• BMB Reports
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• 제42권2호
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• pp.72-80
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• 2009

In mammals, major progress has recently been made with the dissection of early embryonic cell specification, the isolation of stem cells from early embryos, and the production of embryonic-like stem cells from adult cells. These studies have overcome long-standing species barriers for stem cell isolation, have revealed a deeper than expected similarity of embryo cell types across species, and have led to a better understanding of the lineage identities of embryo-derived stem cells, most notably of mouse and human embryonic stem (ES) cells. Thus, it has now become possible to propose a species-overarching classification of embryo stem cells, which are defined here as pre- to early post-implantation conceptus-derived stem cell types that maintain embryonic lineage identities in vitro. The present article gives an overview of these cells and discusses their relationships with each other and the conceptus. Consequently, it is debated whether further embryo stem cell types await isolation, and the study of the earliest extraembryonically committed stem cells is identified as a promising new research field.

• 한국동물생명공학회지
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• 제36권4호
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• pp.175-182
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• 2021

Pluripotent stem cells could self-renew and differentiate into various cells. In particular, porcine pluripotent stem cells are useful for preclinical therapy, transgenic animals, and agricultural usage. These stem cells have naïve and primed pluripotent states. Naïve pluripotent stem cells represented by mouse embryonic stem cells form chimeras after blastocyst injection. Primed pluripotent stem cells represented by mouse epiblast stem cells and human embryonic stem cells. They could not produce chimeras after blastocyst injection. Populations of embryonic stem cells are not homogenous; therefore, reporter systems are used to clarify the status of stem cells and to isolate the cells. For this reason, studies of the OCT4 reporter system have been conducted for decades. This review will discuss the naïve and primed pluripotent states and recent progress in the development of porcine OCT4 reporter systems.

• 한국미생물·생명공학회지
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• 제32권1호
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• pp.11-15
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• 2004

본 연구에서는 C. diphtheriae toxin-A(DTA)를 합성하는 유전자를 tetracycline derivative인 doxycycline에 의해 발현이 유도되는 plasmid('Tet-on' system)에 삽입시켜, 이를 mouse ES cell에 도입시켰으며, 이렇게 제작된 mouse ES cell이 doxycycline의 처리 농도에 따라 mouse ES cell내의 DTA의 발현이 유도되어 이 결과 세포 사별(apoptosis)을 유발시키는 것을 MTT assay를 통해 확인하였다.

• Toxicological Research
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• 제29권4호
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• pp.221-227
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• 2013

Embryonic stem (ES) cells have potential for use in evaluation of developmental toxicity because they are generated in large numbers and differentiate into three germ layers following formation of embryoid bodies (EBs). In earlier study, embryonic stem cell test (EST) was established for assessment of the embryotoxic potential of compounds. Using EBs indicating the onset of differentiation of mouse ES cells, many toxicologists have refined the developmental toxicity of a variety of compounds. However, due to some limitation of the EST method resulting from species-specific differences between humans and mouse, it is an incomplete approach. In this regard, we examined the effects of several developmental toxic chemicals on formation of EBs using human ES cells. Although human ES cells are fastidious in culture and differentiation, we concluded that the relevancy of our experimental method is more accurate than that of EST using mouse ES cells. These types of studies could extend our understanding of how human ES cells could be used for monitoring developmental toxicity and its relevance in relation to its differentiation progress. In addition, this concept will be used as a model system for screening for developmental toxicity of various chemicals. This article might update new information about the usage of embryonic stem cells in the context of their possible ability in the toxicological fields.

• Asian-Australasian Journal of Animal Sciences
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• 제22권3호
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• pp.343-349
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• 2009

This study was designed to simplify a cryopreservation program for embryonic stem cells (ESCs) by selection of cooling method and cryoprotectant. Commercially available mouse E14 embryonic stem cells (ESCs) were cryopreserved with various protocols, and morphology and viability of the frozen-thawed ESCs and their reactive oxygen species (ROS) production were subsequently monitored. Post-thaw colony-formation of ESCs was detected only after a slow freezing using dimethyl sulfoxide (DMSO) by stepwise placement of a freezing container into a $-80^{\circ}C$ deep freezer and subsequently into -$196^{\circ}C$ liquid nitrogen, while no proliferation was detected after vitrification. When the simplified protocol was employed, the replacement of DMSO with a mixture of DMSO and ethylene glycol (EG) further improved the post-thaw survival. ROS generation in ESCs frozen-thawed with the optimized protocol was not increased compared with non-frozen ESCs. The use of fresh mouse embryonic fibroblasts as feeder cells for post-thaw subculture did not further increase post-thaw cell viability. In conclusion, a simplified slow-freezing program without employing programmable freezer but using DMSO and EG was developed which maintains cell viability and colony-forming activity of ESCs during post-thaw subculture.

• Asian-Australasian Journal of Animal Sciences
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• 제23권9호
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• pp.1152-1158
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• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• Animal cells and systems
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• 제13권2호
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• pp.235-245
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• 2009

Embryonic stem (ES) cells have a capability to generate all types of cells. However, the mechanism by which ES cells differentiate into specific cell is still unclear. Using microarray technology, the differentiation process in mouse embryonic stem cells was characterized by temporal gene expression changes of mouse ES cells during differentiation in a monolayer culture. A large number of genes were differentially regulated from 1 day to 14 days, and less number of genes were differentially expressed from 14 days to 28 days. The number of up-regulated genes was linearly increased throughout the 28 days of in vitro differentiation, while the number of down-regulated genes reached the plateau from 14 days to 28 days. Most differentially expressed genes were functionally classified into transcriptional regulation, development, extra cellular matrix (ECM),cytoskeleton organization, cytokines, receptors, RNA processing, DNA replication, chromatin assembly, proliferation and apoptosis related genes. While genes encoding ECM proteins were up-regulated, most of the genes related to proliferation, chromatin assembly, DNA replication, RNA processing, and cytoskeleton organization were down-regulated at 14 days. Genes known to be associated with embryo development or transcriptional regulation were differentially expressed mostly after 14 days of differentiation. These results indicate that the altered expression of ECM genes constitute an early event during the spontaneous differentiation, followed by the inhibition of proliferation and lineage specification. Our study might identify useful time-points for applying selective treatments for directed differentiation of mouse ES cells.

• BMB Reports
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• 제41권10호
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• pp.705-709
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• 2008

NF-${\kappa}B$ is a transcriptional regulator involved in many biological processes including proliferation, survival, and differentiation. Recently, we reported that expression and activity of NF-${\kappa}B$ is comparatively low in undifferentiated human embryonic stem (ES) cells, but increases during differentiation. Here, we found a lower expression of NF-${\kappa}B$ p65 protein in mouse ES cells when compared with mouse embryonic fibroblast cells. Protein levels of NF-${\kappa}B$ p65 and relB were clearly enhanced during retinoic acid-induced differentiation. Furthermore, increased DNA binding activity of NF-${\kappa}B$ in response to TNF-$\alpha$, an agonist of NF-${\kappa}B$ signaling, was seen in differentiated but not undifferentiated mouse ES cells. Taken together with our previous data in human ES cells, it is likely that NF-${\kappa}B$ expression and activity of the NF-${\kappa}B$ signaling pathway is comparatively low in undifferentiated ES cells, but increases during differentiation of ES cells in general.

• Molecules and Cells
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• 제45권11호
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• pp.820-832
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• 2022

As a potential candidate to generate an everlasting cell source to treat various diseases, embryonic stem cells are regarded as a promising therapeutic tool in the regenerative medicine field. Cohesin, a multi-functional complex that controls various cellular activities, plays roles not only in organizing chromosome dynamics but also in controlling transcriptional activities related to self-renewal and differentiation of stem cells. Here, we report a novel role of the α-kleisin subunits of cohesin (RAD21 and REC8) in the maintenance of the balance between these two stem-cell processes. By knocking down REC8, RAD21, or the non-kleisin cohesin subunit SMC3 in mouse embryonic stem cells, we show that reduction in cohesin level impairs their self-renewal. Interestingly, the transcriptomic analysis revealed that knocking down each cohesin subunit enables the differentiation of embryonic stem cells into specific lineages. Specifically, embryonic stem cells in which cohesin subunit RAD21 were knocked down differentiated into cells expressing neural alongside germline lineage markers. Thus, we conclude that cohesin appears to control the fate determination of embryonic stem cells.

• Reproductive and Developmental Biology
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• 제36권4호
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• pp.291-294
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• 2012

Embryonic stem cell classically cultured on feeder layer with FBS contained ES medium. Feeder-free mouse ES cell culture systems are essential to avoid the possible contamination of nonES cells. First we determined the difference between ES cell and MEF by Oct4 population. We demonstrate to culture and to induce differentiation on feeder free condition using a commercially available mouse ES cell lines.