• 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.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.3
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• pp.199-206
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• 2010

Stem-cell therapy has the potential to improve vision in patients with untreatable retinal disease. Various types of cell source including fetal, embryonic and adult stem cells, intrinsic and extrinsic factors for differentiation into retinal progenitors and transplantation mode were discussed in this review. Experimental approaches have successfully induced photoreceptor precursor cells and retinal pigment epithelium. Stem-cell-based therapy is a promising treatment to restore vision in patients with retinal disease, in spite of the challenges.

• Development and Reproduction
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• v.13 no.4
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• pp.271-280
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• 2009

Human placenta is abundant source of adult stem cells. Especially, amniotic epithelial cells have stem cell characteristics, expressing surface markers normally present on embryonic stem cells and germ cells. However, culturing and expanding amniotic epithelial cells in vitro without feeder cells are difficult due to endogenous characteristics of epithelial cells. In the present study, amniotic epithelial cells are isolated and proliferated in several passages by applying dithiothreitol and a Rho-associated kinase inhibitor in culture media. The cultured amniotic epithelial cells showed the epithelial and stem cell characteristics. In conclusion, human placenta-derived amniotic epithelial stem cells can be a major source of stem cells for medical treatment of various diseases without any controversial issues.

• Journal of Veterinary Science
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• v.25 no.3
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• pp.40.1-40.19
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• 2024

Importance: The creation of robust maternal-embryonic interactions and implantation models is important for comprehending the early stages of embryonic development and reproductive disorders. Traditional two-dimensional (2D) cell culture systems often fail to accurately mimic the highly complex in vivo conditions. The employment of three-dimensional (3D) organoids has emerged as a promising strategy to overcome these limitations in recent years. The advancements in the field of organoid technology have opened new avenues for studying the physiology and diseases affecting female reproductive tract. Observations: This review summarizes the current strategies and advancements in the field of 3D organoids to establish maternal-embryonic interaction and implantation models for use in research and personalized medicine in assisted reproductive technology. The concepts of endometrial organoids, menstrual blood flow organoids, placental trophoblast organoids, stem cell-derived blastoids, and in vitro-generated embryo models are discussed in detail. We show the incorportaion of organoid systems and microfluidic technology to enhance tissue performance and precise management of the cellular surroundings. Conclusions and Relevance: This review provides insights into the future direction of modeling maternal-embryonic interaction research and its combination with other powerful technologies to interfere with this dialogue either by promoting or hindering it for improving fertility or methods for contraception, respectively. The merging of organoid systems with microfluidics facilitates the creation of sophisticated and functional organoid models, enhancing insights into organ development, disease mechanisms, and personalized medical investigations.

• Korean Journal of Clinical Laboratory Science
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• v.40 no.2
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• pp.106-112
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• 2008

Bone marrow derived mesenchymal stem cells (BMSCs) are largely studied for their potential clinical use. But it is hard to get enough number of those cells for clinical trials and give serious pain to the patients. Adipose tissue is derived from the embryonic mesenchyme and contains a stroma that is easily isolated with large amount. This cell population (adipose derived stem cells: ADSCs) can be isolated from human lipoaspirates and like MSCs, differentiate toward the osteogenic, adipogenic, myogenic and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the ADSCs extracted from omental or subcutaneous fat tissue were expanded during third to fifth passages. The phenotype of the ADSCs was identified by the conventional cell surface markers using flow cytometry: positive for CD29 and CD44, but negative for CD34, CD45, CD117 and HLA-DR that similar to those observed on BMSCs. The ADSCs were able to differentiate into the osteoblast or adipocytes with induction media. Finally, ADACs expressed multiple CD marker antigens similar to those observed on BMSCs and differentiated into osteoblast, adipocyte. With this, human adipotissue contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

• Journal of Science and Technology Studies
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• v.5 no.1 s.9
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• pp.125-148
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• 2005

This paper deals with public understanding of the stem cell cloning discussed in the Internet, based upon the case study of public discourse about Dr. Hwang's international publication of an advanced research of Stem Cell in Korean context. Public understanding of the stem cell cloning in Korea is characterized as follows: (1) it was defined as therapeutic cloning, (2) it was legitimized as a national pride and a potential vehicle for long-term economic performance, (3) ethical issues were criticized by the exclusion of early embryo from human life and the ubiquity of abortion in Korea.

• 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.

• International Journal of Stem Cells
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• v.17 no.2
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• pp.194-203
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• 2024

Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous, a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult, resulting in inaccuracies. In this study, we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts, human induced PSCs, and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis. We evaluated normalization methods using glucose and selected the most suitable for the metabolic analysis of heterogeneous PSCs during the reprogramming stage. The response of fibroblasts to glucose increased with starvation time, with oxygen consumption rate and extracellular acidification rate responding most effectively to glucose 4 hours after starvation and declining after 5 hours of starvation. Fibroblasts and PSCs achieved appropriate responses to glucose without damaging their metabolism 2~4 and 2~3 hours after starvation, respectively. We developed a novel method for comparing basal metabolic rates of fibroblasts and PSCs, focusing on quantitative analysis of glycolysis and oxidative phosphorylation using glucose without enzyme inhibitors. This protocol enables efficient comparison of energy metabolism among cell types, including undifferentiated PSCs, differentiated cells, and cells undergoing cellular reprogramming, and addresses critical issues, such as differences in basal metabolic levels and sensitivity to normalization, providing valuable insights into cellular energetics.

• 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.

• Biomolecules & Therapeutics
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• v.20 no.3
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• pp.280-285
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• 2012

The developing embryo naturally experiences relatively low oxygen conditions in vivo. Under in vitro hypoxia, mouse embryonic stem cells (mESCs) lose their self-renewal activity and display an early differentiated morphology mediated by the hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$). Previously, we demonstrated that histone deacetylase (HDAC) is activated by hypoxia and increases the protein stability and transcriptional activity of HIF-$1{\alpha}$ in many human cancer cells. Furthermore HDAC1 and 3 mediate the differentiation of mECSs and hematopoietic stem cells. However, the role of HDACs and their inhibitors in hypoxia-induced early differentiation of mESCs remains largely unknown. Here, we examined the effects of several histone deacetylase inhibitors (HDACIs) on the self-renewal properties of mESCs under hypoxia. Inhibition of HDAC under hypoxia effectively decreased the HIF-$1{\alpha}$ protein levels and substantially improved the expression of the LIF-specific receptor (LIFR) and phosphorylated-STAT3 in mESCs. In particular, valproic acid (VPA), a pan HDACI, showed dramatic changes in HIF-$1{\alpha}$ protein levels and LIFR protein expression levels compared to other HDACIs, including sodium butyrate (SB), trichostatin A (TSA), and apicidin (AP). Importantly, our RT-PCR data and alkaline phosphatase assays indicate that VPA helps to maintain the self-renewal activity of mESCs under hypoxia. Taken together, these results suggest that VPA may block the early differentiation of mESCs under hypoxia via the destabilization of HIF-$1{\alpha}$.