• Development and Reproduction
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• v.11 no.2
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• pp.85-96
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• 2007

Mesenchymal stem cells (MSC) are promising candidates for cell-based therapies. One major obstacle for their clinical use is the unsafety of fetal bovine serum (FBS), which is a crucial part of all media currently used for the culture of MSC. We investigated the effect of human cord serum (HCS) on the growth response, mRNA and protein expressions of human amnion-derived stem cells (HAM). HAM were isolated from the amnion after a Caesarean section and cultured in DMEM supplemented with 10% FBS, 5% HCS or 10% HCS. During culture, their biological characteristics at earlier and later passages were analyzed using RT-PCR and immunocytochemistry. Regardless of serum sources, HAM showed the prominent expression of Oct-4, Rex-1, SCF, FGF-5, BMP-4, nestin, GATA-4, NCAM and HLA ABC genes. The expression profile was observed even at later passages. Similarly, HAM cultured in either FBS or HCS exhibited the distinct protein expression of collagen I, II, III and XII, fibronectin, $\alpha$-smooth muscle actin, vimentin, CK18, CD54, FSP, TRA-1-60, SSEA-3, -4 and HLA ABC. However, desmin expression was only observed in HAM cultured in medium supplemented with FBS and vWF expression was only found in HAM cultured in medium supplemented with HCS. Overall pattern of gene and protein expression of HAM was typical of known adult stem cells such as bone marrow-derived MSC. In conclusion, HCS could be as effective as FBS for the culture of HAM.

• Development and Reproduction
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• v.15 no.4
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• pp.281-289
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• 2011

The objective of this study was to investigate the effective cryoprotectants for the cryopreservation of porcine mesenechymal stem cells (pMSCs). In order to understand the effectiveness of various cryoprotectants on pMSCs, we studied the most commonly used cryoprotectants; dimethyl sulfoxide (DMSO), ethylene glycol (EG), DMSO and EG. pMSCs were isolated from bone marrow matrix of piglet (2 month) and characterized by alkaline phopshatase (AP) activity, colony forming, and differentiation to adipocyte. In slow cooling cryopreservation, the pMSCs were exposed to cell medium containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% DMSO, 1.5M EG and 5% DMSO/0.75M EG, respectively, and freezed to $-1^{\circ}C$/min from $25^{\circ}C$ up to $-80^{\circ}C$ in a cryo-container. The proportion of viable cells and the growing rates in fresh pMSCs were significantly (P<0.05) higher than those of other groups, but did not differ between the cryopreserved groups. The expression of Sox-2 and Nanog gene was increased by extending culture time in cryopreserved groups. The expression of Bax gene in cryopreserved groups was similar with fresh pMSCs. Moreover, the gene expression of adipocyte-specific marker as well as chondrogenic/osteogenic factors in cryopreserved groups was similarly to fresh pMSCs. Taken together, our results suggested that all these cryoprotectants of 10% DMSO, 1.5M EG and 5% DMSO/0.75M EG could be used for cryopreservation of the pMSCs.

• 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.

• Radiation Oncology Journal
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• v.20 no.3
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• pp.253-263
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• 2002

Purpose : In order to understand in vivo radiation damage modifying of bFGF on jejunal mucosa, bone marrow and the effect of bFGF on the growth of transplanted mouse sarcoma 180 tumor in mice. Materials and Methods : Mice were treated with $6\;{\mu}g$ of bFGF at 24 hours and 4 hours before exposing to 600 cGy, 800 cGy and 1,000 cGy total body irradiation (TBI), and then exposed to 3,000 cGy local radiation therapy on the tumor bearing thigh. Survival and tumor growth curve were plotted in radiation alone group and combined group of bFGF and irradiation (RT). Histologic examination was performed in another experimental group. Experimental groups consisted of normal control, tumor control, RT (radiation therapy) alone, $6\;{\mu}g$ bFGF alone, combined group of $3\;{\mu}g$ bFGF and irradiation (RT), combined group of $6\;{\mu}g$ bFGF and irradiation (RT). Histologic examination was peformed with H-E staining in marrow, jejunal mucosa, lung and sarcoma 180 bearing tumor. Radiation induced apoptosis was determined in each group with the DNA terminal transferase nick-end labeling method ($ApopTag^{\circledR}$ S7100-kit, Intergen Co.) Results : The results were as follows 1) $6\;{\mu}g$ bFGF given before TBI significantly improved the survival of lethally irradiated mice. bFGF would protect against lethal bone marrow syndrome. 2) $6\;{\mu}g$ bFGF treated group showed a significant higher crypt depth and microvilli length than RT alone group (p<0.05). 3) The bone marrow of bFGF treated group showed less hypocellularity than radiation alone group on day 7 and 14 after TBI (p<0.05), and this protective effect was more evident in $6\;{\mu}g$ bFGF treated group than that of $3\;{\mu}g$ bFGF treated group. 4) bFGF protected against early radiation induced apoptosis in intestinal crypt cell but might have had no antiapoptotic effect in bone marrow stem cell and pulmonary endothelial cells. 5) There was no significant differences in tumor growth rate between tumor control and bFGF alone groups (p>0.05). 6) There were no significant differences in histopathologic findings of lung and mouse sarcoma 180 tumor between radiation alone group and bFGF treated group. Conclusions : Our results suggest that bFGF protects small bowel and bone marrow from acute radiation damage without promoting the inoculated tumor growth in C3H mice. Improved recovery of early responding normal tissue and reduced number of radiation induced apoptosis may be possible mechanism of radioprotective effect of bFGF.

• Journal of Chest Surgery
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• v.39 no.7 s.264
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• pp.511-519
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• 2006

Background: Loss of cardiomyocytes in the myocardial infarction leads to regional contractile dysfunction, and necrotized cardiomyocytes in infracted ventricular tissues are progressively replaced by fibroblasts forming scar tissue. Although cardiomyoplasty, or implantation of ventricular assist device or artificial heart was tried in refractory heart failure, the cardiac transplantation was the only therapeutic modality because these other therapeutic strategies were not permanent. Cell transplantation is tried instead of cardiac transplantation, especially bone marrow is the most popular donated organ. But because bone marrow aspiration procedure is invasive and painful, and it had the fewer amounts of cellular population, the adipose tissue is recommended for harvesting of mesenchymal stem cells. Material and Method: After adipose tissues were extracted from abdominal subcutaneous adipose tissue and intra-abdominal adipose tissue individually, the cellular components were obtained by same method. These cellular components were tried to transformation with the various titers of 5-azacytidine to descript the appropriate concentration of 5-azacytidine and possibility of transformation ability of adipose tissue. Group 1 is abdominal subcutaneous adipose tissue and Group 2 is intra-abdominal adipose tissue-retroperitoneal adipose tissue and omentum. Cellular components were extracted by collagenase and $NH_4Cl$ et al, and these components were cultured by non-induction media - DMEM media containing 10% FBS and inducted by none, $3{\mu}mol/L,\;6{\mu}mol/L,\;and\;9{\mu}mol/L$ 5-azacytidine after the 1st and 2nd subculture. After 4 weeks incubation, tile cell blocks were made, immunostaining was done with the antibodies of CD34, heavy myosin chain, troponin T, and SMA. Result: Immunostaining of the transformed cells for troponin T was positive in the $6{\mu}mol/L\;&\;9{\mu}mol/L$ 5-azacytidine of Group 1 & 2, but CD34 and heavy myosin chain antibodies were negative and SMA antibody was positive in the $3{\mu}mol/L\;&\;6{\mu}mol/L$ 5-azacytidne of Group 2. Conclusion: These observations confirm that adult mesenchymal stem cells isolated from the abdominal subcutaneous adipose tissues and intra-abdominal adipose tissues can be chemically transformed into cardiomyocytes. This can potentially be a source of autologous cells for myocardial repair.

• Molecules and Cells
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• v.38 no.7
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• pp.663-668
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• 2015

hBMSCs are multipotent cells that are useful for tissue regeneration to treat degenerative diseases and others for their differentiation ability into chondrocytes, osteoblasts, adipocytes, hepatocytes and neuronal cells. In this study, biodegradable elastic hydrogels consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(${\varepsilon}$-caprolactone) (PCL) scaffolds were evaluated for tissue engineering because of its biocompatibility and the ability to control the release of bioactive peptides. The primary cultured cells from human bone marrow are confirmed as hBMSC by immunohistochemical analysis. Mesenchymal stem cell markers (collagen type I, fibronectin, CD54, $integrin1{\beta}$, and Hu protein) were shown to be positive, while hematopoietic stem cell markers (CD14 and CD45) were shown to be negative. Three different hydrogel scaffolds with different block compositions (PEG:PCL=6:14 and 14:6 by weight) were fabricated using the salt leaching method. The hBMSCs were expanded, seeded on the scaffolds, and cultured up to 8 days under static conditions in Iscove's Modified Dulbecco's Media (IMDM). The growth of MSCs cultured on the hydrogel with PEG/PCL= 6/14 was faster than that of the others. In addition, the morphology of MSCs seemed to be normal and no cytotoxicity was found. The coating of the vascular endothelial growth factor (VEGF) containing scaffold with Matrigel slowed down the release of VEGF in vitro and promoted the angiogenesis when transplanted into BALB/c nude mice. These results suggest that hBMSCs can be supported by a biode gradable hydrogel scaffold for effective cell growth, and enhance the angiogenesis by Matrigel coating.

• Korean Journal of Clinical Laboratory Science
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• v.49 no.2
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• pp.69-78
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• 2017

Cordyceps militaris has been used in traditional Chinese medicine owing to its anticancer and immunomodulatory activities. Germanium compounds have also been shown to be associated with many pharmacological functions, such as antimicrobial, antiviral, antitumor, antimutagenic, and immunomodulating effects. In this study, we examined the biological properties of hot water extract from mycelial liquid culture of germanium-enriched C. militaris (CMGe). CMGe displayed a concentration-dependent antiproliferation activity against four human cancer cell lines. The antiproliferative activity of CMGe was 2-4-fold lower than that of hot water extract from mycelial liquid culture in C. militaris (CM). However, CM had a concentration-dependent cytotoxicity to human bone marrow-derived mesenchymal stem cells (MSCs). Contrastingly, CMGe did not cause any cellular damage to MSCs. MSCs cultured with CMGe displayed an increased proliferative activity with no cytotoxic effect. The oral administration of CMGe inhibited increased tumor volume and weight compared with the control group. CMGe has the potential to be used as an industrial product in medicinal foods as well as in pharmaceutical products.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.1
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• pp.23-34
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• 2009

Objectives: Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. It is known that long-term in vitro culture of human bone marrow and adipose tissue derived-MSCs lead to a reduction of life span and a change of stem-like characters. The aim of our study was to examine whether stem cell properties of human umbilical cord-derived stem cells (HUC) could be affected by in vitro expansion. Methods: HUC were isolated from human umbilical cord and cultured for 10 passages in vitro. Morphology and population doubling time (PDT) were investigated, and changes of stem cell properties were examined using RT-PCR and immunocytochemistry during serial subcultures. Results: Morphology and PDT of HUC began to change slightly from the 7th passage (p7). Expression level of nestin and vimentin mRNAs increased along with the culture period from p4 until p10. In contrast, expression level of SCF mRNA decreased during the same culture period. Expression level of Oct-4 and HNF-4${\alpha}$ mRNAs was not significantly changed throughout the culture period until p10. Expression level of BMP-4, FGF-5, NCAM and HLA-ABC mRNAs appeared to increase as the culture continued, however, the difference was not significant. Immunocytochemical studies showed that HUC at p3, p6 and p9 positively were stained with antibodies against SSEA-3 and SSEA-4 proteins. Interestingly, staining intensity of HUC for ICAM-1 and HLA-ABC gradually increased throughout the culture period. Intensity against thy-1 and fibronectin antibodies increased at p9 while that against TRA-1-60 and VCAM-1 antibodies began to decrease at p6 until p9. Conclusions: These results suggest that HUC change some of their stem cell characteristics during in vitro culture. Development of culture system might be needed for the maintenance of characteristics.

• Journal of Life Science
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• v.16 no.2 s.75
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• pp.231-239
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• 2006

Human mesenchymal stem cells (hMSCs) in bone marrow (BM) can be induced to differentiate into a variety of mesenchymal tissues, including adipocytes, osteoblasts and chondroblasts, under the influence of certain growth or environmental factors. In this study, we analyzed the differentiation process and the associated gene expression profiles inherent to the process by which hMSCs differentiate into osteoblasts. We conducted a comparison of gene expression profiles of the normal human BM MSCs, using human 8K cDNA microarray, incubated in media containing either a combination of $\beta$-glycerol phosphate, L-ascorbic acid, and dexamethasone, or in medium lacking these osteogenic supplements. During the osteoblastic differentiation process, 36 genes were determined to be up-regulated, and 59 genes were shown to be down-regulated. Osteoprotegerin, LRP5, and metallothionein 2A, all of which are associated with the osteogenetic process, were up-regulated, and genes associated with the differentiation of MSCs into other lineages, including muscle, adipose tissue and vascular structure were down-regulated. The set of differentially expressed genes reported in this work should contribute to our current understanding of the processes inherent to the differentiation of MSCs into osteoblasts.

• Korean Journal of Clinical Laboratory Science
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• v.48 no.3
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• pp.169-175
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• 2016

Saccharin (o-benzoic sulfimide) is the first artificial and non-caloric sweetener that was first synthesized in 1879. In this study, we examined the biological activity of saccharin against various human cancer cell lines and human bone marrow-derived mesenchymal stem cells. A viability assay based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was performed to test for the cytotoxicity of saccharin about the four human cancer cell lines (H460, H157, A549 and SKOV3), one murine cancer cellline (Raw264.7), and MSCs. In order to find the differentially expressed gene in saccharin-treated MSCs against untreated MSCs, we performed annealing control primer (ACP)-based differential display reverse transcriptionp-olymerase chain reaction (DDRT-PCR). All tested cells were treated with saccharin at various concentrations (0.0, 4.8, 7.2, 9.6, 12.0, 14.4 mg/mL) for 48 hr. The number of metabolically active cancer cells decreased when treated with the saccharin at various concentrations for 48 hr as compared with the untreated cells. The decrease in cell survival was more evident with increasing concentrations of saccharin. Moreover, novel candidate genes, which were differentially expressed in MSCs in response to saccharin, were identified in 16 bands on 2% agarose gel. This revealed 16-7 up-regulated and 9 down-regulated-differentially expressed genes indicated by arrows. One of these candidate genes was a FK506-binding protein gene. The functional roles of FK506 binding proteins, with respect to the activities of stem cell proliferation, were not characterized. Further studies are required to get a better understanding of FK506-binding proteins in its roles in increasing stem cell proliferative activities from using saccharin.