• Molecules and Cells
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• 제37권7호
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• pp.562-567
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• 2014

Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-${\beta}1$. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.13-20
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• 2002

Objective: This study was carried out to establish the effectiveness of the vitrification method and the optimal cryoprotectants in the cryopreservation of human embryonic stem cells (ESC). Materials and Methods: Human ESC clumps established at Seoul National University Hospital (SNUhES 1) were cryopreserved with the vitrification method using the EM grid. EDS and EFS40 were used as vitrification solutions. Results: Between the EDS and EFS40 groups, there was no significant difference in the recovery rate after cryopreservation of human ESC. The formation rates of ESC colonies in the vitrified groups were significantly lower than those in the control ESC group (p<0.05, p<0.05). In addition, the formation rate of ESC colonies in the EDS group was significantly higher than that in the EFS40 group (p<0.05). The ESC colonies in the vitrified groups were significantly smaller after culture duration of 2 and 4 days, respectively, compared with the control ESC group (p<0.1, p<0.05). However, these effects could be reduced to nonsignificant level by the additional culture of ESC colonies. The vitrified human ESC retained the properties of pluripotent cells, including the expression of cell surface. markers for the undifferentiated cells such as alkaline phosphatase and SSEA-4 (stage-specific embryonic antigen-4), and the expression of transcription factor Oct-4 (octamer-binding transcription factor-4), and the normal karyotype. Conclusion: The vitrification method using the EM grid and EDS solution was confirmed to be very effective for the cryopreservation of human ESC.

• Clinical and Experimental Reproductive Medicine
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• 제31권4호
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• pp.209-215
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• 2004

Objective: Embryonic stem (ES) cells could be differentiated into the specific cell types by alternation of culture condition and modification of gene expression. This study was performed to evaluate the differentiation protocol for mouse and human ES cells to insulin secreting cells. Methods: Undifferentiated mouse (JH-I) and human (Miz-hESI) ES cells were cultured on STO feeder layer, and embryoid bodies (EBs) were formed by suspension culture. For the differentiation, EBs were cultured by sequential system with three stage protocol. The differentiating ES cells were collected and marker gene expressions were analyzed by seIni-quantitative RT-PCR in each stage. Amount of secreted insulin levels in culture media of human ES cells were measured by human insulin specific RIA kit. Results: During the differentiation process of human ES cells, GATA-4, a-fetoprotein, glucose transporter-2 and Ngn-3 expression were increased whereas OctA was decreased progressively. Insulin and albuInin mRNAs were expressed from stage IT in mouse ES cells and from stage III in human ES cells. We detected 3.0~7.9 IlU/rnl secretion of insulin from differentiated human ES cells by in vitro culture for 36 days. Conclusion: The sequential culture system could induce the differentiation of mouse and human ES cells into insulin secreting cells. This is the fIrst report of differentiation of human ES cells into insulin secreting cells by in vitro culture with serum and insulin free medium.

• Asian-Australasian Journal of Animal Sciences
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• 제28권12호
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• pp.1721-1728
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• 2015

Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (-420/+181) bovine NANOG 5'-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (-420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2004년도 제47차 추계학술대회
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• pp.72.2-73
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• 2004

• 과학기술학연구
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• 제5권1호
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• pp.125-148
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• 2005

본 논문은 황우석 교수의 2005년 5월 배아줄기세포 연구성과 발표에 대한 인터넷상의 공론이 어떻게 이루어졌는지를 통해 일반 공중의 배아복제에 대한 이해를 연구한다. 인터넷 공론에 대한 분석은 일반화의 어려움은 있지만 질적 연구의 장점이 있다는 점을 분명히 하면서 사례연구를 하였다. 5월 20일 주요 신문은 연구성과의 세계적인 의의와 더불어 난치병 치료를 위한 연구로 자리매김을 만들었으며 윤리문제를 부각하는 데서 약간의 차이를 보였다. 그런 영향으로 인터넷상에서는 훨씬 더 분명한 형태로 환호하는 분위기를 보였다. 네이버 게시판은 연구성과에 대한 열광과 더불어 윤리문제제기를 비판하는 경향을 띠었고 연구의 정당성을 묻는 민주노동당에 대해서는 보다 애국주의적 국가주의적 근거에서, 가톨릭의 반대성명에 대해서는 배아는 생명이 아니며 낙태 등이 일상화된 현실적 근거에서 비판되었다.

• International Journal of Stem Cells
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• 제16권4호
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• pp.394-405
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• 2023

The differentiation of pluripotent stem cells has been used to study disease mechanisms and development. We previously described a method for differentiating human pluripotent stem cells (hPSCs) into salivary gland epithelial progenitors (SGEPs). Here, cystic fibrosis transmembrane conductance regulator (CFTR) knockout hPSCs were differentiated into SGEPs derived from CFTR knockout hESCs (CF-SGEPs) using the same protocol to investigate whether the hPSC-derived SGEPs can model the characteristics of CF. CF-a disease that affects salivary gland (SG) function-is caused by mutations of the CFTR gene. Firstly, we successfully generated CFTR knockout hPSCs with reduced CFTR protein expression using the CRISPR-Cas9 system. After 16 days of differentiation, the protein expression of CFTR decreased in SGEPs derived from CFTR knockout hESCs (CF-SGEPs). RNA-Seq revealed that multiple genes modulating SG development and function were down-regulated, and positive regulators of inflammation were up-regulated in CF-SGEPs, correlating with the salivary phenotype of CF patients. These results demonstrated that CFTR suppression disrupted the differentiation of hPSC-derived SGEPs, which modeled the SG development of CF patients. In summary, this study not only proved that the hPSC-derived SGEPs could serve as manipulable and readily accessible cell models for the study of SG developmental diseases but also opened up new avenues for the study of the CF mechanism.

• Reproductive and Developmental Biology
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• 제38권2호
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• pp.79-84
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• 2014

MicroRNAs (miRNAs) are approximately 22 nucleotides of small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The miRNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, metabolism, imprinting, and differentiation. Recently, a paper has shown that expression of the miRNA-302/367 cluster expressed abundantly in mouse and human embryonic stem cells (ESCs) can directly reprogram mouse and human somatic cells to induced pluripotent stem cells (iPSCs) efficiently in the absence of any of the four factors, Oct4, Sox2, c-Myc, and Klf4. To apply this efficient method to porcine, we analyzed porcine genomic sequence containing predicted porcine miRNA-302/367 cluster through ENSEMBL database, generated a non-replicative episomal vector system including miRNA-302/367 cluster originated from porcine embryonic fibroblasts (PEF), and tried to make porcine iPSCs by transfection of the miRNA-302/367 cluster. Colonies expressing EGFP and forming compact shape were found, but they were not established as iPSC lines. Our data in this study show that pig miRNA-302/367 cluster could not satisfy requirement of PEF reprogramming conditions for pluripotency. To make pig iPSC lines by miRNA, further studies on the role of miRNAs in pluripotency and new trials of transfection with conventional reprogramming factors are needed.

• Biomolecules & Therapeutics
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• 제20권3호
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• pp.280-285
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• 2012

The developing embryo naturally experiences relatively low oxygen conditions in vivo. Under in vitro hypoxia, mouse embryonic stem cells (mESCs) lose their self-renewal activity and display an early differentiated morphology mediated by the hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$). Previously, we demonstrated that histone deacetylase (HDAC) is activated by hypoxia and increases the protein stability and transcriptional activity of HIF-$1{\alpha}$ in many human cancer cells. Furthermore HDAC1 and 3 mediate the differentiation of mECSs and hematopoietic stem cells. However, the role of HDACs and their inhibitors in hypoxia-induced early differentiation of mESCs remains largely unknown. Here, we examined the effects of several histone deacetylase inhibitors (HDACIs) on the self-renewal properties of mESCs under hypoxia. Inhibition of HDAC under hypoxia effectively decreased the HIF-$1{\alpha}$ protein levels and substantially improved the expression of the LIF-specific receptor (LIFR) and phosphorylated-STAT3 in mESCs. In particular, valproic acid (VPA), a pan HDACI, showed dramatic changes in HIF-$1{\alpha}$ protein levels and LIFR protein expression levels compared to other HDACIs, including sodium butyrate (SB), trichostatin A (TSA), and apicidin (AP). Importantly, our RT-PCR data and alkaline phosphatase assays indicate that VPA helps to maintain the self-renewal activity of mESCs under hypoxia. Taken together, these results suggest that VPA may block the early differentiation of mESCs under hypoxia via the destabilization of HIF-$1{\alpha}$.

• Clinical and Experimental Reproductive Medicine
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• 제36권4호
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• pp.283-292
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• 2009

목 적: 인간 배아줄기세포 (human embryonic stem cells; hESCs)는 체외에서 오랫동안 증식할 수 있으며, 모든 종류의 세포로 분화할 수 있는 능력을 가진 세포이다. 그러므로, 인간 배아줄기세포는 세포치료의 세포공급원의 역할을 할 수 있을 것으로 기대를 모으고 있다. 인간 배아줄기세포로의 외래 유전자의 도입은 분화경로 규명 및 특정 유전자의 기능 규명 등에 효과적으로 이용될 수 있다. 본 연구에서는 렌티 바이러스를 이용하여 eGFP 유전자를 XY와 XX 핵형을 가진 인간 배아줄기세포주에 도입하고자 하였다. 연구방법: 렌티 바이러스를 이용하여 eGFP 유전자를 인간 배아줄기세포에 도입하였다. 도입된 eGFP의 발현은 형광현미경을 이용하여 확인하였으며, 유세포 분석을 통하여 eGFP 발현세포의 비율을 분석하였다. 또한, eGFP가 도입된 인간 배아줄기세포에서 표지인자인 Oct4, SSEA4 및 Tra-1-81의 발현을 확인하였으며, 배아체의 형성 여부를 확인하여 특성분석을 수행하였다. 결 과: eGFP는 인간 배아줄기세포로 성공적으로 도입되었다. eGFP의 발현은 40 계대 이상 안정적으로 지속되었다. eGFP를 발현하는 인간 배아줄기세포는 eGFP 도입 후에도, 배아줄기세포의 특성을 유지하고 있음이 확인되었다. 또한, 자연적 분화 동안 발현이 감소하는 현상이 관찰되었다. 결 론: 본 연구에서는 렌티 바이러스를 이용하여 eGFP가 도입된 인간 배아줄기세포주를 확립하였으며, 그 특성이 유지되고 있음을 확인하였다. 표지 유전자가 도입된 인간 배아줄기세포주는 분화 및 다른 연구에 활용될 수 있을 것으로 기대된다.