• 대한생식의학회:학술대회논문집
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• 대한불임학회 2006년도 The 5th Biannual Meeting of Pacific Rim Society for Fertility and Sterility
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• pp.160.1-160.1
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• 2006

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2006년도 The 5th Biannual Meeting of Pacific Rim Society for Fertility and Sterility
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• pp.159.2-159.2
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• 2006

• Reproductive and Developmental Biology
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• 제34권3호
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• pp.135-141
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• 2010

Embryoid bodies (EBs) generated from human embryonic stem cells (hESCs) include spontaneously induced endodermal lineage cells (ELCs). Activin-A plays important roles in the endoderm differentiation of hESCs. Despite studies on the generation of ELCs from hESCs with treatment of Actvin-A, it was unclear for localization and pattern of ELCs by Activin-A during differentiation of hESCs. Accordingly in this study, we knew that Actvin-A increased the cystic EBs formation, including the highly enriched AFP (endoderm lineage specific marker)-expressing cells in the surface of cystic EBs. To induce the EBs formation from undifferentiated hESCs, cells were transferred onto petri-dish and cultured in suspension condition without bFGF removed hESC media (EB media) for 3 days. Next to investigate the effect of Activin-A, EBs were subsequently cultured in EB media supplement with 100 ng/ml Activin-A for 3 days. After 5~7 days of Activin-A treatment, cystic EBs began to appear which increased in numbers reaching ~60% of initially formed EBs over 5 days. Endoderm lineage marker, AFP were highly expressed and specifically localized at the surface region of cystic EBs comparison with normal EBs. We next attached the cystic EBs onto gelatin-coated plates and cultured for 5 days. In the results of real-time PCR and immunocytochemistry analysis, AFP-expressing cells migrated and localized at the outgrowth region of attached cystic EBs. To obtain the AFP-expressing cells of the outgrowth region, we manually isolated by using micro-dissection and cultured them. These cells strongly express AFP over 70% of isolated cells post re-plating. Here, we first showed an expression pattern of specifically localized ELCs by Activin-A during differentiation of hESCs. From this observation, we could highly purified ELCs from undifferentiated hESCs. Taken together, our system will provide a novel and efficient option to generate ELCs from hESCs.

• Animal Bioscience
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• 제36권12호
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• pp.1905-1917
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• 2023

Objective: Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. Methods: A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. Results: In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. Conclusion: Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 1993년도 학술대회지
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• pp.127-131
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• 1993

The effects of total ginseng saponin. extracts of Panax ginseng C.A. Meyer, on the differentiation of F9 teratocarcinoma stem cells were studied. F9 stem cells cultured in the presence of ginseng saponin together with dibutyric cAMP became parietal endoderm - like cells. Moreover, the expressions of differentiation marker genes. laminin. type IV collagen. and retinoic acid $receptor-{\beta}(RAR{\beta})$ were increased after treatment of ginseng saponin. Among various ginsenosides purified from crude ginseng saponin, $Rh_1\;and\;Rh_2$ caused the differentiation of F9 cells most effectively. Since ginsenosides and steroid hormone show resemblance in chemical structure. we studied the possibility of the involvement of a steroid receptor in the differentiation process induced by ginsenosides. According to Southwestern blot analysis, a 94 kDa protein regarding as a steroid receptor was detected in F9 cells cultured in the medium containing ginseng saponin. Based on these data, we suggest that ginseng saponin, especially ginsenosides $Rh_1\;and\;Rh_2$ cause the differentiation of F9 cells and the effects of ginsenosides might be exerted via binding with a steroid receptor or its analogous nuclear receptor.

• 약학회지
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• 제43권3호
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• pp.378-384
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• 1999

Retinoic acid (RA) and dibutyryl cyclic AMP (dbcAMP) induce the differentiation of the multipotent embryonic carcinoma cell line, F9 cells, into parietal endoderm like cell. The F9 cells are highly proliferative doubling approximately 12 hourse. S Phase is predominant, lasting 10 hours and G2/M phase occupies most of the remaining cycle (2 hours) and G1 phase is nearly non-existent. In this study, we showed the effect of RA and dbcAMPon the cell cycle associated molecules (especially around G1 phase) during F9 cell differentiation. Differentiation of F9 cells was induced by the combined addition of RA ($10^{-7}M$) and dbcAMP (0.5mM), and cells were harvested daily up to 4 days. Flow cytometric analysis showed the prolongation of G1 phase around 30 hours after induction. Western blot analysis revealed that the amount of cyclin D1 and cdk2 were increased at day 4. However, histone H1 kinase activity of cdk2 was decreased. These data strongly suggest that RA and dbcAMP induce the growth arrest of F9 cells at G1 phase by decreasing the activity of cdk2, although they have increased the protein contents of cyclin D1 and cdk2. The reason for the discrepancy between the H1 kinase activity and protein contents are not clear yet.

• Molecules and Cells
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• 제23권1호
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• pp.49-56
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• 2007

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential.

• Reproductive and Developmental Biology
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• 제35권1호
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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.

• 한국발생생물학회지:발생과생식
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• 제6권1호
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• pp.55-66
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• 2002

배아줄기세포(embryonic stem cell, ESC)는 미분화상태로 지속적인 계대가 가능하며, 정상 핵형과 전 분화능(pluripotency)을 가져 생체내-외에서 분화 유도시 삼배엽성의 모든 세포로 분화 가능하다. ESC를 feeder 세포 없이 부유배양하면 배아체(embryoid body, EB)를 형성하고, 초기 배아 발생과 유사한 분화 양상을 갖는다. ESC의 분화 유도가 초기배아 발생처럼 생식호르몬(GTH: FSH, LH; steroids)의 영향을 받는지는 불명하다. 본 연구는 ESC가 분화과정중 생식호르몬처리에 의해 그들 수용체가 발현되는가를 알아보고자 하였다. 순계혈통 생쥐인 C57BL/6J에서 과배란 유도후 포배를 수획하고, 유사분열적으로 불활성화된 feeder 세포와 공배양하여, 계대배양 하는 중 배아줄기세포주(JHYl)를 확립하였다. JHY1의 alkaline phosphatase 활성과 SSEA-1, 3, 4 발현을 통해 ESC임을 확인하였다. Feeder 세포 없이 ESC를 계대배양 후 호르몬처리(FSH LH E$_2$, P$_4$, T)하에서 5일 동안 부유배양하여 배아체를 형성시키고, 이후 7일 동안 부착배양하여 분화를 유도하였다. GTH와 스테로이드의 수용체 발현 실험에서 ESC에 E$_2$ 처리에 의한 LHR의 발현 증가를 제외한 나머지 호르몬 처리군에서 ESC보다 낮은 생식호르몬의 수용체 발현이 관찰되었다. 생식호르몬을 농도별 수용체 발현 정도는 증감되지 않았다. 미분화 ESC 표지유전자인 Oct-4는 호르몬 처리군에서도 발현되었다. 각 배엽의 표지유전자들(영양세포, handl; 외배엽성, keratin와fgf-5; 중배엽성, enolase와 $\alpha$ -globin; 내배엽성, gata-4와 $\alpha$ -fetoprotein) 등의 발현 양상을 조사한 결과 호르몬 처리후 내배엽성 표지유전자외에는 발현 증가가 관찰되지 않았다. 즉 생식호르몬에 의해 gata-4, $\alpha$-fetoprotein의 발현이 증가되는 것으로 보아 내배엽성 계열로의 분화 유도가 이루어진 것으로 사료된다.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.263-271
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• 2009

Cardiovascular diseases (CVDs) are one of the most cause of death around the world and fields of interest for cardiac stem cells. Also, current use of terminally differentiated adult cardiomyocytes for CVDs has limited regenerative capacity therefore any significant cell loss may result in the development of progressive heart failure. Human embryonic stem cells (hESCs) derived from blastocyst-stage embryos spontaneously have ability to differentiate via embryo-like aggregates (endoderm, ectoderm and mesoderm) in vitro into various cell types including cardiomyocyte. However, most effective molecule or optimized condition which can induce cardiac differentiation of hESCs is rarely studied. In this study, we developed both spontaneous and inductive cardiomyocyte-like cells differentiation from hESCs by treatment of induced-factors, 5-azacytidine, BMP-4 and cardiogenol C. On the one hand, spontaneous and inductive cardiomyocyte-like cells showed that cardiac markers are expressed for further analysis by RT-PCR and immunocytochemistry. Interestingly, BMP-4 greatly improved homogeneous population of the cardiomyocyte-like cells from hESCs CHA15 and H09. In conclusion, we verified that spontaneously differentiated cells showed cardiac specific markers which characterize cardiac cells, treated extrinsic factors can manage cellular signals and found that hESCs can undergo differentiation into cardiomyocytes better than spontaneous group. This finding offers an insight into the inductive factor of differentiated cardiomyocytes and provides some helpful information that may offer the potential of cardiomyocytes derived from hESCs using extrinsic factors.