• BMB Reports
• /
• 제51권10호
• /
• pp.500-507
• /
• 2018

Cell reprogramming has been considered a powerful technique in the regenerative medicine field. In addition to diverse its strengths, cell reprogramming technology also has several drawbacks generated during the process of reprogramming. Telomere shortening caused by the cell reprogramming process impedes the efficiency of cell reprogramming. Transcription factors used for reprogramming alter genomic contents and result in genetic mutations. Additionally, defective mitochondria functioning such as excessive mitochondrial fission leads to the limitation of pluripotency and ultimately reduces the efficiency of reprogramming. These problems including genomic instability and impaired mitochondrial dynamics should be resolved to apply cell reprograming in clinical research and to address efficiency and safety concerns. Sirtuin (NAD+-dependent histone deacetylase) has been known to control the chromatin state of the telomere and influence mitochondria function in cells. Recently, several studies reported that Sirtuins could control for genomic instability in cell reprogramming. Here, we review recent findings regarding the role of Sirtuins in cell reprogramming. And we propose that the manipulation of Sirtuins may improve defects that result from the steps of cell reprogramming.

• 한국발생생물학회지:발생과생식
• /
• 제20권2호
• /
• pp.141-147
• /
• 2016

Rad51 is a key component of homologous recombination (HR) to repair DNA double-strand breaks and it forms Rad51 recombinase filaments of broken single-stranded DNA to promote HR. In addition to its role in DNA repair and cell cycle progression, Rad51 contributes to the reprogramming process during the generation of induced pluripotent stem cells. In light of this, we performed reprogramming experiments to examine the effect of co-expression of Rad51 and four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, on the reprogramming efficiency. Co-expression of Rad51 significantly increased the numbers of alkaline phosphatase-positive colonies and embryonic stem cell-like colonies during the process of reprogramming. Co-expression ofRad51 significantly increased the expression of epithelial markers at an early stage of reprogramming compared with control cells. Phosphorylated histone H2AX (${\gamma}H2AX$), which initiates the DNA double-strand break repair system, was highly accumulated in reprogramming intermediates upon co-expression of Rad51. This study identified a novel role of Rad51 in enhancing the reprogramming efficiency, possibly by facilitating mesenchymal-to-epithelial transition and by regulating a DNA damage repair pathway during the early phase of the reprogramming process.

• 한국가축번식학회지
• /
• 제27권4호
• /
• pp.349-357
• /
• 2003

Although nuclear transfer (NT) techniques are used to clone animals, its efficiency is very low. Moreover, nuclear transfer has resulted in offspring with severe developmental problems, probably due to incomplete nuclear reprogramming. Nuclear reprogramming is characterized by functional modification of the transferred nucleus to allow it to direct normal embryo development with the potential to grow to term. Although the nature of the reprogramming factor(s) in mammals is not clear, various nuclear as well as cytoplasmic components are involved in the processes. In this article we review recent data on factors involved in the nuclear reprogramming of cloned embryos.

• BMB Reports
• /
• 제52권5호
• /
• pp.324-329
• /
• 2019

Recent progress in cellular reprogramming technology and lineage-specific cell differentiation has provided great opportunities for translational research. Because virus-based gene delivery is not a practical reprogramming protocol, protein-based reprogramming has been receiving attention as a safe way to generate reprogrammed cells. However, the poor efficiency of the cellular uptake of reprogramming proteins is still a major obstacle. Here, we reported key factors which improve the cellular uptake of these proteins. Purified red fluorescent proteins fused with 9xLysine (dsRED-9K) as a cell penetrating peptide were efficiently delivered into the diverse primary cells. Protein delivery was improved by the addition of amodiaquine. Furthermore, purified dsRED-9K was able to penetrate all cell lineages derived from mouse embryonic stem cells efficiently. Our data may provide important insights into the design of protein-based reprogramming or differentiation protocols.

• 한국수정란이식학회지
• /
• 제26권1호
• /
• pp.85-89
• /
• 2011

Somatic cell nuclear transfer (SCNT) is a useful tool for reproducing genetically identical animals or producing transgenic animals. Many reports have demonstrated that the efficiency of animal cloning by SCNT requires reprogramming of the somatic nucleus to a totipotent like-state. The SCNT-related reprogramming might mimic the natural reprogramming process that occurs during normal mammalian development. However, recent evidence indicates that the reprogramming event by SCNT is incomplete. In this study, the traditional SCNT procedure (TNT) was modified by injecting donor nuclei into recipient cytoplasm prior to the enucleation process to expose the donor nucleus before removing the karyoplast containing the chromosomes of the oocytes which might possess additional reprogramming factors, and this modified technique was named as reversing the usual order of SCNT (RONT). Other procedures including activation and in vitro culture were the same as TNT. Contrary to expectations, the rate of blastocyst development was not different significantly between RONT and TNT (8.6% and 7.9%, respectively). However, duration of micromanipulation performed by the same technician and equipments was remarkably reduced because the ruptured oocytes after nuclear injection were excluded from the enucleation process. This study suggests that RONT, a simplified SCNT protocol, shortens the duration of SCNT procedure and this less time-costing protocol may enable the researchers to perform murine SCNT easier.

• 한국발생생물학회지:발생과생식
• /
• 제14권2호
• /
• pp.43-50
• /
• 2010

최근에 체세포 리프로그래밍 기법을 사용하여 체세포에 몇 가지 전사인자(리프로그래밍 인자)를 넣어줌으로써 유도만능줄기세포(induced pluripotent stem cell, iPS)를 만드는데 성공하였다. 유도만능줄기세포는 배아줄기세포와 유사하게 자가재생 할 수 있는 능력이 있으며, 신체의 모든 타입의 세포로 분화할 수 있는 특징을 가지고 있다. 배아줄기세포와는 달리 면역거부반응이 없다는 점과 윤리적인 문제로부터 자유롭다는 장점이 있어 2006년 Yamanaka 팀이 유도만능줄기세포에 관해 처음 보고한 이후로 이 분야 연구의 급속한 발전이 이루어지고 있다. 하지만 안전성의 문제점 때문에 세포치료제로 사용되기 위해서는 리프로그래밍 인자의 도입 방법 및 새로운 리프로그래밍 인자의 발굴 등 몇 가지 해결해야 할 점들이 남아 있다. 본 종설에서는 유도만능줄기세포를 만드는데 사용된 몇 가지 리프로그래밍 인자에 대해 보고된 연구 내용을 리프로그래밍 인자가 존재하는 세포인 배아줄기세포 및 난자와 배아에서 정리하고자 하며, 리프로그래밍 인자의 연구에 관한 방향에 대해 논의하고자 한다.

• Reproductive and Developmental Biology
• /
• 제32권2호
• /
• pp.135-140
• /
• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• 한국위성정보통신학회논문지
• /
• 제12권3호
• /
• pp.93-97
• /
• 2017

위성의 임무 수행 도중에 발생하는 소프트웨어 버그는 치명적인 문제점을 야기하여 위성 전체의 임무 실패를 초래할 수도 있다. 이를 막기 위해 개발단계부터 수많은 테스트와 검증을 수행하여 비행소프트웨어가 높은 신뢰도를 지니도록 만들어 준다 [1]. 하지만 위성이 발사 후 궤도에 올라갔을 때 하드웨어 측면에서의 문제, 혹은 미 발견 버그 등의 예상치 못한 문제들이 발견될 수도 있다. 이 경우 비행소프트웨어를 궤도 상에서 수정해야만 위성이 지속적으로 임무를 수행할 수 있다. 본 논문에서는 저궤도 위성의 reprogramming capability를 확인하고 reprogramming 절차에 대해 알아보고 검증한다.

• Molecules and Cells
• /
• 제41권7호
• /
• pp.631-638
• /
• 2018

Spermatogonial stem cells (SSCs) derived from mouse testis are unipotent in regard of spermatogenesis. Our previous study demonstrated that SSCs can be fully reprogrammed into pluripotent stem cells, so called germline-derived pluripotent stem cells (gPS cells), on feeder cells (mouse embryonic fibroblasts), which supports SSC proliferation and induction of pluripotency. Because of an uncontrollable microenvironment caused by interactions with feeder cells, feeder-based SSC reprogramming is not suitable for elucidation of the self-reprogramming mechanism by which SSCs are converted into pluripotent stem cells. Recently, we have established a Matrigel-based SSC expansion culture system that allows longterm SSC proliferation without mouse embryonic fibroblast support. In this study, we developed a new feeder-free SSC self-reprogramming protocol based on the Matrigel-based culture system. The gPS cells generated using a feeder-free reprogramming system showed pluripotency at the molecular and cellular levels. The differentiation potential of gPS cells was confirmed in vitro and in vivo. Our study shows for the first time that the induction of SSC pluripotency can be achieved without feeder cells. The newly developed feeder-free self-reprogramming system could be a useful tool to reveal the mechanism by which unipotent cells are self-reprogrammed into pluripotent stem cells.

• 한국동물생명공학회지
• /
• 제38권4호
• /
• pp.199-208
• /
• 2023

Background: Canine induced pluripotent stem cells (iPSCs) are an attractive source for veterinary regenerative medicine, disease modeling, and drug development. Here we used vitamin C (Vc) to improve the reprogramming efficiency of canine iPSCs, and its functions in the reprogramming process were elucidated. Methods: Retroviral transduction of Oct4, Sox2, Klf4, c-Myc (OSKM), and GFP was employed to induce reprogramming in canine fetal fibroblasts. Following transduction, the culture medium was subsequently replaced with ESC medium containing Vc to determine the effect on reprogramming activity. Results: The number of AP-positive iPSC colonies dramatically increased in culture conditions supplemented with Vc. Vc enhanced the efficacy of retrovirus transduction, which appears to be correlated with enhanced cell proliferation capacity. To confirm the characteristics of the Vc-treated iPSCs, the cells were cultured to passage 5, and pluripotency markers including Oct4, Sox2, Nanog, and Tra-1-60 were observed by immunocytochemistry. The expression of endogenous pluripotent genes (Oct4, Nanog, Rex1, and telomerase) were also verified by PCR. The complete silencing of exogenously transduced human OSKM factors was observed exclusively in canine iPSCs treated with Vc. Canine iPSCs treated with Vc are capable of forming embryoid bodies in vitro and have spontaneously differentiated into three germ layers. Conclusions: Our findings emphasize a straightforward method for enhancing the efficiency of canine iPSC generation and provide insight into the Vc effect on the reprogramming process.