• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.451-455
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• 2000

Gene targeting are very useful tools for the research on the gene function in vivo, mass production of foreign materials and biomedical approach of therapeutic process. But this process is very complicated and necessary highly skilled technique, because it is very different from ES cell origin, genetic background of embryo, and experimental conditions. We investigated the productivity ability of chimeric mouse after aggregation with TT2 ES cells. Increse of ES cell density caused gradual decrease in embryo development in vitro and in th $\varepsilon$ production of chimeric mice in vivo. One million ES cell density for the aggregation was very efficient to produce high percentage chimeric mice in their coat color. These results suggested that appropriate cell density plays a key role in the development and production of chimeric mice by a 8-cell aggregation method.

• Development and Reproduction
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• v.17 no.1
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• pp.9-16
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• 2013

Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.

• Development and Reproduction
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• v.9 no.2
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• pp.105-114
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• 2005

Embryonic stem(ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decision and to develop methods for getting enough cell numbers for clinical applications. Hematopoiesis has been widely studied, and hematopoietic differentiation from ES cells is a good model to study lineage commitment. In this study, we investigated stemness and compared the efficiency of hematopoietic differentiation using two different mouse embryonic stem cell lines TC-1 and B6-1. Although the two cell lines showed known stem cell properties with minor differences, the embryoid body formation efficiency in methylcellulose was much higher in TC-1 than B6-1. When measured potentials of hematopoietic differentiation using functional(colony-forming cell) and phenotypic(specific marker expression) assays, we found that TC-1 can differentiate into hematopoietic cells in methylcellulose culture but B6-1 cannot. These results imply that we can improve the efficiency of hematopoietic cell differentiation by selection of proper cell lines and this may be also applied in the differentiation of human embryonic stem cells.

• Journal of Microbiology
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• v.45 no.6
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• pp.547-552
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• 2007

Previously, we generated monoclonal antibodies (MAbs) that bound to the surface of human embryonic stem cells (hESCs) in an attempt to discover new hESC-specific surface markers. In this study, MAb 47-235 (IgG1, ${\kappa}$) was selected for further characterization. The MAb bound to the surface of undifferentiated hESCs but did not bind to mouse ESCs or mouse embryonic fibroblast cells in flow cytometric analysis. The antibody immunoprecipitated a 47 kDa protein from the lysates of cell surface-biotinylated hESCs. Identification of the protein by quadrupole time of flight tandem mass spectrometry revealed that 47-235 binds to Ag 243-5 protein of Mycoplasma arginini. BM-Cyclin treatment of the hESCs that reacted with 47-235 resulted in loss of mycoplasma DNA and the reactivity to 47-235. Nevertheless, the hESCs that were reactive to 47-235 maintained self-renewal and pluripotency and thus could be differentiated into three embryonic germ layers.

• Korean Journal of Veterinary Service
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• v.31 no.4
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• pp.449-456
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• 2008

The differentiation of mouse embryonic stem(ES) cell into smooth muscle cells(SMC) may play a major role in cardiovascular development and under pathophysiological conditions. Therefore, in the present study, we have examined the differentiation of ES cells and its related gene expression. SMC differentiation was indicated by cellular morphology and time-dependent induction of dibutyryl adenosine 3,5-cyclic monophosphate(DBcAMP)and retinoic acid(RA) on smooth muscle ${\alpha}$-actin($SM{\alpha}A$), smooth muscle myosin heavy chain(SMMHC) gene expression. The control was undifferentiated ES cells(protein expressions represent 50-60kDaOct-4). The results of this study show that morphology of embryoid body and confirmation of $SM{\alpha}A$ expression by immunocytochemistry. Moreover, SMMHC and desmin expression was significantly increased by time dependent manner(5, 7, 15 days), in contrast to $SM{\alpha}A$ expression was slightly decreased on 15days. In conclusion, DBcAMP and RA stimulate mouse ES cells differentiation into SMC and enhanced $SM{\alpha}A$, SMMHC and desmin expression.

• Development and Reproduction
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• v.13 no.3
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• pp.155-161
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• 2009

It has been reported that ganglioside GT1b is expressed during neuronal cell differentiation from undifferentiated mouse embryonic stem cells (mESCs), which suggests that ganglioside GT1b has a direct effect on neuronal cell differentiation. Therefore, this study was conducted to evaluate the effect of exogenous addition of ganglioside GT1b to an in vitro model of neuronal cell differentiation from undifferentiated mESCs. The results revealed that a significant increase in the expression of ganglioside GT1b occurred during neuronal differentiation of undifferentiated mESCs. Next, we evaluated the effect of retinoic acid (RA) on GT1b-treated undifferentiated mESCs, which was found to lead to increased neuronal differentiation. Taken together, the results of this study suggest that ganglioside GT1b plays a crucial role in neuronal differentiation of mESCs.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.06a
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• pp.19-25
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• 2002

Researches on manipulation pluripotent stem cells derived from blastocysts or promordial germ cells (PGCs) have a great advantages for developing innovative technologies in various fields of life science including medicine, pharmaceutics, and biotechnology. Since the first isolation in the mouse embryos, stem cells or stem cell-like colonies have been continuously established in the mouse of different strains, cattle, pig, rabbit, and human. In the animal species, stem cell biology is important for developing transgenic technology including disease model animal and bioreactor production. ES cell can be isolated from the inner cell mass of blastocysts by either mechanical operation or immunosurgery. So, mass production of blastocyst is a prerequisite factor for successful undertaking ES cell manipulation. In the case of animal ES cell research, various protocol of gamete biotechnology can be applied for improving the efficiency of stem cell research. Somatic cell nuclear transfer technique can be applied to researches on animal ES cells, since it is powerful tool for producing clone embryos containing genes of interest. In this presentation, a brief review was made for explaining how somatic cell nuclear transfer technology could contribute to improving stem cell manipulation technology.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.21-26
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• 2012

Suspension culture is a useful tool for culturing embryonic stem (ES) cells in large-scale, but the stability of pluripotency and karyotype has to be maintained $in$ $vitro$ for clinical application. Therefore, we investigated whether the chromosomal abnormality of ES cells was induced in suspension culture or not. The ES cells were cultured in suspension as a form of aggregate with or without mouse embryonic fibroblasts (MEFs), and 0 or 1,000 U/ml leukemia inhibitory factor (LIF) was treated to suspended ES cells. After culturing ES cells in suspension, their karyotype, DNA content, and properties of pluripotency and differentiation were evaluated. As a result, the formation of tetraploid ES cell population was significantly increased in suspension culture in which ES cells were co-cultured with both MEFs and LIF. Tetraploid ES cell population was also generated when ES cells were cultured alone in suspension regardless of the existence of LIF. On the other hand, the formation of tetraploid ES cell population was not detected in LIF-free condition, in which MEFs were included. The origin of tetraploid ES cell population was turned out to be E14 ES cells and not MEFs by microsatellite analysis and the basic properties of them were still maintained despite ploidy-conversion to tetraploidy. Furthermore, we identified the ploidy shift from tetraploidy to near-triploidy as tetraploid ES cells were differentiated spontaneously. From these results, we demonstrated that suspension culture system could induce ploidy-conversion generating tetraploid ES cell population. Moreover, optimization of suspension culture system may make possible mass-production of ES cells.

• BMB Reports
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• v.51 no.2
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• pp.92-97
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• 2018

B cell leukemia/lymphoma 3 (Bcl3) plays a pivotal role in immune homeostasis, cellular proliferation, and cell survival, as a co-activator or co-repressor of transcription of the $NF-{\kappa}B$ family. Recently, it was reported that Bcl3 positively regulates pluripotency genes, including Oct4, in mouse embryonic stem cells (mESCs). However, the role of Bcl3 in the maintenance of pluripotency and self-renewal activity is not fully established. Here, we report the dynamic regulation of the proliferation, pluripotency, and self-renewal of mESCs by Bcl3 via an influence on Nanog transcriptional activity. Bcl3 expression is predominantly observed in immature mESCs, but significantly decreased during cell differentiation by LIF depletion and in mESC-derived EBs. Importantly, the knockdown of Bcl3 resulted in the loss of self-renewal ability and decreased cell proliferation. Similarly, the ectopic expression of Bcl3 also resulted in a significant reduction of proliferation, and the self-renewal of mESCs was demonstrated by alkaline phosphatase staining and clonogenic single cell-derived colony assay. We further examined that Bcl3-mediated regulation of Nanog transcriptional activity in mESCs, which indicated that Bcl3 acts as a transcriptional repressor of Nanog expression in mESCs. In conclusion, we demonstrated that a sufficient concentration of Bcl3 in mESCs plays a critical role in the maintenance of pluripotency and the self-renewal of mESCs via the regulation of Nanog transcriptional activity.

• Animal cells and systems
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• v.13 no.2
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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.