• Clinical and Experimental Reproductive Medicine
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• 제39권2호
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• pp.87-93
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• 2012

Objective: Lin28 has been known to control the proliferation and pluripotency of embryonic stem cells. The purpose of this study was to determine the downstream effectors of Lin28 in mouse embryonic stem cells (mESCs) by RNA interference and microarray analysis. Methods: The control siRNA and Lin28 siRNA (Dharmacon) were transfected into mESCs. Total RNA was prepared from each type of transfected mESC and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to confirm the downregulation of Lin28. The RNAs were labeled and hybridized with an Affymetrix Gene-Chip Mouse Genome 430 2.0 array. The data analysis was accomplished by GenPlex 3.0 software. The expression levels of selected genes were confirmed by quantitative real-time RT-PCR. Results: According to the statistical analysis of the cDNA microarray, a total of 500 genes were altered in Lin28-downregulated mESCs (up-regulated, 384; down-regulated, 116). After differentially expressed gene filtering, 31 genes were selected as candidate genes regulated by Lin28 downregulation. Among them, neuropeptide Y5 receptor and oocyte-specific homeobox 5 genes were significantly upregulated in Lin28-downregulated mESCs. We also showed that the families of neuropeptide Y receptor (Npyr) and oocyte-specific homeobox (Obox) genes were upregulated by downregulation of Lin28. Conclusion: Based on the results of this study, we suggest that Lin28 controls the characteristics of mESCs through the regulation of effectors such as the Npyr and Obox families.

• BMB Reports
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• 제44권12호
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• pp.799-804
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• 2011

Gangliosides play an important role in neuronal differentiation processes. The regulation of ganglioside levels is related to the induction of neuronal cell differentiation. In this study, the ST8Sia5 gene was transfected into mESCs and then differentiated into neuronal cells. Interestingly, ST8Sia5 gene transfected mESCs expressed GQ1b by HPTLC and immunofluorescence analysis. To investigate the effects of GQ1b over-expression in neurogenesis, neuronal cells were differentiated from GQ1b expressing mESCs in the presence of retinoic acid. In GQ1b expressing mESCs, increased EBs formation was observed. After 4 days, EBs were co-localized with GQ1b and nestin, and GFAP. Moreover, GQ1b co-localized with MAP-2 expressing cells in GQ1b expressing mESCs in 7-day-old EBs. Furthermore, GQ1b expressing mESCs increased the ERK1/2 MAP kinase pathway. These results suggest that the ST8Sia5 gene increases ganglioside GQ1b and improves neuronal differentiation via the ERK1/2 MAP kinase pathway.

• 한국발생생물학회지:발생과생식
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• 제20권2호
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• pp.149-155
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• 2016

Sprouty (Spry) genes encode inhibitors of the receptor tyrosine kinase signaling cascade, which plays important roles in stem cells. However, the role of Spry4 in the stemness of embryonic stem cells has not been fully elucidated. Here, we used mouse embryonic stem cells (mESCs) as a model system to investigate the role of Spry4 in the stem cells. Suppression of Spry4 expression results in the decreases of cell proliferation, EB formation and stemness marker expression, suggesting that Spry4 activity is associated with stemness of mESCs. Teratoma assay showed that the cartilage maturation was facilitated in Spry4 knocked down mESCs. Our results suggest that Spry4 is an important regulator of the stemness and differentiation of mESCs.

• BMB Reports
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• 제49권10호
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• pp.527-528
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• 2016

In embryonic stem cells (ESCs), cell cycle regulation is deeply connected to pluripotency. Especially, core transcription factors (CTFs) which are essential to maintaining the pluripotency transcription programs should be reset during M/G1 transition. However, it remains unknown about how CTFs are governed during cell cycle progression. Here, we describe that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) axis during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle related target genes in determining the identity of ESCs. Aurkb starts to phosphorylate Oct4(S229) at the onset of G2/M phase, inducing the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Furthermore, Aurkb phosphormimetic and PP1 binding-deficient mutations in Oct4 disrupt the pluripotent cell cycle, lead to the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Based on our findings, we suggest that the cell cycle is directly linked to pluripotency programs in ESCs.

• 한국동물생명공학회지
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• 제35권1호
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• pp.21-27
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• 2020

Somatic cell nuclear transfer derived embryonic stem cells (NT-ESCs) have significant advantages in various fields such as genetics, embryology, stem cell science, and regenerative medicine. However, the poor establishment of NT-ESCs hinders various research. Here, we applied fasudil, a Rho-associated kinase (ROCK) inhibitor, to develop somatic cell nuclear transfer (SCNT) embryos and establish NT-ESCs. In the study, MII oocytes were isolated from female B6D2F1 mice and performed SCNT with mouse embryonic fibroblasts (MEFs). The reconstructed NT-oocytes were activated artificially, and cultured to blastocysts in KSOM supplemented with 10 μM fasudil. Further, the blastocysts were seeded on inactivated MEFs in embryonic stem cell medium supplemented with 10 μM fasudil. A total of 26% of embryos formed into blastocysts in the fasudil treated group, while this ratio was 44% in the fasudil free control group. On the other hand, 30% of blastocysts were established NT-ESCs after exposure of fasudil, which was significantly higher than the control group (10%). The results suggest that fasudil reduced blastocyst development after SCNT due to inhibition of 2 cell cleavage while improved the establishment of NT-ESCs through the anti-apoptotic pathway.

• 대한화장품학회지
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• 제45권4호
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• pp.415-422
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• 2019

피부 기저막에 위치하고 있는 인간 상피 줄기 세포는 피부상피층의 항상성 유지에 중요한 역할을 담당하고 있다. 비록 상피 줄기 세포가 조직손상에 대한 반응으로 상처 회복에 필요한 새로운 세포들을 공급하고 있지만 일부 세포는 정지 상태로 남아 생존을 위해 분화와 노화로부터 보호된다. 이러한 관점에서 적소세포와 외부세포기질 단백질로 구성된 특정 미세환경인 줄기세포 적소는 줄기세포를 보호하기 위해 적절한 자극을 제공해 준다. 줄기세포 마커는 상피 줄기세포의 표면에 발현되며, 기저막의 세포외기질과 부착하여 장기간의 성장 잠재력을 가지며 외부 자극에 대한 세포 사멸의 저항성을 가진다. 본 연구에서는 외부자극의 주요 인자로써 자외선 조사가 인테그린 α2, β1 와 α6 의 발현을 저하시킴을 확인하였으며 붉나무 추출물이 자외선에 의해 유도되어지는 인테그린 발현 저하를 억제하는 것을 확인 할 수 있었다. 또한 붉나무 추출물은 콜라겐 IV와 라미닌과 같은 상피 줄기세포의 부착과 연관된 분자들의 발현을 상향조절 하였다. 이러한 결과는 붉나무 추출물이 줄기세포 표면의 인테그린의 발현을 증가시키고 적소에서의 세포외기질 성분의 발현을 증가시킴으로써 자외선 조사에 대한 보호효과가 있음을 확인하였다.

• 천문학회보
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• 제39권2호
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• pp.55.1-55.1
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• 2014

on the observation with the Gemini Multi-Object Spectrograph on the Gemini-South 8.1 m telescope. The radial velocities of four ESCs do not display any sign of systematic motion, unlike the intermediate age carbon stars in NGC 6822. The ages and metallicities derived using the Lick indices show that the ESCs are old (>=8 Gyr) and metal poor ([Fe/H] <= -1.5). NGC 6822 is found to have both metal poor ($[Fe/H]{\approx}-2.0$) and metal rich ($[Fe/H]{\approx}-0.9$) star clusters within 15' (2 kpc) from the center, whereas only metal poor clusters are observed in the outer halo with r >= 20'(2.6 kpc). Based on the kinematics, old ages, and low metallicities of ESCs, we discuss the possible origin of ESCs and the formation of the outer halo of a small dwarf irregular galaxy NGC6822.

• Journal of Veterinary Science
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• 제22권4호
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• pp.54.1-54.13
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• 2021

Background: Hypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase. Objectives: This study aims to investigate the correlation between melatonin and hypoxia induction in cardiomyocytes differentiation. Methods: Mouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated. Results: Under hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects. Conclusions: This study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway.

• Molecules and Cells
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• 제25권4호
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• pp.487-493
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• 2008

HoxB4 has been shown to enhance hematopoietic engraftment by hematopoietic stem cells (HSC) from differentiating mouse embryonic stem cell (mESC) cultures. Here we examined the effect of ectopic expression of HoxB4 in differentiated human embryonic stem cells (hESCs). Stable HoxB4-expressing hESCs were established by lentiviral transduction, and the forced expression of HoxB4 did not affect stem cell features. HoxB4-expressing hESC-derived CD34+ cells generated higher numbers of erythroid and blast-like colonies than controls. The number of CD34+ cells increased but CD45+ and KDR+ cell numbers were not significantly affected. When the hESC derived CD34+ cells were transplanted into $NOD/SCID{\beta}2m-/-$ mice, the ectopic expression of HoxB4 did not alter their repopulating capacity. Our findings show that overexpression of HoxB4 in differentiating hESCs increases hematopoietic colony formation and hematopoietic cell formation in vitro, but does not affect in vivo repopulation in adult mice hosts.

• Asian Pacific Journal of Cancer Prevention
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• 제14권11호
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• pp.6341-6345
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• 2013

To clarify the role of stem cells in hepatocarcinogenesis, glypican-3 (GPC-3) and E-cadherin expression was investigated in embryonic cell lineages. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs) and hepatocyte like cells (HCs), representing 0, 22 and 40 days of differentiation, respectively, were treated in vitro with diethylnitrosamine (DEN) at four doses (0, 1, 5 and 15 mM; G1, G2, G3 and G4, respectively) for 24 h and GPC-3 and E-cadherin expression was examined by relative quantitative real-time PCR and immunocytochemistry. GPC-3 mRNA expression was significantly different for G4 at day 0 (p<0.001) and for G4 at day 22 (p<0.01) compared with the control (G1). E-cadherin mRNA expression was significantly different for G3 and G4 at day 0 (p<0.05 and p<0.001, respectively), for G2 and G4 (p<0.05 and p<0.001, respectively) at day 22 and for G2 and G4 (p<0.01 and p<0.001, respectively) at day 40 compared with G1. Immunofluorescence staining for GPC-3 showed a membranous and/or granular expression in cytoplasm of ESCs and HPCs and granular and/or diffuse expression in cytoplasm of HCs, which were also stained by E-cadherin. DEN treatment increased GPC-3 expression in ESCs, HPCs and HCs, with increase of E-cadherin expression. Taken together, the expression of GPC-3 was altered by DEN treatment. However, its expression pattern was different at the stage of embryo stem cells and its derived hepatic lineage cells. This suggests that GPC-3 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, associated with E-cadherin expression.