인간태아 섬유아세포와 생쥐배아 섬유아세포를 기저세포로 활용한 인간 배아줄기세포의 확립

조혜원,고경래,김미경,이재익,신수일,이동형,김기형,이규섭,Cho, Hye Won,Ko, Kyoung Rae,Kim, Mi Kyoung,Lee, Jae Ik,Sin, Su Il,Lee, Dong Hyung,Kim, Ki Hyung,Lee, Kyu Sup 대한생식의학회 2005 Clinical and Experimental Reproductive Medicine Vol.32 No.2

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.

체외수정 유래 생쥐 배아줄기세포와 유사한 특성을 보유한 단위발생 유래 생쥐 배아줄기세포

박세필,김은영,이금실,이영재,신현아,민현정,이훈택,정길생,임진호,Park, Se-Pill,Kim, Eun-Young,Lee, Keum-Si,Lee, Young-Jae,Shin, Hyun-Ah,Min, Hyun-Jung,Lee, Hoon-Taek,Chung, Kil-Saeng,Lim, Jin-Ho 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.2

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.

체외수정 생쥐 배아에서의 배아 줄기세포 확립

문신용,박용빈,김희선,오선경,천대우,서창석,최영민,김정구,이진용,김석현,Shin, Yong-Moon,Park, Yong-Bin,Kim, Hee-Sun,Oh, Sun-Kyung,Chun, Dae-Woo,Suh, Chang-Suk,Choe, Young-Min,Kim, Jung-Gu,Lee, Jin-Yong,Kim, Seok-Hyun 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.1

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. Materials and Methods: After Fl hybrid (C57BL female $\times$ CBA mael) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). Results: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. Conclusion: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

동결-융해된 인간 배반포기 배 유래의 배아 간(幹) 세포 배양

김은영,남화경,이금실,박세영,박은미,윤지연,허영태,조현정,박세필,정길생,임진호,Kim, Eun-Young,Nam, Hwa-Kyung,Lee, Keum-Sil,Park, Sae-Young,Park, Eun-Mi,Yoon, Ji-Yeon,Heo, Young-Tae,Cho, Hyun-Jung,Park, Se-Pill,Chung, Kil-Saeng,Lim, Jin 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.1

Objective: This study was to establish the human embryonic stem (ES) cells derived from frozen-thawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. Methods: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. Results: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and ${\beta}$-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. Conclusion: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.

인간배아줄기세포연구의 민사법적 의미

김민중(KIM Min-Joong) 한국법학원 2008 저스티스 Vol.- No.103

인간배아줄기세포연구에 대한 사회적 논쟁이 뜨겁다. 인간배아줄기세포연구가 척수장애, 파킨슨, 알츠하이머, 뇌졸중, 당뇨와 같은 난치병을 퇴치할 수 있다고 하는 의미에서는 이보다 인류에 더 공헌할 수 있는 일을 찾기 어렵다고 할 수 있다. 그러나 인간배아줄기세포연구가 사회적, 윤리적, 법적으로 아무런 문제점도 없다고 할 수는 없다. 인간배아줄기세포의 연구나 이용과 관련하여 선결하여야 할 과제도 적지 않고, 인간배아줄기세포연구에는 과학적인 문제뿐만 아니라 여러 가지 해결하여야 할 윤리적, 법적 문제가 내포되어 있다는 사실을 부인할 수 없다. 현재 인간배아줄기세포의 연구나 이용에 관한 생명윤리의 문제는 국내에서 뿐만 아니라, 전세계적으로도 대단한 논란이 되고 있다. 또한 인간배아줄기세포연구가 야기하는 문제는 생명윤리의 문제에 국한되지 아니한다. 법적으로도 인간배아줄기세포연구와 관련한 다양한 문제가 제기된다. 인간배아줄기세포연구에 관한 근본적인 법적 문제로서는 인간의 존엄성와 관련한 인권문제가 제기된다. 그리고 예를 들어 인간배아 줄기세포연구를 형법적으로 금지하여야 하는가 하는 형법상의 문제도 제기된다. 역시 인간배아줄기세포연구에서의 법적 문제에 관한 핵심적인 내용의 하나는 인간배아줄기세포연구를 둘러싼 민사법적 과제라고 할 수 있다. 인간배아줄기세포연구와 관련하여 제기되는 민사법적 과제는 매우 다양하리라고 예상되며, 계약법적 문제를 비롯하여 책임법적 문제, 가족법적 문제에까지 민법 전반에 걸쳐 문제가 제기될 수 있다. 우선 인간배아줄기세포연구와 관련하여 다양한 계약관계가 성립할 수 있다. 예를 들어 인간배아줄기세포연구를 위하여는 배아, 특히 난자가 필요하므로, 배아나 난자를 제공받기 위하여는 배아제공자 및 난자 제공자와의 사이에 배아제공계약 또는 난자제공계약이 성립한다. 또한 줄기세포를 난치성 질환을 치료하기 위한 세포치료의 재료로 사용하는 경우에는 환자와의 사이에 첨단의료행위로서의 임상실험이나 줄기세포치료를 실시하기 위한 임상실험계약 또는 의료계약이 성립한다. 인간배아줄기세포연구와 관련한 임상실험을 통하여 피실험자나 제3자가 손해를 입을 수 있다. 만약 줄기세포연구를 적용한 임상실험에서 피실험자나 제3자에게 손해가 발생하면 손해배상책임이 문제되고, 보통 임상실험으로 생긴 손해에 따른 피실험자에 대한 책임은 계약책임이 되고, 제3자에 대한 책임은 불법행위책임으로 된다. The debate over the treatment of human stem cell is new. Ever since human stem cells were first isolated, the possible applications of stem cell research and the moral and legal issues surrounding human stem cell research have created much controversy. Now human stem cell research is a controversial and divisive topic. Research on embryonic stem cells has generated great intrigue in the scientific community. Many medical researchers consider stem cell-based therapies to have the potential of treating a host of human illnesses and yielding a number of medical benefits. However, the embryonic stem cell research raises numerous ethical and legal concerns. The embryonic stem cell research destroys the human embryo. This has generated a storm of debate about if this research can be legally and ethically justified. Human embryonic stem cell is 'master cell', able to develop into almost any cell in the human body. The research with human embryonic stem cells raises complex and sensitive legal questions that should be considered carefully and discussed widely. The civil legal debate surrounding human embryonic stem cell research, will be the focus of this paper.

Effects of Various Addition and Exclusion Time of Glucose on Development of Mouse Two-Cell Embryos

Park, S. B.,Park, K. S.,Lee, T. H.,Chun, S. S.,Kim, K. S.,Song, H. B. 한국동물생명공학회(구 한국동물번식학회) 2004 Reproductive & developmental biology Vol.28 No.4

This study was conducted to investigate the effect of various addition and exclusion time of glucose (Control: no addition, A: 24~72 h, B: 24~48 h, C: 48~72 h, D: 0~72 h, E: 0~48 h, F: 0~24 h and 48~72 h, G: 0~24 h) on embryonic developmental capacity of 2-cell embryos in mice. Developed blastocysts were assessed for mean cell number by differential staining. The zona-intact blastocyst (ZiB) rates were higher (p<0.05) in group B than control. However, the zona-escape blastocyst (ZeB) rates were not significantly different in all groups. At 72 h, total blastocyst (ZiB + ZeB) formation rates were not significantly different in all groups. The mean cell number was not significantly different among all groups. The inner cell mass (ICM) cell number was higher (p<0.05) in group F than control, group A, B and G. The trophectoderm (TE) cell number was higher (p<0.05) in control than group A and D. The %ICM was higher (p<0.05) in group C, D and F than control. The ICM : TE ratio was not significantly different in all groups. Between control and glucose group, no significant difference was observed in the total blastocysts (ZiB + ZeB) formation rates. Also, no significant difference was observed in the mean cell number, ICM cell number and ICM : TE ratio. However the TE cell number was higher (p<0.05) in control than glucose group and %ICM was higher (p<0.05) in glucose group than control. In conclusion, glucose added in culture medium was not inhibitory on blastocyst formation but glucose added for 48 ~72 h in culture medium increases %ICM of blastocysts in mice.

Effects of Various Addition and Exclusion Time of Glucose on Development of Mouse Two-Cell Embryos

Park, S. B.,Park, K. S.,Lee, T. H.,Chun, S. S.,Kim, K. S.,Song, H. B. 한국동물번식학회 2004 Reproductive & developmental biology Vol.28 No.4

This study was conducted to investigate the effect of various addition and exclusion time of glucose (Control: no addition, A: 24~72 h, B: 24~48 h, C: 48~72 h, D: 0~72 h, E: 0~48 h, F: 0~24 h and 48~72 h, G: 0~24 h) on embryonic developmental capacity of 2-cell embryos in mice. Developed blastocysts were assessed for mean cell number by differential staining. The zona-intact blastocyst (ZiB) rates were higher (p<0.05) in group B than control. However, the zona-escape blastocyst (ZeB) rates were not significantly different in all groups. At 72 h, total blastocyst (ZiB + ZeB) formation rates were not significantly different in all groups. The mean cell number was not significantly different among all groups. The inner cell mass (ICM) cell number was higher (p<0.05) in group F than control, group A, B and G. The trophectoderm (TE) cell number was higher (p<0.05) in control than group A and D. The %ICM was higher (p<0.05) in group C, D and F than control. The ICM : TE ratio was not significantly different in all groups. Between control and glucose group, no significant difference was observed in the total blastocysts (ZiB + ZeB) formation rates. Also, no significant difference was observed in the mean cell number, ICM cell number and ICM : TE ratio. However the TE cell number was higher (p<0.05) in control than glucose group and %ICM was higher (p<0.05) in glucose group than control. In conclusion, glucose added in culture medium was not inhibitory on blastocyst formation but glucose added for 48 ~72 h in culture medium increases %ICM of blastocysts in mice.

Potential functional roles of follistatin on bovine somatic cell nuclear transfer embryos

Lee, Kyung-Bon,Woo, Jae-Seok,Lee, Bo-Myoung,Park, Kang-Sun,Han, Kil-Woo,Kim, Min Kyu Institute of Agricultural Science 2013 Korean Journal of Agricultural Science Vol.40 No.4

To demonstrate that follistatin treatment enhances the efficiency of nuclear transfer (SCNT), cell allocation and preimplantational development were determined in bovine SCNT embryos in the present study. Treatment of activated SCNT embryos with 10 ng/ml follistatin significantly increased the proportion of blastocyst development compared to untreated SCNT embryos. In addition, an increase in trophectoderm (TE) cell numbers and relatively higher proportion of TE cells to total cells were observed, but the number of inner cell mass (ICM) cell and total cell numbers were not changed (P < 0.05). No significant effect of other doses of follistatin was observed for the above endpoints. However, treatment with 1 and 10 ng/ml follistatin reduced the proportion of nuclear transfer blastocysts with an ICM ratio of > 60% relative to untreated nuclear transfer blastocysts at Day 7. No significant effect of follistatin treatment on proportions of nuclear transfer blastocysts with ICM ratio of 20-40% or 40-60% was observed. Taken together, these results suggested that follistatin can be used to increase developmental competence of SCNT embryos in terms of cell allocation, particularly TE cells, during preimplantation stages, subsequently enhancing placentation and birth of live offspring.

Potential functional roles of follistatin on bovine somatic cell nuclear transfer embryos

Kyung-Bon Lee,Jae-Seok Woo,Bo-Myoung Lee,Kang-Sun Park,Kil-Woo Han,Min Kyu Kim 충남대학교 농업과학연구소 2013 농업과학연구 Vol.40 No.4

To demonstrate that follistatin treatment enhances the efficiency of nuclear transfer (SCNT), cell allocation and preimplantational development were determined in bovine SCNT embryos in the present study. Treatment of activated SCNT embryos with 10 ng/ml follistatin significantly increased the proportion of blastocyst development compared to untreated SCNT embryos. In addition, an increase in trophectoderm (TE) cell numbers and relatively higher proportion of TE cells to total cells were observed, but the number of inner cell mass (ICM) cell and total cell numbers were not changed (P < 0.05). No significant effect of other doses of follistatin was observed for the above endpoints. However, treatment with 1 and 10 ng/ml follistatin reduced the proportion of nuclear transfer blastocysts with an ICM ratio of > 60% relative to untreated nuclear transfer blastocysts at Day 7. No significant effect of follistatin treatment on proportions of nuclear transfer blastocysts with ICM ratio of 20-40% or 40-60% was observed. Taken together, these results suggested that follistatin can be used to increase developmental competence of SCNT embryos in terms of cell allocation, particularly TE cells, during preimplantation stages, subsequently enhancing placentation and birth of live offspring.

마우스에서 배반포 형성, 세포 수 및 ICM의 비율에 미치는 배양액의 효과

박기상,이택후,전상식,송해범,Park, Kee-Sang,Lee, Taek-Hoo,Chun, Sang-Sik,Song, Hai-Bum 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.4

Objective : The aim of this study was to evaluate the influence of different media on blastulation, mean cell number, percentage of inner cell mass (ICM) of total cells and ICM : trophectoderm (TE) ratio in mice. Materials and methods: A total 552 two cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection (mated just after hCG injection) and cultured in MEM (n=276) or TCM (n=276) supplemented with 20% hFF. The grading of blastocysts from zona-intact (ZiB) to -escape (hatching and hatched, ZeB) was performed at 72 hours after culture. Total, TE and ICM cell numbers were analyzed by differential staining of blastocyst. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values < 0.05 were accepted as statistically significant. Results: The blastulation rate in MEM ($64.9{\pm}4.95%$) was significantly higher (p=0.0031) than that in TCM ($57.2{\pm}5.22%$). No differences were found in the number of ZiB and ZeB between MEM ($31.9{\pm}2.62$, $33.0{\pm}4.58%$), and TCM ($27.2{\pm}4.28$, $30.1{\pm}4.58%$). A total 314 blastocysts (MEM=166; TCM=148) were stained differentially. Mean cell number of blastocysts was significantly higher (p=0.0002) in TCM ($73.1{\pm}3.3$) than in MEM ($61.7{\pm}2.5$). Differential staining was successfully performed in 155 blastocysts (MEM=77; TCM=78). The percentage of ICM was significantly higher in MEM than in TCM ($20.9{\pm}1.3$ vs. $17.1{\pm}1.2%$, p=0.0281). The ICM : TE ratio was higher in TCM than in MEM (1 : $4.85{\pm}0.68$ vs. 1 : $3.78{\pm}0.78$, NS). Conclusion: These results show that MEM increase the blastocyst formation and percentage of ICM of total cells comparing with TCM in mice.