• BMB Reports
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• v.54 no.10
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• pp.505-515
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• 2021

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.12
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• pp.1641-1646
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• 2004

The objective of this study was to generate transgenic mice expressing human resistin gene by using the tetraploidembryonic stem (ES) cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR, cloned into $pCR^{(R)}$ 2.1 $TOPO^{(R)}$ vector and constructed in pCMV-Tag4C vector. Mammalian expression plasmid containing human resistin was transfected into D3-GL ES cells by Lipofectamine 2,000, and then after 10-12 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec (fusion rate: 2,114/2,256, 93.5%) and cultured up to the blastocyst stage (development rate: 1,862/2,114, 94.6%). The selected 15-20 ES cells were injected into tetraploid blastocysts, and then transferred into the uteri of E 2.5 d pseudopregnant recipient mice. To investigate the gestation progress, two E 19.5 mused fetuses were recovered by Cesarean section of which one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, our findings demonstrate that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mice for the rapid analysis of gene function in vivo.

• Journal of Korean Neurosurgical Society
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• v.42 no.2
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• pp.118-124
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• 2007

Objective : Adipose tissue is derived from the embryonic mesoderm and contains a heterogenous stromal cell population. Authors have tried to verify the characteristics of stem cell of adipose derived stromal cells (ADSCs) and to investigate immunohistochemical findings after transplantation of ADSC into rat brain to evaluate survival, migration and differentiation of transplanted stromal cells. Methods : First ADSCs were isolated from human adipose tissue and induced adipose, osseous and neuronal differentiation under appropriate culture condition in vitro and examined phenotypes profile of human ADSCs in undifferentiated states using flow cytometry and immunohistochemical study. Human ADSCs were transplanted into the healthy rat brain to investigate survival, migration and differentiation after 4 weeks. Results : From human adipose tissue, adipose stem cells were harvested and subcultured for several times. The cultured ADSCs were differentiated into adipocytes, osteoctye and neuron-like cell under conditioned media. Flow cytometric analysis of undifferentiated ADSCs revealed that ADSCs were positive for CD29, CD44 and negative for CD34, CD45, CD117 and HLA-DR. Transplanted human ADSCs were found mainly in cortex adjacent to injection site and migrated from injection site at a distance of at least 1 mm along the cortex and corpus callosum. A few transplanted cells have differentiated into neuron and astrocyte. Conclusion : ADSCs were differentiated into multilineage cell lines through transdifferentiation. ADSCs were survived and migrated in xenograft without immunosuppression. Based on this data, ADSCs may be potential source of stem cells for many human disease including neurologic disorder.

• Journal of Science and Technology Studies
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• v.8 no.1
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• pp.55-95
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• 2008

This research was performed with the aim of analyzing the 'expectation dynamics' of embryonic stem cell research which was revealed throughout the establishment process of Stem Cell Research Center from 2000 to 2002. Expectation dynamics is a chained process: expectation construction - raising fund - performing research. Normally, researchers are considerably circumspect and politically neutral in assessing the result of research. However, some researchers are very involved in building the expectation dynamics by developing an overestimated impact of the result, which can be understood as a kind of strategy for solving the financial problem and defending the criticism in terms of bioethics. Nowadays Biotechnology R&D costs a big budget and requires large site human resources, so building the expectation dynamics is a decisive element for a successful R&D performance, which makes the strategy-development in the political context much more important. By analyzing the actors-network of embryonic stem cell research in term of 'expectation dynamics', we can clarify the identify of embryonic stem cell researchers and draw a conclusion which is very helpful for decision makers and the public to make a decision related with embryonic stem cells.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.78-78
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• 2003

Embryonic stem (ES) cells, derived from preimplantation embryo, are able to differentiate into various types of cells consisting the whole body, or pluripotency. In contrast, terminally differentiated cells do not usually alter their nature but frequently die or transform if they are exposed to inappropriate external stimulations. In addition to the plasticity, ES cells are expected to be different from terminally differentiated cells in very many ways, such as patterns of gene expressions, ability and response of the cells in confronting environmental stimulations, metabolism, and growth rate. As a model system to differentiate these two types of cells, human ES cells (MB03) and terminally differentiated cells (HeLa), we examined the ability of these two types of cells in confronting a severe oxidative insult, that is $H_2O$$_2$. Approximately 1$\times$10$^4$ cells were plated in 96 well plate and serum starved for overnight. The conditioned cells were exposed to a various concentration of $H_2O$$_2$ fur 24 hrs and loaded with neutral red (50$\mu\textrm{g}$/ml) for 4 hrs, washed with PBS for 2 min three times, and entrapped dye was dissolved out using acetic ethanol. Cytotoxicity was determined by reading the amount of dye in the medium using microplate reader. equipped with 575 nm filter. Relative amount of the dye entrapped within MB03 or HeLa were not significantly different when cells were exposed up to 0.4 mM $H_2O$$_2$. However, this sharply decreased down to 0.12% in HeLa cells when the cells were exposed to 0.8 mM $H_2O$$_2$, while it was approximately 54% in MB03 suggesting that this concentration of $H_2O$$_2$ is the defensive threshold for HeLa cells. The resistance to oxidative stimulation reversed, however, when cells were co-treated with BSO (L-buthionine- 〔S, R〕-sulfoximine) which chelates intracellular GSH. This result suggests that cellular GSH is the major defensive mechanism of human ES cells. Induction of enzymes involved in GSH metabolism and type of cell death is currently being studied.

• BMB Reports
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• v.48 no.5
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• pp.256-265
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• 2015

Cardiovascular and neurodegenerative diseases are major health threats in many developed countries. Recently, target tissues derived from human embryonic stem (hES) cells and induced pluripotent stem cells (iPSCs), such as cardiomyocytes (CMs) or neurons, have been actively mobilized for drug screening. Knowledge of drug toxicity and efficacy obtained using stem cell-derived tissues could parallel that obtained from human trials. Furthermore, iPSC disease models could be advantageous in the development of personalized medicine in various parts of disease sectors. To obtain the maximum benefit from iPSCs in disease modeling, researchers are now focusing on aging, maturation, and metabolism to recapitulate the pathological features seen in patients. Compared to pediatric disease modeling, adult-onset disease modeling with iPSCs requires proper maturation for full manifestation of pathological features. Herein, the success of iPSC technology, focusing on patient-specific drug treatment, maturation-based disease modeling, and alternative approaches to compensate for the current limitations of patient iPSC modeling, will be further discussed. [BMB Reports 2015; 48(5): 256-265]

• Biomedical Science Letters
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• v.10 no.4
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• pp.391-395
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• 2004

The demand for the production of gene-defective mice from embryonic stem (ES) cells is increasing to clarify decisive gene function in vivo. Although blastocyst injection is widely used to generate ES cell-mediated knockout mice, coculture method has been alternatively used because of several advantages, such as low cost and simple procedure. Thus, this experiment was designed to demonstrate the feasibility of the coculture method using J1 ES cells, which are known to be efficient for blastocyst injection. Eight-cell embryos were harvested from 2.5 days post-coitum (dpc), denuded with acid tyrode's solution, and transferred onto trypsinized J1 ES cells. Aggregation was carried out following two typical methods, which are simple coculture method and aggregation in groove prepared by aggregation needle. Successfully aggregated-embryos were developed to blastocysts for 24 h and transferred into uterus of pseudo-pregnant foster mother. Chimeric offspring was judged by coat pigmentation. In this study, we could obtain chimeric mice from all the two aggregation methods, but the chimera production efficiencies in coculture using groove were three times higher at least than those in the other group. In conclusion, these observations suggest that coculture method should be available for production of knockout mice from J1 ES cells. Presently, the germ-line transmission rates of the chimeras produced from the two methods are under investigation.

• BMB Reports
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• v.55 no.3
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• pp.142-147
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• 2022

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.211-215
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• 2008

The HMG box containing protein (HBP) has a high mobility group domain and involved in the regulation of proliferation and differentiation of tissues. We screened HBP2 in glioblastoma using Suppression Subtractive Hybridization (SSH) and isolated human spermatogonial stem cell-like cells (hSSC-like cells) derived from patients of nonobstructive azoospermia (NOA). Expression of HBP2 was analyzed by RT-PCR in undifferentiated stem cells (human Embryonic Stem Cells, hSSC-like cells 2P) and spontaneous differentiated stem cells (hSSC-like cells 4P). It was overexpressed in hESC and hSSC-like cells 2P but not in hSSC-like cells 4P. Also, the expression level of HBP2 was downregulated in colon tumor tissues compared to normal tissues. Specifically in synchronized WI-38 cells, HBP2 was highly upregulated until the G1 phase of the cell cycle and gradually decreased during the S phase. Our results suggest that HBP2 was downregulated during the spontaneous differentiation of hSSC-like cells. HBP2 was differently expressed in colon tissues and was related to G1-progression in WI-38 cells. It may playa role in the maintenance of an undifferentiated hSSC-like cell state and transits from G1 to S in WI-38 cells. This research was important that it identified a biomarker for an undifferentiated state of hSSC-like cells and characterized its involvement to arrest during cell cycle in colon cancer.