• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.98-98
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• 2003

Recently, human embryonic stem (hES) cells have become very important resources for ES cell basic research, cell replacement therapy, and other medical applications; thus, efficient cryopreservation methods for these cells are needed. This study examined whether a newly developed minimum volume cooling (MVC) vitrification method, which was tested through cryopreservation of sensitive bovine oocytes, can be used for freezing hES cells. Feeder-free cultured hES cell (MB03) colonies were mechanically dissected into several small clumps following enzymatic treatment. We compared the freezing efficiency of a slow-cooling method using a cryo-module (0.4-0.6C/min, 20-30 clumps/vial) and MVC vitrification using a modified 0.5-ml French mini-straw designated as a MVC straw (>$20,000{\circ}C$/min, 10 clumps/straw) After thawing, in vitro survival of hES cell clumps was higher for MVC-vitrified cells (80.8%, 97/120) than for slow-cooled cells (38.2%, 39/102). Further, the proliferation rate of surviving MVC-vitrified cells was similar to that of control hES cells from 2 weeks after thawing. In addition, vitrified-thawed hES cells demonstrated a normal karyotype, were positively immunostained for surface marker antibodies (AP, SSEA-4 and TRA-1-60) and the Oct-4 antibody, and could differentiate into all three embryonic germ layer cells in vitro. This result demonstrates that hES cell clumps can be successfully cryopreserved by a newly developed MVC vitrification method without loss of human cell characteristics.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.209-215
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• 2004

Objective: Embryonic stem (ES) cells could be differentiated into the specific cell types by alternation of culture condition and modification of gene expression. This study was performed to evaluate the differentiation protocol for mouse and human ES cells to insulin secreting cells. Methods: Undifferentiated mouse (JH-I) and human (Miz-hESI) ES cells were cultured on STO feeder layer, and embryoid bodies (EBs) were formed by suspension culture. For the differentiation, EBs were cultured by sequential system with three stage protocol. The differentiating ES cells were collected and marker gene expressions were analyzed by seIni-quantitative RT-PCR in each stage. Amount of secreted insulin levels in culture media of human ES cells were measured by human insulin specific RIA kit. Results: During the differentiation process of human ES cells, GATA-4, a-fetoprotein, glucose transporter-2 and Ngn-3 expression were increased whereas OctA was decreased progressively. Insulin and albuInin mRNAs were expressed from stage IT in mouse ES cells and from stage III in human ES cells. We detected 3.0~7.9 IlU/rnl secretion of insulin from differentiated human ES cells by in vitro culture for 36 days. Conclusion: The sequential culture system could induce the differentiation of mouse and human ES cells into insulin secreting cells. This is the fIrst report of differentiation of human ES cells into insulin secreting cells by in vitro culture with serum and insulin free medium.

• 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.

• BMB Reports
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• v.41 no.10
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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.

• Toxicological Research
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• v.29 no.4
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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.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.2
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• pp.139-147
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• 2002

Objective : This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. Materials and Methods: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid body (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to detect cardiomyocytes (anti-sarcomeric ? -actinin Ab, 1 : 100; anti-cardiac troponin I Ab, 1 : 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. Results: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3 % at 13 days and 69.8% at 15 days, respectively. Also, the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and P-mES02, 4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric ? -actinin Ab and cardiac specific anti-cardiac troponin I Ab. Conclusion: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.

• 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)

• Development and Reproduction
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• v.16 no.4
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• pp.353-361
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• 2012

Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.102-102
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• 2003

Embryonic stem (ES) cells proliferate extensively in the undifferentiated state and have the potential to differentiate into a variety of cell types in response to various environmental cues. The generation of functional dopaminergic neurons from ES cells is promising for cell replacement therapy to treat Parkinson's disease. We compared the in vitro differentiation potential of pluripotent human embryonic stem (hES, MB03) cells induced with basic fibroblast growth factor (bFGF) or retinoic acid (RA). Both types of treatment resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)- $\alpha$ during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression, compared to control (P<0.05). In contrast, no effect was observed on the rate of mature or glutamic acid decarboxylase-positive neurons. Immunostaining and HPLC analyses revealed the higher levels of TH (20.3%) and dopamine in bFGF and TGF-$\alpha$ treated hES cells than in RA or BDNF treated hES cells. The results indicate that TGF-$\alpha$ may be successfully used in the bFGF induction protocol to yield higher numbers of functional dopaminergic neurons from hES cells.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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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)