• 한국수정란이식학회지
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• 제32권3호
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• pp.81-86
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• 2017

The Somatic cell nuclear transfer (SCNT) method can be applied to various fields such as species conservation, regenerative medicine, farming industries and drug production. However, the efficiency using SCNT is very low for many reasons. One of the troubles of SCNT is that it is highly dependent on the researcher's competence. For that reason, four somatic cell nuclear injection methods were compared to evaluate the effect of hole-sealing process and existence of cytochalasin B (CB) on efficiency of murine SCNT protocol. As a results, the microinjection with the hole-sealing process, the oocyte plasma membrane is inhaled with injection pipette, in HCZB with CB was presented to be the most efficient for the reconstructed in SCNT process. In addition, we demonstrated that the oocytes manipulated in Hepes-CZB medium (HCZB) with CB does not affect the developmental rate and the morphology of the blastocyst during the pre-implantation stage. For this reason, we suggest the microinjection involving hole-sealing in HCZB with CB could improve SCNT process efficiency.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.223-228
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• 2009

We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum-starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or $150\;{\mu}M$ roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine-treated groups (27.6%) than that of the roscovitine-treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine-treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine-treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine-treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine-treatment creates a more suitable condition for nuclear reprogramming after SCNT.

• 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.

• 생명과학회지
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• 제30권4호
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• pp.394-401
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• 2020

배아줄기세포는 만능세포이기 때문에 동물에게 주입되면 종양으로 발달할 수도 있다. 따라서 연구자들은 종양 형성으로부터 비교적 자유로운 성체세포로부터 세포 특이적 줄기세포(성체줄기세포)를 확보하는데 관심을 두고 있다. 성체줄기세포는 제한적으로 세포분열을 할 수 있고 지정된 특정 세포로만 발달할 수 있다. 포유동물에서 각 조직의 세포들은 자연적 생리조건하에서는 역분화 혹은 교차분화에 의해 성체줄기세포로 전환되지 않는다. 따라서 일본 연구자들에 의하여 2006년 성체세포의 리프로그램에 의한 유도만능줄기세포(iPSCs) 기술이 소개되어 성체줄기세포 연구의 새로운 장을 열었다. 비록 연구현장에서 iPSCs 기술이 폭 넓게 이용되지만, 리프로그램의 안정성뿐만 아니라 유전체에 외래유전자의 도입 등의 문제점도 있다. Reversine은 iPSCs 보다 2년 앞서 발견된 작은 화학적 합성 분자인 퓨린 유사체이다. Reversine은 분화된 세포를 리프로그램에 의한 역분화를 유도하여 다능성 줄기세포로 전환시킬 수 있으며, 적절한 분화조건하에서 다른 세포로 교차분화를 유도할 수도 있다. 따라서 reversine은 iPSCs가 가지고 있는 문제점을 극복하고 화학적인 방법을 이용하여 성체세포를 다능성 줄기세포로 전환시킬 수 있는 물질로 활용될 수 있다. Reversine이 백색지방세포를 갈색지방형세포(beige cell)로 전변시켜 열발산에 의한 에너지소비를 촉진함을 제시하여 항비만인자로서 그 가능성을 열어 놓았다. Reversine은 세포 역분화의 기능적 역할 이외에 항암 인자로서 또 다른 기능들이 보고되고 있어 앞으로 여러 분야에서 그 이용성이 기대되는 물질이다.

• Journal of Genetic Medicine
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• 제9권2호
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• pp.67-72
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• 2012

The generation of induced pluripotent stem cells (iPSCs) derived from patients' somatic cells provides a new paradigm for studying human genetic diseases. Human iPSCs which have similar properties of human embryonic stem cells (hESCs) provide a powerful platform to recapitulate the disease-specific cell types by using various differentiation techniques. This promising technology has being realized the possibility to explore pathophysiology of many human genetic diseases at the molecular and cellular levels. Furthermore, disease-specific human iPSCs can also be used for patient-based drug screening and new drug discovery at the stage of the pre-clinical test in vitro. In this review, we summarized the concept and history of cellular reprogramming or iPSC generation and highlight recent progresses for disease modeling using patient-specific iPSCs.

• International Journal of Stem Cells
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• 제16권1호
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• pp.27-35
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• 2023

Background and Objectives: Spermatogonial stem cells (SSCs) are the most primitive cells in spermatogenesis and are the only adult stem cells capable of passing on the genome of a given species to the next generation. SSCs are the only adult stem cells known to exhibit high Oct4 expression and can be induced to self-reprogram into pluripotent cells depending on culture conditions. Epigenetic modulation is well known to be involved in the induction of pluripotency of somatic cells. However, epigenetic modulation in self-reprogramming of SSCs into pluripotent cells has not been studied. Methods and Results: In this study, we examined the involvement of epigenetic modulation by assessing whether selfreprogramming of SSCs is enhanced by treatment with epigenetic modulators. We found that second-generation selective class I HDAC inhibitors increased SSC reprogramming efficiency, whereas non-selective HDAC inhibitors had no effect. Conclusions: We showed that pluripotent stem cells derived from adult SSCs by treatment with small molecules with epigenetic modulator functions exhibit pluripotency in vitro and in vivo. Our results suggest that the mechanism of SSC reprogramming by epigenetic modulator can be used for important applications in epigenetic reprogramming research.

• International Journal of Oral Biology
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• 제39권4호
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• pp.177-185
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• 2014

Transfection is a gene delivery tool that is a popular means of manipulating cellular properties, such as induced pluripotent stem cell (iPSC) generation by reprogramming factors (Yamanaka factors). However, the efficiency of transfection needs to be improved. In the present study, three transfection protocols - non-liposomal transfection (NLT), magnetofection and electroporation - were compared by analysis of their transfection efficiencies and cell viabilities using human dental pulp cells (hDPC) and bovine fetal fibroblasts (bFF) as cell sources. Enhanced green fluorescent protein gene was used as the delivery indicator. For magnetofection, Polymag reagent was administrated. NLT, FuGENE-HD and X-treme GENE 9 DNA transfection reagents were used for NLT. For electroporation, the $Neon^{TM}$ and $NEPA21^{TM}$ electroporators were tested. $Neon^{TM}$ electroporation showed highest transfection efficiency when compared with NLT, magnetofection, and $NEPA21^{TM}$ electroporation, with transfection efficiency of about 33% in hDPC and 50% in bFF, based on viable cell population in each cell type. These results suggest that transfection by $Neon^{TM}$ electroporation can be used to deliver foreign genes efficiently in human and bovine somatic cells.

• International Journal of Stem Cells
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• 제16권2호
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• pp.156-167
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• 2023

Background and Objectives: Cellular reprogramming in regenerative medicine holds great promise for treating patients with neurological disorders. In this regard, small molecule-mediated cellular conversion has attracted special attention because of its ease of reproducibility, applicability, and fewer safety concerns. However, currently available protocols for the direct conversion of somatic cells to neurons are limited in clinical application due of their complex nature, lengthy process, and low conversion efficiency. Methods and Results: Here, we report a new protocol involving chemical-based direct conversion of human fibroblasts (HF) to matured neuron-like cells with a short duration and high conversion efficiency using temporal and strategic dual epigenetic regulation. In this protocol, epigenetic modulation by inhibition of histone deacetylase and bromodomain enabled to overcome "recalcitrant" nature of adult fibroblasts and shorten the duration of neuronal reprogramming. We further observed that an extended epigenetic regulation is necessary to maintain the induced neuronal program to generate a homogenous population of neuron-like cells. Conclusions: Therefore, our study provides a new protocol to produce neurons-like cells and highlights the need of proper epigenetic resetting to establish and maintain neuronal program in HF.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.7-8
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• 2003

Since it was first reported in 1997, somatic cell cloning has been demonstrated in several other mammalian species. On the mouse, it can be cloned from embryonic stem (ES) cells, fetus-derived cells, and adult-derived cells, both male and female. While cloning efficiencies range from 0 to 20%, rates of just 1-2% are typical (i.e. one or two live offspring per one hundred initial embryos). Recently, abnormalities in mice cloned from somatic cells have been reported, such as abnormal gene expression in embryo (Boiani et al., 2001, Bortvin et al., 2003), abnormal placenta (Wakayama and Yanagimachi 1999), obesity (Tamashiro et ai, 2000, 2002) or early death (Ogonuki et al., 2002). Such abnormalities notwithstanding, success in generating cloned offspring has opened new avenues of investigation and provides a valuable tool that basic research scientists have employed to study complex processes such as genomic reprogramming, imprinting and embryonic development. On the other hand, mouse ES cell lines can also be generated from adult somatic cells via nuclear transfer. These 'ntES cells' are capable of differentiation into an extensive variety of cell types in vitro, as well assperm and oocytes in vivo. Interestingly, the establish rate of ntES cell line from cloned blastocyst is much higher than the success rate of cloned mouse. It is also possible to make cloned mice from ntES cell nuclei as donor, but this serial nuclear transfer method could not improved the cloning efficiency. Might be ntES cell has both character between ES cell and somatic cell. A number of potential agricultural and clinical applications are also are being explored, including the reproductive cloning of farm animals and therapeutic cloning for human cell, tissue, and organ replacement. This talk seeks to describe both the relationship between nucleus donor cell type and cloning success rate, and methods for establishing ntES cell lines. (중략)

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2001년도 제40차 춘계학술대회
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• pp.49.1-49.1
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• 2001