• Development and Reproduction
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• v.12 no.1
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• pp.1-8
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• 2008

Specific protocols to increase the differentiation of neuronal cells from embryonic stem (ES) cells have been well established, such as retinoic acid induction and lineage selection of neuronal cells. For the neuropathological studies, ES-derived neurons (ES neurons) must show normal physiological characteristics related to cell death and survival and should be maintained in vitro for a sufficient time to show insults-specific cell death without spontaneous death. When mouse ES cells were plated onto astrocytes monolayer after retinoic acid induction, most ES cells differentiated into neuronal cells, which were confirmed by the presence of specific neuronal markers, and the cultures were viable for at least four weeks. When these cultures were examined for vulnerability to glutamate excitotoxicity, ES neurons were vulnerable to excitotoxic insults mediated by agonist-specific receptors. The vulnerability to excitotoxic death increased with developmental age of ES neurons in vitro. Specific receptors for Neurotrophin and GDNF family ligands were present in ES neurons. GDNF and NT-3 could modulate the survival and excitotoxic vulnerability of ES neurons. The vulnerability and resistance to toxic insults, which are essential requirements of model culture systems for neuropathological studies, make ES neurons to a useful model culture system. Especially ES cell are highly amenable to genetic modification unlikely to primary neuronal cells, which will give us a chance to answer more complicated neurophysiological questions. Recently there was an outstanding attempt to explore the cellular toxicity using human ES cells (Schrattenholz & Klemm, 2007) and it suggested that ES cells could be a new model system for neurophysiological studies soon and go further a large-scale screening system for pharmacological compounds in the future.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.283-292
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• 2009

Objective: Human embryonic stem cells (hESCs) can proliferate indefinitely and differentiate into all kinds of cell types in vitro. Therefore, hESCs can be used as a cell source for cell-based therapy. Transduction of foreign genes to hESCs could be useful for tracing differentiation processes of hESCs and elucidation of gene function. Thus, we tried to introduce enhanced green fluorescent protein (eGFP) gene to hESCs, XX and XY cell lines in this study. Methods: Lentivirus containing eGFP was packaged in 293T cells and applied to hESCs to transduce eGFP. Expression of transduced eGFP was evaluated under the fluorescence microscope and eGFP positive population was analyzed by FACS. Expression of undifferentiation state markers such as Oct4, Nanog, SSEA4 and Tra-1-81 was examined by RT-PCR and/or immunofluorescence in eGFP-hESCs after transduction. In addition, the ability of eGFP-hESCs to form embryoid bodies (EBs) was tested. Results: eGFP was successfully transduced to hESCs by lentivirus. eGFP expression was stably maintained up to more than 40 passages. eGFP-hESCs retained expression patterns of undifferentiation state markers after transduction. Interestingly, disappearance of transduced eGFP was notably observed during spontaneous differentiation of eGFP-hESCs. Conclusion: We established eGFP expressing hESC lines using lentivirus and showed the maintenance of undifferentiation characteristics of these eGFP-hESCs. This reporter-containing hESCs could be useful for tracing the processes of differentiation of hESCs and other studies.

• The Science & Technology
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• no.4 s.419
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• pp.24-27
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• 2004

국내 연구진이 세계 최초로 사람의 체세포와 난자를 이용해 인간배아줄기세포를 만들어내는데 성공했다. 연구책임자인 황우석(서울대 수의대), 문신용(서울대 의대) 교수는 세계적인 과학학술지 사이언스의 초청으로 지난 2월 미국 시애틀에서 국제기자회견을 가졌다. 한국 과학자가 뉴욕타임스, BBC, 워싱턴포스트 등 세계 유수의 언론사를 대상으로 대규모 기자회견을 가진 것은 처음 있는 일이다. 이 연구는 전세계 언론에 대서특필되면서 세계 생명공학계에‘태극기를 휘날린’대단한 사건으로 받아들여지고 있다. 황 교수팀의 배아줄기세포연구를 간단히 요약하면‘사람의 난자에 사람의 체세포핵을 넣어 복제배아를 만들고 배양을 통해 인간배아 줄기세포로 분화시킨 것’이다. 그런데 왜 이 문장 하나에 수많은 사람들이 놀라고 열광하는 것일까. 귀국 이후 쏟아지는 인터뷰 요청을 사양하고 다시 실 험실로 돌아가 연구에 몰두하고 있는 황 교수를 어렵게 만났다. 인간배아줄기세포 생산 성공까지의 중요한 과정들을 황 교수와 문답으로 다시 풀어본다.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.311-319
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• 2009

Objective: Human embryonic stem cells (hESCs) have the capacity to differentiate into all of the cell types and therefore hold promise for cell therapeutic applications. In order to utilize this important potential of hESCs, enhancement of currently used technologies for handling and manipulating the cells is required. The cryopreservation of hESC colonies was successfully performed using the vitrification and slow freezing-rapid thawing method. However, most of the hESC colonies were showed extremely spontaneous differentiation after freezing and thawing. In this study, we were performed to rapidly collect of early passage hESCs, which was thawed and had high rate of spontaneously differentiation of SNUhES11 cell line. Methods: Four days after plating, partially spontaneously differentiated parts of hESC colony were cut off using finely drawn-out dissecting pipette, which is mechanical separation method. Results: After separating of spontaneously differentiated cells, we observed that removed parts were recovered by undifferentiated cells. Furthermore, mechanical separation method was more efficient for hESCs expansion after thawing when we repeated this method. The recovery rate after removing differentiated parts of hESC colonies were 55.0%, 74.5%, and 71.1% when we have applied this method to three passages. Conclusion: Mechanical separation method is highly effective for rapidly collecting and large volumes of undifferentiated cells after thawing of cryopreserved early passage hESCs.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.1
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• pp.39-48
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• 2008

Objective: The aim of this study was to investigate whether multipotent germline stem cells (MGSCs) can be established from neonatal mouse testis. Methods: Various cells containing MGSCs were collected from neonatal testis of ICR mice and allocated to plates for in vitro culture. After 7 days in culture, the cells were passed to a fresh culture plate and continuously cultured. From the third or fourth passage, the presumed MGSCs were cultured and maintained on mitomycin C-inactivated STO feeder cells. The MGSCs were cultured in a condition where mouse embryonic stem cells (ESCs) are cultured. Characteristics of the MGSCs were evaluated by RT-PCR, immunocytochemistry, alkaline phosphatase activity, karyotyping, and transmission electron microscopy. Results: Two MGSCs lines were established from 9 pooled sets of neonatal testicular cells. MGSCs colonies were morphologically undistinguishable from ESCs colonies and both MGSC lines as well as ESCs expressed undifferentiated stem cell markers, such as Thy-1, Oct-4, Nanog, Sox2 and alkaline phosphatase. Fine structure of undifferentiated MGSCs were similar to those of ESCs and 60% of MGSCs (12/20) had normal karyotype at passage 10. They were able to form embryoid bodies (EBs) and MGSC-derived EBs expressed marker genes of three germ layers. Conclusion: We could establish the MGSCs from neonatal mouse testis and they were differentiated to multipotent lineages of three germ layers. Molecular characteristics of MGSCs were similar to those of ESCs. Our results suggest a possibility that multipotent stem cells derived from testis, the MGSCs, could replace the ESCs in biotechnology and regenerative medicine.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.270-270
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• 2004

이 연구는 Ⅰ) 혈관 폐색에 의한 인지 및 기억장애 동물모델에서 뇌졸중 치료제로써 인간배아줄기 세포의 신경세포 보호효과 및 작용기간을 밝히며, Ⅱ) 행동 약리학적 연구를 통해 기억력증진에 미치는 효과에 대해 밝히고 혈관 폐색에 의한 동물모델에서의 기억기능 증진 및 세포효과를 검증하고자 실시하였다. 중대 뇌동맥 폐색에 의한 쥐의 동물모델은 Sprague Dawley계 흰 쥐(260∼300 g)의 국소 중대뇌동맥을 일시적으로 폐색시켜 만들었다. 본 연구(미국 국립보건원에 등록된 MB03세포)에 사용된 인간배아줄기세포는 3×10⁴ cells/㎠ 밀도의 배양접시 내에서 4일 동안 embryoid bodies(EBs)의 형성을 위해 집합체를 이루도록 유도하였다. (중략)

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.271-271
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• 2004

본 실험은 TGF-a를 처리하여 분화가 유도된 인간배아 줄기세포를 파킨슨 동물모델에 이식하여 숙주세포에서의 생존 및 이식효과를 검토하고자 실시하였다. TGF-a로 분화된 세포의 이식효과를 판정하고자 배양시 TGF-a처리군과 처리하지 않은 군으로 나누어 분화를 유도한 인간배아 줄기세포를 hoechst33342로 표지 하여 병변 유발과 동일한 방법으로 동측 선조체내에 4×10⁴개/2ul가 되도록 이식하고(이식 위치: AP 0.7, ML 2.0, DV3.4) 이식 후 2, 4주에서 행동학적 변화를 관찰하고 4주에 동물을 희생시켜 4% PFA를 이용하여 뇌 조직을 고정하고 뇌 조직은 40㎛ 두께로 동결 절편을 만들어 면역조직화학염색을 시행하여 신경세포로의 분화 및 TH 발현 여부를 관찰하였고 분화의 표지물질로 nestin, NF200, GFAP, TH를 사용하여 형태학적 변화를 관찰하였다. (중략)

• Journal of Life Science
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• v.23 no.10
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• pp.1223-1229
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• 2013

Ursolic acid (UA) a bio-active ingredient found in a variety of fruits and vegetables, and it has potent antioxidant activity. However, the role of UA in mouse embryonic stem (ES) cells is poorly understood. This study investigated the functional role of UA in regulating the development of mouse ES cells under hypoxia. Hypoxia did not exert a significant effect on the undifferentiated state of mouse ES cells. However, it induced reactive oxygen species (ROS) generation and increased the level of lactate dehydrogenase (LDH) production at 48 h of hypoxic exposure. Conversely, oxidative stress induced by hypoxia was significantly inhibited by UA ($30{\mu}M$) pretreatment. Hypoxia significantly decreased cell survival and the level of [$^3H$] thymidine incorporation, both of which recovered following pretreatment of UA. In addition, UA decreased the apoptotic effect of hypoxia by attenuating caspase-3 cleavage or by recovering cellular inhibition of the apoptotic protein (cIAP)-2 and Bcl-2 expression. We further found that UA decreased senescence-associated beta-galactosidase activity. We suggest that UA is a natural antioxidant and one of the functional modulators of hypoxia-induced survival, apoptosis, proliferation, and aging in mouse ES cells.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.4
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• pp.293-298
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• 2003

목 적: 인간의 배아줄기세포는 전분화능과 영속성을 가지고 있어 발생 및 분화에 관련된 기초 연구 뿐 만 아니라 재생의학, 약물검색 등에서도 매우 유용한 재료로 이용될 수 있다.본 연구에서는 유전체의 변형이 배아줄기세포주의 확립 효율에 미치는 영향을 살펴보고자 비정상적인 포배기 배아에서 내세포괴를 분리하여 배양하였다. 연구 방법: 인간의 체외수정 및 배아이식술에서 공여 받은1개 또는3개의 전핵이 관찰되는 비정상 수정란 (n=20)과 착상전 유전진단에서 이수성이 확인된 배아 (n=27)를 대상으로 하였다. 일반적인 immunosurgery 방법으로 영양배엽세포들을 제거하고 내세포괴를 분리한 후 PMEF 혹은 STO feeder 세포위에서 배양하였다. 배아줄기세포의 배양시스템을 검증하기 위해서 이미 확립된 Miz-hES1 cell line을 동시에 같은 조건 하에서 계대배양하였다. 결 과: 비정상 수정란에서 발생된 포배기 배아에서 분리한 1개의 내세포괴가 배아줄기세포와 유사한 colony를 형성하였으나, 계대배양에는 실패하였다. 이수성 배아에서 발생된 포배기 배아의 내세포괴 배양에서는 두개의 colony가 계대배양 중에 영양배엽세포의 형태로 분화되어 미분화 상태를 유지하지 못하였다. 동일한 시기와 조건 하에서 계대배양된 Miz-hES1 cell line이 미분화상태로 유지됨을 karyotyping (46, XY)과 immunophenotyping (positive in SSEA-3 and -4)으로 확인하였다. 결 론: 본 연구의 결과에서 비정상 수정란과 이수성 배아에서 발생된 포배기 배아에서 유래한 내세포괴는 배아줄기세포주 확립 및 미분화 상태 유지 능력이 매우 저조한 것으로 여겨진다. 따라서, 인간의 배아줄기세포주를 확립하는데 있어 배아의 정상여부가 중요한 요소로 작용할 것으로 생각된다.

• Development and Reproduction
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• v.11 no.2
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• pp.111-119
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• 2007

Differentiation of cells can be induced through modulation of endogenous regulators using exogenous factors. Useful transfection systems to transport a specific exogenous regulator into cell have been tried but still there are many obstacles to overcome. In this study, we examined the transfection efficiency of cell permeable peptides (CPPs) in mouse embryonic stem cell under the various conditions. To identify the CPP-mediated translocation of a protein, we employed recombinant CPP-enhanced green fluorescent protein (EGFP). Viability of R1 cells was different between experimental groups depending on the kind of CPP and the concentration of CPP-EGFP. Translocation of CPP-EGFPs into the R1 cells was not detected until 30 min after CPP-EGFPs treatment in all groups. After 1 hr, translocation of pEP-1-EGFP-N was detected, but it could not be detected in the other group. Transfection of pEP-1EGFP-N was independent on its concentration. The time course did not show saturation even after 24 hr in pEP-1-EGFP-N. These results showed that the permeability depended on the kind of CPP and the location of His-tag in the case of examined CPPs, and did not need biological energy. On summary, the efficiency of transfection of CPP-EGFP depends on the CPP sequences but the culture time is not a key factor in transfection for the mouse embryonic stem cell. For the future studies to improve the efficiency of translocation of protein into embryonic stem cells, it is needed to develop modified CPP or mediator. The studies would be very useful to induce the differentiation of embryonic stem cells.