• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.648-656
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• 2008

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-N1) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for $30{\mu}s$ in $Ca^{2+}$ and $Mg^{2+}-containing$ 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.209-220
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• 2017

The estrogen-mediated effect of mesenchymal stem cells (MSCs) is a highly critical factor for the clinical application of MSCs. However, the present study is conducted on MSCs derived from adult donors, which have different physiological status with steroid hormonal changes. Therefore, we explores the important role of $17{\beta}$-estradiol (E2) in MSCs derived from female and male newborn piglets (NF- and NM-pBMSCs), which are non-sexually matured donors with steroid hormones. The results revealed that in vitro treatment of MSCs with E2 improved cell proliferation, but the rates varied according to the gender of the newborn donors. Following in vitro treatment of newborn MSCs with E2, mRNA levels of Oct3/4 and Sox2 increased in both genders of MSCs and they may be correlated with both estrogen receptor ${\alpha}$ ($ER{\alpha}$) and $ER{\beta}$ in NF-pBMSCs, but NM-pBMSCs were only correlated with $ER{\alpha}$. Moreover, E2-treated NF-pBMSCs decreased in ${\beta}$-galactosidase activity but no influence on NM-pBMSCs. In E2-mediated differentiation capacity, E2 induced an increase in the osteogenic and chondrogenic abilities of both pBMSCs, but adipogenic ability may increased only in NF-pBMSCs. These results demonstrate that E2 could affect both genders of newborn donor-derived MSCs, but the regulatory role of E2 varies depending on gender-dependent characteristics even though the original newborn donors had not been affected by functional steroid hormones.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.4
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• pp.299-305
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• 2010

Purpose: Adipose tissue is located beneath the skin, around internal organs, and in the bone marrow in humans. Its main role is to store energy in the form of fat, although it also cushions and insulates the body. Adipose tissue also has the ability to dynamically expand and shrink throughout the life of an adult. Recently, it has been shown that adipose tissue contains a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells that, in cell culture conditions, have extensive proliferative capacity and are able to differentiate into several lineages, including, osteogenic, chondrogenic, endothelial cells, and myogenic lineages. Materials and Methods: This study focused on endothelial cell culture from the adipose tissue. Adipose tissues were harvested from buccal fat pad during bilateral sagittal split ramus osteotomy for surgical correction of mandibular prognathism. The tissues were treated with 0.075% type I collagenase. The samples were neutralized with DMEM/and centrifuged for 10 min at 2,400 rpm. The pellet was treated with 3 volume of RBC lysis buffer and filtered through a 100 ${\mu}m$ nylon cell strainer. The filtered cells were centrifuged for 10 min at 2,400 rpm. The cells were further cultured in the endothelial cell culture medium (EGM-2, Cambrex, Walkersville, Md., USA) supplemented with 10% fetal bovine serum, human EGF, human VEGF, human insulin-like growth factor-1, human FGF-$\beta$, heparin, ascorbic acid and hydrocortisone at a density of $1{\times}10^5$ cells/well in a 24-well plate. Low positivity of endothelial cell markers, such as CD31 and CD146, was observed during early passage of cells. Results: Increase of CD146 positivity was observed in passage 5 to 7 adipose tissue-derived cells. However, CD44, representative mesenchymal stem cell marker, was also strongly expressed. CD146 sorted adipose tissue-derived cells was cultured using immuno-magnetic beads. Magnetic labeling with 100 ${\mu}l$ microbeads per 108 cells was performed for 30 minutes at $4^{\circ}C$ a using CD146 direct cell isolation kit. Magnetic separation was carried out and a separator under a biological hood. Aliquous of CD146+ sorted cells were evaluated for purity by flow cytometry. Sorted cells were 96.04% positivity for CD146. And then tube formation was examined. These CD146 sorted adipose tissue-derived cells formed tube-like structures on Matrigel. Conclusion: These results suggest that adipose tissue-derived cells are endothelial cells. With the fabrication of the vascularized scaffold construct, novel approaches could be developed to enhance the engineered scaffold by the addition of adipose tissue-derived endothelial cells and periosteal-derived osteoblastic cells to promote bone growth.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.4
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• pp.243-249
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• 2010

Tissue engineered bone (TEB) can replace an autogenous bone graft requiring an secondary operation site as well as avoid complications like inflammation or infection from xenogenic or synthetic bone graft. Adult mesenchymal stem cells (MSC) for TEB are considered to have various ranges of differentiation capacity or multipotency by the donor site and age. This study examined the effect of age on proliferation capacity, differentiation capacity and bone morphogenetic protein-2 (BMP-2) responsiveness of human bone marrow stromal cells (hBMSC) according to the age. In addition, to evaluate the effect on enhancement for osteoblast differentiation, the hBMSC were treated with Trichostatin A (TSA) and 5-Azacitidine (5-AZC) which was HDAC inhibitors and methyltransferase inhibitors respectively affecting chromatin remodeling temporarily and reversibly. The young and old group of hBMSC obtained from the iliac crest from total 9 healthy patients, showed similar proliferation capacity. Cell surface markers such as CD34, CD45, CD90 and CD105 showed uniform expression regardless of age. However, the young group showed more prominent transdifferentiation capacity with adipogenic differentiation. The osteoblast differentiation capacity or BMP responsiveness was low and similar between young and old group. TSA and 5-AZC showed potential for enhancing the BMP effect on osteoblast differentiation by increasing the expression level of osteogenic master gene, such as DLX5, ALP. More study will be needed to determine the positive effect of the reversible function of HDAC inhibitors or methyltransferase inhibitors on enhancing the low osteoblast differentiation capacity of hBMSC.

• BMB Reports
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• v.44 no.12
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• pp.793-798
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• 2011

Recently, pluripotency induction or cellular reprogramming by introducing critical transcription factors has been extensively studied, but has been demonstrated only in vitro. Based on reports that Oct4 is critically involved in transforming neural stem cells into pluripotent cells, we used the lentiviral vector to introduce the Oct4 gene into the hippocampal dentate gyrus (DG) of adult mice. We examined whether this manipulation led to cellular or behavioral changes, possibly through processes involving the transformation of NS cells into pluripotent cells. The Oct4 lentivirus-infused group and the green fluorescent protein lentivirus-infused group showed a similar thickness of the DG and a comparable level of synaptophysin expression in the DG. Furthermore, our behavioral analyses did not show any differences between the groups concerning exploratory activity, anxiety, or memory abilities. This first trial for pluripotency induction in vivo, despite negative results, provides implications and information for future studies on in vivo cellular reprogramming.

• Journal of Ginseng Research
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• v.46 no.3
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• pp.408-417
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• 2022

Background: Korean Red Ginseng extract (KRGE) has been used as a health supplement and herbal medicine. Astrocytes are one of the key cells in the central nervous system (CNS) and have bioenergetic potential as they stimulate mitochondrial biogenesis. They play a critical role in connecting the brain vasculature and nerves in the CNS. Methods: Brain samples from KRGE-administered mice were tested using immunohistochemistry. Treatment of human brain astrocytes with KRGE was subjected to assays such as proliferation, cytotoxicity, Mitotracker, ATP production, and O₂ consumption rate as well as western blotting to demonstrate the expression of proteins related to mitochondria functions. The expression of hypoxia-inducible factor-1α (HIF-1α) was diminished utilizing siRNA transfection. Results: Brain samples from KRGE-administered mice harbored an increased number of GFAP-expressing astrocytes. KRGE triggered the proliferation of astrocytes in vitro. Enhanced mitochondrial biogenesis induced by KRGE was detected using Mitotracker staining, ATP production, and O₂ consumption rate assays. The expression of proteins related to mitochondrial electron transport was increased in KRGE-treated astrocytes. These effects were blocked by HIF-1α knockdown. The factors secreted from KRGE-treated astrocytes were determined, revealing the expression of various cytokines and growth factors, especially those related to angiogenesis and neurogenesis. KRGE-treated astrocyte conditioned media enhanced the differentiation of adult neural stem cells into mature neurons, increasing the migration of endothelial cells, and these effects were reduced in the background of HIF-1α knockdown. Conclusion: Our findings suggest that KRGE exhibits prophylactic potential by stimulating astrocyte mitochondrial biogenesis through HIF-1α, resulting in improved neurovascular function.

• Journal of Acupuncture Research
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• v.23 no.5
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• pp.69-77
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• 2006

Background : Mesenchymal stem cells (MSCs) are present in most of the tissue matrix, taking part in their regeneration when injury or damage occurs. The aim of this study was to investigate the presence of cells with pluripotential characteristics in human subchondral bone and the capacity of these cells to differentiate to osteoblast. Methods : Human subchondral bone were digested with collagenase. Isolated cells were cultured with a-MEM, 15% FBS, 10-8M dexamethasone and 50 ng/mL ascoric acid. Cells from 0 day(isolated cells), 7 day (first subculture) and 14 days (third subculture) were used to carry out phenotypic characterization experiments flowcytometry analysis with 11 monoclonal antibodies) and osteogenic differentiation experiments. Osteogenic differentiation of cells was assessment by quantification of bone extracellular matrix components by following analysis: alkaline phosphatase(ALP) stains to detect ALP activity, RT-PCR and western blot to detect osteocalcin (OCN), osteopontin (OPN) and type I collagen(Col I), and Alizarin red stains to detect calcium deposition. Results : Flowcytometry analyses showed that in our population more than 98% of cells were positive for MSC markers: SH-2(CD105, 99%), CD29 (95%), CD73 (95%). Cells were negative for hematopoietic markers (CD11b, CD34, and CD45). Furthermore, cells showed positive stain to multipotent markers such as CDl17 (c-kit) (15.1%), and CD166 (74.9%), and cell adhesion molecules such as CD54 (78.1%) and CD106 (63.5%). The osteogenic specific marker analyses showed that the culture of these cells for 7 and 14 days stimulates ALP, OCN, OPN and Col I synthesis by RT-PCR and Western blot analysis. Also, after 14 days in the culture of MSCs induces mineralization by Arizarin red stain. Conclusion : In this work, we demonstrated a new and efficient method for osteoblastic differentiation of human subchondral bone stem cells. As MSCs takes part in reparative processes of adult tissues, these cells could play an important role in osteogenesis.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.8
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• pp.1171-1177
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• 2015

The objective of this study was to establish the optimum protocol for the isolation and culture of porcine muscle satellite cells. Mononuclear muscle satellite cells are a kind of adult stem cell, which is located between the basal lamina and sarcolemma of muscle fibers and is the primary source of myogenic precursor cells in postnatal muscle. Muscle satellite cells are a useful model to investigate the mechanisms of muscle growth and development. Although the isolation and culture protocols of muscle satellite cells in some species (e.g. mouse) have been established successfully, the culture system for porcine muscle satellite cells is very limited. In this study, we optimized the isolation procedure of porcine muscle satellite cells and elaborated the isolation and culture process in detail. Furthermore, we characterized the porcine muscle satellite cells using the immunofluorecence. Our study provides a reference for the isolation of porcine muscle satellite cells and will be useful for studying the molecular mechanisms in these cells.

• Biomolecules & Therapeutics
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• v.24 no.2
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• pp.123-131
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• 2016

Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway.

• International Journal of Oral Biology
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• v.35 no.4
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• pp.209-214
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• 2010

Cells that have endogenous multipotent properties can be used as a starting source for the generation of induced pluripotent cells (iPSC). In addition, small molecules associated with epigenetic reprogramming are also widely used to enhance the multi- or pluripotency of such cells. Skinderived precursor cells (SKPs) are multipotent, sphereforming and embryonic neural crest-related precursor cells. These cells can be isolated from a juvenile or adult mammalian dermis. SKPs are also an efficient starting cell source for reprogramming and the generation of iPSCs because of the high expression levels of Sox2 and Klf4 in these cells as well as their endogenous multipotency. In this study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was tested in the generation of iPSCs as a potential enhancer of the reprogramming potential of SKPs. SKPs were isolated from the back skins of 5-6 week old C57BL/6 X DBA/2 F1 mice. After passage 3, the SKPs was treated with 2 mM of VPA and the quantitative real time RT-PCR was performed to quantify the expression of Oct4 and Klf4 (pluripotency specific genes), and Snai2 and Ngfr (neural crest specific genes). The results show that Oct4 and Klf4 expression was decreased by VPA treatment. However, there were no significant changes in neural crest specific gene expression following VPA treatment. Hence, although VPA is one of the most potent of the HDAC inhibitors, it does not enhance the reprogramming of multipotent skin precursor cells in mice.