• 한국발생생물학회지:발생과생식
• /
• 제14권2호
• /
• pp.123-129
• /
• 2010

배아 줄기세포는 미분화상태에서 자가 재생을 유지할 수 있다. 자가 재생은 OCT4, SOX2와 NANOG와 같은 많은 인자들이 작용한다. 생쥐 배아 줄기세포에서 OCT4와 SOX2가 Nanog 프로모터에 결합하여 Nanog 유전자의 발현을 촉진한다는 사실은 생쥐 promoter에 관한 정밀분석으로 알려져 있다. 본 연구에서는 인간 Nanog promoter를 정밀 분석하기 위해 연속적인 결손 돌연변이를 가진 promoter-reporter construct를 제조하였다. Promoter의 최대 활성은 0.6 kb(-253/+365) promoter-reporter construct에서 발견되었으며, 이 construct에는 OCT4 및 SOX2의 결합부위가 포함된다. OCT4와 SOX2의 기여도를 확인하기 위하여 OCT4 및 SOX2의 결합부위에 자리 특이적 돌연변이를 유도하고 promoter 활성에 미치는 영향을 조사한 결과, OCT4나 SOX2 어느 한 군데라도 돌연변이가 존재하면 promoter 활성이 현저히 저해되었다. 본 연구 결과를 통해 인간 Nanog 유전자 발현에 있어 OCT4 및 SOX2가 필수적임을 직접적으로 확인할 수 있었다.

• Clinical and Experimental Reproductive Medicine
• /
• 제29권2호
• /
• pp.129-138
• /
• 2002

Objective: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Materials and Methods: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and $5{\mu}g$/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal sperm of hybrid F1 male mice ($1{times}10^6/ml$). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, b1astocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identify ES cells, the surface markers alkaline phosphatase, SSEA-1, 3,4 and Oct4 staining were examined in rep1ated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. Results: Although the cleavage rate (${\geq}$2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic b1astocysts ($9.6{\pm}3.1,\;35.1{\pm}5.2$) were signficantly lower than those of IVF blastocysts ($19.5{\pm}4.7,\;63.2{\pm}13.0$) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-l and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac cell differentiation derived from mES or P-mES cells was confirmed. Conclusion: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.

• Reproductive and Developmental Biology
• /
• 제34권3호
• /
• pp.143-151
• /
• 2010

Pluripotency of human embryonic stem cell (hESC) is one of the most valuable ability of hESCs for applying cell therapy field, but also showing side effect, for example teratoma formation. When transplant multipotent stem cell, such as mesnchymal stem cell (MSC) which retains similar differentiation ability, they do not form teratoma in vivo, but there exist limitation of cellular source supply. Accordingly, differentiation of hESC into MSC will be promising cellular source with strong points of both hESC and MSC line. In this study, we described the derivation of MSC like cell population from feeder free cultured hESC (hESC-MSC) using direct differentiation system. Cells population, hESC-MSC and bone marrow derived MSC (BM-MSC) retained similar characteristics in vitro, such as morphology, MSC specific marker expression and differentiation capacity. At the point of differentiation of both cell populations, differentiation rate was slower in hESC-MSC than BM-MSC. As these reason, to verify differentially expressed molecular condition of both cell population which bring out different differentiation rate, we compare the molecular condition of hESC-MSC and BM-MSC using 2-D proteomic analysis tool. In the proteomic analysis, we identified 49 differentially expressed proteins in hESC-MSC and BM-MSC, and they involved in different biological process such as positive regulation of molecular function, biological process, cellular metabolic process, nitrogen compound metabolic process, macromolecule metabolic process, metabolic process, molecular function, and positive regulation of molecular function and regulation of ubiquitin protein ligase activity during mitotic cell cycle, cellular response to stress, and RNA localization. As the related function of differentially expressed proteins, we sought to these proteins were key regulators which contribute to their differentiation rate, developmental process and cell proliferation. Our results suggest that the expressions of these proteins between the hESC-MSC and BM-MSC, could give to us further evidence for hESC differentiation into the mesenchymal stem cell is associated with a differentiation factor. As the initial step to understand fundamental difference of hESC-MSC and BM-MSC, we sought to investigate different protein expression profile. And the grafting of hESC differentiation into MSC and their comparative proteomic analysis will be positively contribute to cell therapy without cellular source limitation, also with exact background of their molecular condition.

• Clinical and Experimental Reproductive Medicine
• /
• 제32권2호
• /
• pp.133-147
• /
• 2005

Objectives: This study was carried out to establish human embryonic stem cells derived from frozen-thawed embryos using mouse embryonic fibroblasts (mEFs), human fetal skin and muscle fibroblasts as feeder cells, and to identify the characteristic of embryonic stem cells. Methods: When primary mEFs, human fetal skin and muscle fibroblasts were prepared, passaging on 4 days from replating could have effective trypsinization and clear feeder layers. Eight of 23 frozenthawed 4~8 cell stage embryos donated from consenting couples developed to blastocysts. Inner cell mass (ICM) was isolated by immunosurgery. ICM was co-cultured on mEFs, human fetal skin or muscle fibroblasts. The ICM colonies grown on mEFs, human fetal skin or muscle fibroblasts were tested the expression of stage specific embryonic antigen-3, -4 (SSEA-3, -4), octamer binding transcription factor-4 mRNA (Oct-4) and alkaline phosphatase surface marker. Results: We obtained 1 ICM colony from 2 ICM co-cultured on mEFs as feeder cells and did not obtain any ICM colony from 6 ICM clumps co-cultured on human fetal skin or muscle fibroblasts. The colony formed on mEFs could be passaged 30 times every 5 days with sustaining undifferentiated colony appearance. When the colonies cultured on mEFs were grown on human fetal skin or muscle fibroblasts, the colonies could be passaged 15 times every 9 days with sustaining undifferentiated colony appearance. The colonies grown on mEFs and human fetal fibroblasts expressed SSEA-4 and alkaline phosphatase surface markers and positive for the expression of Oct-4 by reverse transcription-polymerase chain reaction (RT-PCR). The produced embryoid body differentiated spontaneously to neural progenitorlike cells, neuron-like cells and beating cardiomyocyte-like cells, and frozen-thawed embryonic stem cells displayed normal 46,XX karyotype. Conclusions: The human embryonic stem cells can be established by using mEFs and human fetal fibroblasts produced in laboratory as feeder cells.

• 한국수정란이식학회지
• /
• 제27권3호
• /
• pp.171-174
• /
• 2012

Various small molecules can be used to control major signaling pathways to enhance stemness and inhibit differentiation in murine embryonic stem cell (mESC) culture. Small molecules inhibiting the fibroblast growth factor (FGF)/ERK pathway can preserve pluripotent cells from stimulation of differentiation. In this study, we aimed to evaluate the effect of pluripotin (SC-1), an inhibitor of the FGF/ERK pathway, on the colony formation of outgrowing presumptive mESCs. After plating the zona pellucida-free blastocyst on the feeder layer, attached cell clumps was cultured with SC-1 until the endpoint of the experiment at passage 10. In this experiment, when the number of colonies was counted at passage 3, SC-1-treated group showed 3.4 fold more mESC colonies when compared with control group. However, after passage 4, there was no stimulating effect of SC-1 on the colony formation. In conclusion, SC-1 treatment can be used to promote mESC generation by increasing the number of early mESC colonies.

• 한국발생생물학회지:발생과생식
• /
• 제12권2호
• /
• pp.141-149
• /
• 2008

This study was conducted to develop an efficient cryopreservation method of human embryonic stem (ES) cells using vitrification. In an initial experiment, sub-clumps of human ES cells (CHA-hES3 and CHA-hES4) were vitrified using grids after incubation with STO feeder cells for 1 or 16 h (Groups 1-1 and 1-2, respectively). After storage for $2{\sim}4$ months, thawed clumps were re-plated on a fresh feeder layer. The survival rates of warmed CHA-hES3 and CHA-hES4 cells of Group 1-2 were significantly higher than those of the corresponding Group 1-1 cells. In the second experiment, human ES cells were vitrified after incubation with feeder or feeder-conditioned medium (Groups 2-1 to -7). Relative mRNA expression of BM proteins and survival rates were increased following incubation of ES cells with fresh feeder cells for 16 h. In conclusion, increasing of tight adhesion between ES cells by extended incubation with feeder could reduce cryoinjury after vitrifying/warming.

• Reproductive and Developmental Biology
• /
• 제34권3호
• /
• pp.229-233
• /
• 2010

Pluripotency and self-renewal capacity of human embryonic stem cells (hESCs) are retained by hESCs related genes as OCT4, SOX2 and NANOG. These genes are shown high expression level in diverse cancer cells and have potential role in the carcinogenesis. On the contrary to this, several genes which are up-regulated in the differentiated hESCs are involved to suppress the carcinogenesis or proliferation of cells. We discovered several genes in immortalized lung fibroblast (WI-38 VA13) by suppression subtractive hybridization. Among them, we focused chromosome 6 open reading frame 62 (C6orf62) which is uncharacterized, mapped to 6p22.3 and generated to Hepatitis B virus X-transactivated proteins (HBVx-transactivated proteins, XTP). Aim of this study was to characterize C6orf62 through analyzing of expression pattern in various cell lines. Expression of C6orf62 was significantly upregulated in diverse normal cell lines than cancer cell lines. And C6orf62 was up-regulated in differentiated hESCs (endothelial cells, neural cells) compared to those of undifferentiated hESCs. Also, C6orf62 in WI-38 cells was highly up-regulated during G1/S transition of the cell cycle. Taken together, C6orf62 is shown expression pattern similar to differentiated hESCs-associated genes which down-regulated in cancer cells. Therefore, we assume that C6orf62 may participate to suppress the proliferation and to induce differentiation through regulating the cell cycle.

• 한국발생생물학회지:발생과생식
• /
• 제14권4호
• /
• pp.215-223
• /
• 2010

배아줄기세포를 이용한 형질전환동물의 제조는 유전자의 기능 연구에 필수적이다. 특히 유전자 파괴 생쥐는 유전자의 기능 연구뿐만 아니라 사람 질병 연구에 중요한 모델이 되어 왔다. 유전자 적중법(gene targeting)과 유전자 함정법(gene trapping)은 ES 세포에서 녹아웃(knockout) 생쥐를 제조하는 대표적인 방법이다. 20여 년 전 유전자 적중법과 함정법이 최초로 개발된 이후에 이 기술은 많은 변화를 거쳤다. 특히 상동재조합에 기초한 전통적 유전자 적중법은 대량 제조기반의 조건부 유전자 적중법의 개발로 이어졌고, 유전자 적중법 및 유전자 함정법의 장점 요소의 조합은 유전자를 파괴하는 범위를 넓혔고, 유전자 적중을 더욱 효율적으로 만들었다. 이런 기술은 특정 유전자를 표적으로 하는 다양한 종류의 돌연변이 형질전환동물을 제조할 수 있게 하여 포스트게놈 시대에 요구되는 전체 유전체의 기능 연구를 더욱 효과적으로 진행시켜 줄 것이다.

• 한국발생생물학회지:발생과생식
• /
• 제12권2호
• /
• pp.159-168
• /
• 2008

Nanog is a newly identified member of the homeobox family of DNA binding transcription factors that functions to maintain the undifferentiated state of stem cells. However, molecular mechanisms underlying the function of Nanog remain largely unknown. To elucidate the regulatory roles of Nanog involved in maintenance of P19 embryonal carcinoma (EC) stem cells, we transfected three small interfering RNA (siRNA) duplexes targeted against different regions of the Nanog gene into P19 cells. The Nanog siRNA-100 duplexes effectively decreased the expression of Nanog up to 30.7% compared to other two Nanog siRNAs, the Nanog siRNA-400 (67.9 %) and -793 (53.0%). When examined by RT-PCR and real-time PCR, the expression of markers for pluripotency such as Fgf4, Oct3/4, Rex1, Sox1 and Yes was downregulated at 48 h after transfection with Nanog siRNA-100. Furthermore, expression of the ectodermal markers, Fgf5 and Isl1 was reduced by Nanog knockdown. By contrast, the expression of other markers for pluripotency such as Cripto, Sox2 and Zfp57 was not affected by Nanog knockdown at this time. On the other hand, the expression of Lif/Stat3 pathway molecules and of the endoderm markers including Dab2, Gata4, Gata6 and the germ cell nuclear factor was not changed by Nanog knockdown. The results of this study demonstrated that the knockdown of Nanog expression by RNA interference in P19 cells was sufficient to modulate the expression of pluripotent markers involved in the self-renewal of EC stem cells. These results provide the valuable information on potential downstream targets of Nanog and add to our understanding of the function of Nanog in P19 EC stem cells.

• 한국수정란이식학회지
• /
• 제28권1호
• /
• pp.1-6
• /
• 2013

A lot of works have been dedicated to clarify the reasons why the establishment of embryonic stem cells (ESCs) from pig is more difficult than that from mouse and human. Several concomitant factors such as culture condition including feeder layer, sensitivity of cell to cell contact, definitive markers of pluripotency for evaluation of the validity and optimal timing of derivation have been suggested as the disturbing factors in the establishment of porcine ESCs Traditionally, attempts to derive stem cells from porcine embryos have depend on protocols established for mouse ESCs using inner cell mass (ICM) for the isolation and culture. And more recently, protocols used for primate ESCs were also applied. However, there is no report for the establishment of porcine ESCs. Indeed, ungulate species including pigs have crucial developmental differences unlike rodents and primates. Here we will review recent studies about issues for establishment of porcine ESCs and discuss the promise and strategies focusing on the timing for derivation and pluripotent state of porcine ESCs.