• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.133-137
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• 2009

Pluripotent embryonic stem (ES) cells isolated from inner cell mass (ICM) of blastocyst-stage embryos are capable of differentiating into various cell lineages and demonstrate germ-line transmission in experimentally produced chimeras. These cells have a great potential as tools for transgenic animal production, screening of newly-developed drugs, and cell therapy. Miniature pigs, selectively bred pigs for small size, offer several advantages over large breed pigs in biomedical research including human disease model and xenotransplantation. In the present study, factors affecting primary culture of somatic cell nuclear transfer blastocysts from miniature pigs for isolation of ES cells were investigated. Formation of primary colonies occurred only on STO cells in human ES medium. In contrast, no ICM outgrowth was observed on mouse embryonic fibroblasts (MEF) in porcine ES medium. Plating intact blastocysts and isolated ICM resulted in comparable attachment on feeder layer and primary colony formation. After subculture of ES-like colonies, two putative ES cell lines were isolated. Colonies of putative ES cells morphologically resembled murine ES cells. These cells were maintained in culture up to three passages, but lost by spontaneous differentiation. The present study demonstrates factors involved in the early stage of nuclear transfer ES cell isolation in miniature pigs. However, long-term maintenance and characterization of nuclear transfer ES cells in miniature pigs are remained to be done in further studies.

• International Journal of Oral Biology
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• v.39 no.4
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• pp.177-185
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• 2014

Transfection is a gene delivery tool that is a popular means of manipulating cellular properties, such as induced pluripotent stem cell (iPSC) generation by reprogramming factors (Yamanaka factors). However, the efficiency of transfection needs to be improved. In the present study, three transfection protocols - non-liposomal transfection (NLT), magnetofection and electroporation - were compared by analysis of their transfection efficiencies and cell viabilities using human dental pulp cells (hDPC) and bovine fetal fibroblasts (bFF) as cell sources. Enhanced green fluorescent protein gene was used as the delivery indicator. For magnetofection, Polymag reagent was administrated. NLT, FuGENE-HD and X-treme GENE 9 DNA transfection reagents were used for NLT. For electroporation, the $Neon^{TM}$ and $NEPA21^{TM}$ electroporators were tested. $Neon^{TM}$ electroporation showed highest transfection efficiency when compared with NLT, magnetofection, and $NEPA21^{TM}$ electroporation, with transfection efficiency of about 33% in hDPC and 50% in bFF, based on viable cell population in each cell type. These results suggest that transfection by $Neon^{TM}$ electroporation can be used to deliver foreign genes efficiently in human and bovine somatic cells.

• International Journal of Stem Cells
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• v.16 no.3
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• pp.269-280
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• 2023

Background and Objectives: The colonic epithelial layer is a complex structure consisting of multiple cell types that regulate various aspects of colonic physiology, yet the mechanisms underlying epithelial cell differentiation during development remain unclear. Organoids have emerged as a promising model for investigating organogenesis, but achieving organ-like cell configurations within colonic organoids is challenging. Here, we investigated the biological significance of peripheral neurons in the formation of colonic organoids. Methods and Results: Colonic organoids were co-cultured with human embryonic stem cell (hESC)-derived peripheral neurons, resulting in the morphological maturation of columnar epithelial cells, as well as the presence of enterochromaffin cells. Substance P released from immature peripheral neurons played a critical role in the development of colonic epithelial cells. These findings highlight the vital role of inter-organ interactions in organoid development and provide insights into colonic epithelial cell differentiation mechanisms. Conclusions: Our results suggest that the peripheral nervous system may have a significant role in the development of colonic epithelial cells, which could have important implications for future studies of organogenesis and disease modeling.

• Development and Reproduction
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• v.20 no.1
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• pp.63-71
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• 2016

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KO-SR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xeno-free conditions for clinical grade hESCs culture will be useful data in future clinical studies.

• International Journal of Stem Cells
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• v.16 no.2
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• pp.215-233
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• 2023

Background and Objectives: MYC, also known as an oncogenic reprogramming factor, is a multifunctional transcription factor that maintains induced pluripotent stem cells (iPSCs). Although MYC is frequently upregulated in various cancers and is correlated with a poor prognosis, MYC is downregulated and correlated with a good prognosis in lung adenocarcinoma. MYC and two other MYC family genes, MYCN and MYCL, have similar structures and could contribute to tumorigenic conversion both in vitro and in vivo. Methods and Results: We systematically investigated whether MYC family genes act as prognostic factors in various human cancers. We first evaluated alterations in the expression of MYC family genes in various cancers using the Oncomine and The Cancer Genome Atlas (TCGA) database and their mutation and copy number alterations using the TCGA database with cBioPortal. Then, we investigated the association between the expression of MYC family genes and the prognosis of cancer patients using various prognosis databases. Multivariate analysis also confirmed that co-expression of MYC/MYCL/MYCN was significantly associated with the prognosis of lung, gastric, liver, and breast cancers. Conclusions: Taken together, our results demonstrate that the MYC family can function not only as an oncogene but also as a tumor suppressor gene in various cancers, which could be used to develop a novel approach to cancer treatment.

• 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.

• Archives of Plastic Surgery
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• v.34 no.5
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• pp.537-542
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• 2007

Purpose: Adipose tissue contains a population of pluripotent stem cells capable of differentiating along multiple mesenchymal cell lineages. It is well known that fat depots from different part of our body shows different nature not only in morphological aspect but also physiologic aspect. The authors compared the adipogenic potentials and osteogenic potentials of adipose stem cells from different anatomical sites of human. Methods: After laparotomy by surgery team, the authors isolated these adipose stem cells successfully from 7 men with an average age of 58, and induced differentiation along adipogenic and osteogenic lineages in vitro. On the 14th day, cells cultured in adipogenic media differentiated into adipocytes in vitro, as evidenced by positive Oil Red O staining of lipid vacuoles. On the 21st day, cells cultured in osteogenic media differentiated into osteoblasts in vitro as demonstrated by Alizarin red staining of a calcified extracellular matrix. Results: After exposure to adipogenic and osteogenic differentiation medium, subcutaneous adipose stem cells were found to possess greater adipogenic and osteogenic potentials than cells isolated from visceral adipose tissues. Conclusion: This study indicates that adipogenic and osteogenic potentials of adipose stem cells vary by their anatomical sites, with subcutaneous adipose stem cells exhibiting higher adipogenic and osteogenic potential than those isolated from visceral fat.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.243-248
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• 2003

Sperrnatogenesis, the process by which the male germ-line stem cells(GSCs; type A spermatogonia) divide and differentiate to produce the mature spermatozoa, occurs in the seminiferous tubules of the testis. The GSCs proliferate actively to produce two types of cells: other GSCs and differentiating spermatogonia. GSCs have unipotentcy, devoted solely to the generation of sperm. The function of GSCs has broad implications for development, disease, and evolution. Spermatogenesis is fundamental for propagation of species and the defects of this system can result in infertility or disease. The ability to identify, isolate, culture, and alter GSCs will allow powerful new approaches in animal transgenesis and human gene therapy relating to infertility. Until recently, research on stem cells in the testis has been limited because of technical difficulties in isolating and identifying these cell populations. Here, we were trying to find out optimal conditions for in vitro culture of GSCs for identifying and isolating GSCs. We collected mouse GSCs from 3-days old mouse by two-step enzyme digestion method. GSCs were plated and grown on mouse embryonic fibroblasts in Dulbecco's modified Eagle's medium (DMEM) containing 15％ fatal bovine serum, 10 mM 2-mercaptoethanol, 1％ non-essential amino acids, 1 ng/$m\ell$ bFGF, 10 $\mu$M forskolin, 1500 U/$m\ell$ human recombinant leukemia inhibitory factor (LIF). Over a period 3∼5 days, GSCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells. After 5th passages, cells within the colonies continued to be alkaline phosphatase and Oct-4 positive and tested positive against a panel of two immunological markers(Integrin $\alpha$ 6 and Integrin $\beta$ 1) that have been recognized generally to characterize GSCs. SSEA-1, SSEA-3, and SSEA-4 also showed positive signals. Based on our data, these GSCs-derived cultures meet the criteria for GSCs itself and even other pluripotent stem cells. We reported here the establishment of in vitro cultures from mouse male GSCs.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.1
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• pp.26-34
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• 2009

Porcine embryonic stem (ES) cells have a great potential as tools for transgenic animal production and studies of regulation of differentiation genes. Although several studies showed successful derivation of porcine ES-like cells, these cells were not maintained long-term in culture. Therefore, this study was conducted to establish porcine pluripotent ES-like cells using in vivo fertilized embryos and to maintain these cells in long term culture. Porcine ES-like cells from in vivo embryos obtained by immunosurgery or whole explant culture were successfully cultured for over 56 passages. Morphology of porcine ES-like cells was flat-shaped with a monolayer type colony. These cells stained for alkaline phosphatase throughout the culture. Furthermore, porcine ES-like cells reacted with antibodies against Oct-4, SSEA-1, SSEA-4, Tra-1-60, and Tra-1-81, which are typical markers of undifferentiated stem cells. To characterize the ability of porcine ES-like cells to differentiate into three germ layers, embryoid body formation was induced. After plating of these cells, porcine ES-like cells were spontaneously differentiated into various cell types of all three germ layers. In addition, porcine ES-like cells were successfully derived from IVF blastocysts in media containing human recombinant basic fibroblast growth factor.

• Molecules and Cells
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• v.42 no.11
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• pp.739-746
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• 2019

Significant knowledge about the pathophysiology of Alzheimer's disease (AD) has been gained in the last century; however, the understanding of its causes of onset remains limited. Late-onset AD is observed in about 95% of patients, and APOE4-encoding apolipoprotein E4 (ApoE4) is strongly associated with these cases. As an apolipoprotein, the function of ApoE in brain cholesterol transport has been extensively studied and widely appreciated. Development of new technologies such as human-induced pluripotent stem cells (hiPSCs) and CRISPR-Cas9 genome editing tools have enabled us to develop human brain model systems in vitro and readily manipulate genomic information. In the context of these advances, recent studies provide strong evidence that abnormal cholesterol metabolism by ApoE4 could be linked to AD-associated pathology. In this review, we discuss novel discoveries in brain cholesterol dysregulation by ApoE4. We further elaborate cell type-specific roles in cholesterol regulation of four major brain cell types, neurons, astrocytes, microglia, and oligodendrocytes, and how its dysregulation can be linked to AD pathology.