laddering. Co-incubation of neuro 2A cells with 50mM KCl prevented it from the cytotoxicity induced by rotenone. Intracellular accumulation of reactive oxygen species[ROS] resulting by rotenone were significantly reduced by 50mM KCl. Potassium exhibited significantly similar potency compared to the antioxidants. Conclusion : The present findings showed that potassium attenuated rotenone-induced cytotoxicity, intracellular accumulation of ROS, and fragmentation of DNA in Neuro 2A cells. These findings suggest the therapeutic potential of potassium ion in neuronal apoptosis, but the practical application of high concentration of potassium ion remains to be settled.

• 한국안광학회지
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• 제5권2호
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• pp.15-20
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• 2000

성체 소의 망막에 존재하는 doparminergic 신경세포의 형태를 연구하였다. Dopaminerglc 세포는 항체면역세포화학적 방법을 이용하여 확인하였다. Tyrosine hydroxylase 면역 반응 신경 세포의 대부분은 내핵층의 가장 깊은 부분에 위치했다. 이들 세포들의 돌기들은 단층이었으며 그리고 내망상층의 1층내에 위치했다. Tyrosine-hydroxylase 면역 반응되는 두 번째 주요 세포 집단은 치환된 amacrine 세포들이다. 치환된 tyrosine-hydroxylase 면역 반응 amacrine 세포의 돌기들도 역시 내망상층의 1층내에 나타났다. 소수의 신경 세포의 돌기들은 외망상층에서 나타났다. 매우 낮은 밀도의 신경세포들은 내망상층의 중간과 깊은 층에서 tyrosine-hydroxylase 면역 반응 돌기들의 추가적인 층을 가졌다. Doparminergic 신경세포의 돌기들은 방사선형으로 넓게 신장되어 큰 모양을 형성하였고 수상돌기의 가지들은 적당하게 뻗어 있었다. 이러한 돌기들은 때때로 varicosity를 가지지만 "dendritic rings"을 형성하지는 않았다. 본연구의 결과는 doparminergic 세포는 소의 망막내 특이 신경세포 형태를 구성하는 것을 알 수 있었다.

• 대한본초학회지
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• 제23권3호
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• pp.53-60
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• 2008

Objectives : Oxidative stress has a critical role in neurodegenerative diseases. In this study, we investigated the antioxidant and neuroprotective effects of the ethanolic extract of Banryong-hwan (BRHE) in SH-SY5Y cells and primary rat mesencephalic dopaminergic neurons. Methods : To assess the antioxidant effects, we carried out 1,1-diphenyl-2-picrylhydrazyl(DPPH) free radical scavenging assay, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)(ABTS) radical cation decolorization assay, and determination of total polyphenolic content. We evaluated the effect of BRHE treatment on neuroprotection against 6-hydroxydopamine(6-OHDA) toxicity using thiazolyl blue tetrazolium bromide(MTT) assay, nitric oxide(NO) assay, reactive oxygen species(ROS) assay in SH-SY5Y cells and tyrosine hydroxylase(TH) immunocytochemistry in primary rat mesencephalic dopaminergic neurons. Results : BRHE showed IC50 values of 328.10 ${\mu}g/mL$ and 43.12 ${\mu}g/mL$ in DPPH assay and in ABTS assay, respectively. Total polyphenolic content was 180.76 ${\mu}g/mL$. In SH-SY5Y cells, BRHE significantly attenuated the toxicity induced by 6-OHDA at the concentrations of 25-100 ${\mu}g/mL$ pre- and post- treatment in MTT assay. While 6-OHDA increased the NO and ROS contents, BRHE decreased them in a dose dependent manner. Moreover, in primary dopaminergic neuron culture, BRHE significantly protect-ed the dopaminergic cell loss against 6-OHDA toxicity up to 136% at the concentration of 75 ${\mu}g/mL$. Conclusions : These results demonstrate that BRHE has neuroprotective effect against 6-OHDA induced neurotoxicity through decreasing NO and ROS generation.

• 한국발생생물학회지:발생과생식
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• 제12권1호
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• pp.31-39
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• 2008

손상된 뇌신경조직내에서 신경줄기세포로부터 새로운 신경세포로의 분화가 상당히 제한되어 있어 이것이 손상된 뇌신경조직의 복구가 잘 이루어지지 않는 원인이라 여겨지고 있다. 본 연구에서는 세포배양을 통해 지방조직 중간엽 줄기세포를 도파민성 신경세포와 콜린성 신경세포로 분화를 유도하였다. 중간엽 줄기세포를 신경세포로 분화시키기 위해 N2배양액에 bFGF, EGF, dimethyl sulphoxide (DMSO)와 butylated hydroxyanisole (BHA)를 첨가하여 유도하였다. DMSO와 BHA에 처리된 중간엽 줄기세포가 빠르게 신경세포 모양으로 분화하는 것을 관찰하였으며, 이것은 면역조직학적 염색에서 신경세포 특이 표지인 $\beta$-tubulin III, 별아교세포에 대한 특이 표지인 GFAP, 흰돌기아교세포에 대한 특이 표지인 Gal-C에 대해 양성반응을 나타내었다. RT-PCR 분석에서 배양 단계에 따라 신경세포에 특이적인 표지 인자인 neuro D1, $\beta$-tubulin III, GFAP, nestin 등의 발현을 통해, 중간엽 줄기세포가 신경세포로 분화됨을 확인하였다. 그러나 중간엽줄기세포가 신경세포로 분화된 이후에는 줄기세포 표지인 SCF, C-kit와 stat-3 등은 발현되지 않았다. 또한, 중간엽줄기세포에 bFGF, SHH와 FGF8 등을 처리하면 도파민 신경세포로 분화하였다. 중간엽 줄기세포에 bFGF, RA, Shh를 처리하여 콜린성 신경세포로 분화시켰을 때, 신경세포 특이 표지인 $\beta$-tubulin III와 콜린성 신경 특이 표지인 ChAT에 양성반응를 보였다. 결론적으로 사람 지방조직의 중간엽 줄기세포가 도파민성과 콜린성 신경세포로 분화가 가능하고 이러한 잠재성을 가진 지방 유래 중간엽 줄기세포는 퇴행성 신경질환에 대한 세포 치료제로서 가능성을 제시한다.

• Molecules and Cells
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• 제41권7호
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• pp.646-652
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• 2018

Neurodegeneration can result in memory loss in the central nervous system (CNS) and impairment of taste and smell in the peripheral nervous system (PNS). The neurodegeneration seen in Parkinson's disease (PD) is characterized by functional loss of dopaminergic neurons. Recent studies have also found a role for dopaminergic neurons in regulating taste memory rewards in insects. To investigate how taste memories and sugar sensitivity can be affected in PD, we utilized the $DJ-1{\beta}$ mutant fruit fly, $DJ-1{\beta}^{ex54}$, as a PD model. We performed binary choice feeding assays, electrophysiology and taste-mediated memory tests to explore the function of the $DJ-1{\beta}$ gene in terms of sugar sensitivity as well as associative taste memory. We found that PD flies exhibited an impaired ability to discriminate sucrose across a range of sugar concentrations, with normal responses at only very high concentrations of sugar. They also showed an impairment in associative taste memory. We highlight that the taste impairment and memory defect in $DJ-1{\beta}^{ex54}$ can be recovered by the expression of wild-type $DJ-1{\beta}$ gene in the dopaminergic neurons. We also emphasized the role of dopaminergic neurons in restoring taste memory function. This impaired memory property of $DJ-1{\beta}^{ex54}$ flies also allows them to be used as a model system for finding supplementary dietary foods that can improve memory function. Here we provide evidence that the associative taste memory of both control and $DJ-1{\beta}^{ex54}$ flies can be enhanced with dietary supplementation of the medicinal plant, omija.