• Development and Reproduction
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• v.17 no.4
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• pp.379-387
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• 2013

Embryonic stem (ES) cells can self-renew and differentiate to various cells depending on the culture condition. Although ES cells are a good model for cell type specification and can be useful for application in clinics in the future, studies on ES cells have many experimental restraints including low transfection efficiency and transgene expression. Here, we observed that transgene expression after transfection was enhanced by treatment with histone deacetylse (HDAC) inhibitors such as trichostatin A, sodium butyrate, and valproic acid. Transfection was performed using conventional transfection reagents with a retroviral vector encoding GFP under the control of CMV promoter as a reporter. Treatment of ES cells with HDAC inhibitors after transfection increased population of GFP positive cells up to 180% compared with untreated control. ES cells showed normal expression of stem cell markers after treatment with HDAC inhibitors. Transgene expression was further enhanced by modifying transfection procedure. GFP positive cells selected after transfection were proved to have the stem cell properties. Our improved protocol for enhanced gene delivery and expression in mouse ES cells without hampering ES cell properties will be useful for study and application of ES cells.

• Development and Reproduction
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• v.12 no.2
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• pp.141-149
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• 2008

This study was conducted to develop an efficient cryopreservation method of human embryonic stem (ES) cells using vitrification. In an initial experiment, sub-clumps of human ES cells (CHA-hES3 and CHA-hES4) were vitrified using grids after incubation with STO feeder cells for 1 or 16 h (Groups 1-1 and 1-2, respectively). After storage for $2{\sim}4$ months, thawed clumps were re-plated on a fresh feeder layer. The survival rates of warmed CHA-hES3 and CHA-hES4 cells of Group 1-2 were significantly higher than those of the corresponding Group 1-1 cells. In the second experiment, human ES cells were vitrified after incubation with feeder or feeder-conditioned medium (Groups 2-1 to -7). Relative mRNA expression of BM proteins and survival rates were increased following incubation of ES cells with fresh feeder cells for 16 h. In conclusion, increasing of tight adhesion between ES cells by extended incubation with feeder could reduce cryoinjury after vitrifying/warming.

• Development and Reproduction
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• v.14 no.4
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• pp.253-259
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• 2010

DNA methylation is one of the major epigenetic regulations of gene expression. The DNA methylation patterns are dramatically changed during gametogenesis and embryogenesis, and especially, it has been known that embryonic stem cells show a distinct methylation pattern. In this study, we examined the methylation patterns of imprinting genes, H19, Igf2r, and Snrpn, in stem cells induced from fertilized embryo (fES) and somatic cell nuclear transferred embryo (ntES). The methylation pattern of H19 gene in both fES and ntES were similar. However, the methylation patterns of Igf2r and Snrpn in ntES (hypermethylated) were slightly different from fES cells.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.121-130
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• 2023

Background: Coating a culture plate with molecules that aid in cell adhesion is a technique widely used to produce animal cell cultures. Extracellular matrix (ECM) is known for its efficiency in promoting adhesion, survival, and proliferation of adherent cells. Gelatin, a cost-effective type of ECM, is widely used in animal cell cultures including feeder-free embryonic stem (ES) cells. However, the optimal concentration of gelatin is a point of debate among researchers, with no studies having established the optimal gelatin concentration. Methods: In this study, we coated plastic plates with gelatin in a concentration-dependent manner and assessed Young's modulus using atomic force microscopy (AFM) to investigate the microstructure of the surface of each plastic plate. The adhesion, proliferation, and differentiation of the ESCs were compared and analyzed revealing differences in surface microstructure dependent on coating concentration. Results: According to AFM analysis, there was a clear difference in the microstructure of the surface according to the presence or absence of the gelatin coating, and it was confirmed that there was no difference at a concentration of 0.5% or more. ES cell also confirmed the difference in cell adhesion, proliferation, and differentiation according to the presence or absence of gelatin coating, and also it showed no difference over the concentration of 0.5%. Conclusions: The optimum gelatin-coating for the maintenance and differentiation of ES cells is 0.5%, and the gelatin concentration-mediated microenvironment and ES cell signaling are closely correlated.

• 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.

• BMB Reports
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• v.42 no.2
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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.

• Reproductive and Developmental Biology
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• v.31 no.4
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• pp.267-272
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• 2007

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the capacity to differentiate into various types of cells in the body. Hence, these cells may potentially be an indefinite source of cells for cell therapy in various degenerative diseases including neuronal disorders. For clinical applications of human ES cells, directed differentiation of these cells would be necessary. The objective of this study is to develop the culture condition for the expansion of neural precursor cells derived from human ES cells. Human ES cells were able to differentiate into neural precursor cells upon a stepwise culture condition. Neural precursor cells were propagated up to 5000-fold in cell numbers over 12-week period of culture and evaluated for their characteristics. Expressions of sox1 and pax6 transcripts were dramatically up-regulated along the differentiation stages by RT-PCR analysis. In contrast, expressions of oct4 and nanog transcripts were completely disappeared in neural precursor cells. Expressions of nestin, pax6 and sox1 were also confirmed in neural precursor cells by immunocytochemical analysis. Upon differentiation, the expanded neural precursor cells differentiated into neurons, astrocytes, and oligodendrocytes. In immunocytochemical analysis, expressions of type III ${\beta}$-tubulin and MAP2ab were observed Presence of astrocytes and oligodendrocytes were also confirmed by expressions of GFAP and O4, respectively. Results of this study demonstrate the feasibility of long-term expansion of human ES cell-derived neural precursor cells in vitro, which can be a potential source of the cells for the treatment of neurodegenerative disorders.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.179-186
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• 2003

Human embryonic stem (hES) cell lines have been derived from human blastocysts and are expected to have far-reaching applications in regenerative medicine. The objective of this study is to improve freezing method with less cryo-injuries and best survival rates in hES cells by comparing various vitrification conditions. For the vitrifications, ES cells are exposed to the 4 different cryoprotectants, ethylene glycol (EG), 1,2-propanediol (PROH), EG with dime-thylsulfoxide (DMSO) and EG with PROH. We compared to types of vehicles, such as open pulled straw (OPS) or electron microscopic cooper grids (EM grids). Thawed hES cells were dipped into sequentially holding media with 0.2 M sucrose for 1 min, 0.1 M sucrose for 5 min and holding media for 5 min twice and plated onto a fresh feeder layer. Survival rates of vitrified hES cells were assessed by counting of undifferentiated colonies. It shows high survival rates of hES cells frozen with EG and DMSO (60.8％), or EG and PROH(65.8％) on EM grids better than those of OPS, compared to those frozen with EG alone (2.4％) or PROH alone (0％) alone. The hES cells vitrified with EM grid showed relatively constant colony forming efficiency and survival rates, compared to those of unverified hES cells. The vitrified hES cells retained the normal morphology, alkaline phosphates activity, and the expression of SSEA-3 and 4. Through RT-PCR analysis showed Oct-4 gene expression was down-regulated and embryonic germ layer markers were up-regulated in the vitrified hES cells during spontaneous differentiation. These results show that vitrification method by using EM grid supplemented with EG and PROH in hES cells may be most efficient at present to minimize cyto-toxicity and cellular damage derived by ice crystal formation and furthermore may be employed for clinical application.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.85-85
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• 2002

As an effort to direct differentiation of human embryonic stem cells (hES, MB03) to dopamine-producing neuronal cells, we expressed Nurr-l in hES and examined the expression of tyrosine hydroxylase (TH) after bFGF induction. To introduce Nurr-l, hES cells were maintained in humidified chamber with 5% CO₂ and 95% air in DMEM/Fl2 supplemented with FBS (10%), penicillin (100U/㎖), and streptomycin (100㎍/㎖). (omitted)

• 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.