Somatic cell nuclear transfer (SCNT) in pigs has been used as a very important tool to produce transgenic for the pharmaceutical protein, xenotransplantation, and disease model and basic research of cloned animals. However, the production efficiency of SCNT embryos is very low in pigs and miniature pigs. The type of donor cell is an important factor influencing the production efficiency of these cloned pigs. Here, we investigated the developmental efficiency of SCNT embryos to blastocysts and full term development using fetal fibroblasts (FF) and mesenchymal stem cells (MSCs) to identify a suitable cell type as donor cell. We isolated each MSCs and FF from the femoral region and fetus. Cultured donor cell was injected into matured embryos for cloning. After that, we transferred cloned embryos into surrogate mothers. In term of in vitro development, the SCNT embryos that used MSCs had significantly higher in cleavage rates than those of FF (81.5% vs. 72%) (p<0.05), but the blastocyst formation rates and apoptotic cell ratio was similar (15.1%, 6.18% vs. 20.8%, 9.32%). After embryo transferred to surrogates, nine and nineteen clone piglets were obtained from the MSCs and FF group, respectively, without significant differences in pregnancy and birth rate (50%, 40% vs. 52.3%, 45.4%) (p>0.05). Moreover, there was no significant difference in the corpus hemorrhagicum numbers of ovary, according to pregnancy, abortion, and delivery of surrogate mothers between MSCs and FF groups. Therefore, the MSCs and FF are useful donor cells for production of clone piglets through SCNT, and can be used as important basic data for improving the efficiency of production of transgenic clone pigs in the future.

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.

Telomeres are known as a specialized region in the end of chromosomes to protect DNA destruction, but their lengths are shortened by repetition of cell division. This telomere shortening can be preserved or be elongated by telomerase and TERT expression. Although a certain condition in the cells may affect to the cellular and molecular characteristics, the effect of differentiation induction to telomere length and telomerase activity in mesenchymal stem cells (MSCs) has been less studied. Therefore, the present study aimed to uncover periodical alterations of telomere length, telomerase activity and TERT expression in the dental pulp-derived MSCs (DP-MSCs) under condition of differentiation inductions into adipocytes and osteoblasts on a weekly basis up to 3 weeks. Shortening of telomere was significantly (p < 0.05) identified from early-middle stages of both differentiations in comparison with undifferentiated DP-MSCs by non-radioactive chemiluminescent assay and qRT-PCR method. Telomere length in undifferentiated DP-MSCs was 10.5 kb, but the late stage of differentiated DP-MSCs which can be regarded as the adult somatic cell exhibited 8.1-8.6 kb. Furthermore, the relative-quantitative telomerase repeat amplification protocol or western blotting presented significant (p < 0.05) decrease of telomerase activity since early stages of differentiations or TERT expression from middle stages of differentiations than undifferentiated state, respectively. Based on these results, it is supposed that shortened telomere length in differentiated DP-MSCs was remained along with prolonged differentiation durations, possibly due to weakened telomerase activity and TERT expression. We expect that the present study contributes on understanding differentiation mechanism of MSCs, and provides standardizing therapeutic strategies in clinical application of MSCs in the animal biotechnology.

Because mesenchymal stem cells (MSCs) maintain distinct capacities with respect to self-renewal, differentiation ability and immunomodulatory function, they have been highly considered as the therapeutic agents for cell-based clinical application. Of particular, differentiation condition alters characteristics of MSCs, including cellular morphology, expression of gene/protein and cell surface molecule, immunological property and apoptosis. However, the previous results for differentiation-related apoptosis in MSCs have still remained controversial due to varied outcomes. Therefore, the present study aimed to disclose periodical alterations of pro- and anti-apoptosis in MSCs under differentiation inductions. The human dental pulp-derived MSCs (DP-MSCs) were differentiated into adipocytes and osteoblasts during early (1 week), middle (2 weeks) and late (3 weeks) stages, and were investigated on their apoptosis-related changes by Annexin V assay, qRT-PCR and western blotting. The ratio of apoptotic cell population was significantly (p < 0.05) elevated during the early to middle stages of differentiations but recovered up to the similar level of undifferentiated state at the late stage of differentiation. In the expression of mRNA and protein, whereas expressions of pro-apoptosis-related makers (BAX and BAK) were not altered in any kind and duration of differentiation inductions, anti-apoptosis marker (BCL2) was significantly (p < 0.05) elevated even at the early stage of differentiations. The recovery of apoptotic cell population at the late stage of differentiation is expected to be associated with the response by elevation of anti-apoptotic molecules. The present study may contribute on understanding for cellular mechanism in differentiation of MSCs and provide background data in clinical application of MSCs in the animal biotechnology to develop effective and safe therapeutic strategy.

The objective of this study was to identify the proteins actively involved in the protection and repair of damaged cells, secreted by canine adipose derived mesenchymal stem cells (AT-MSCs) into the conditioned media. For this purpose, conditioned media (CM) was recovered from passage three stage canine AT-MSCs and skin fibroblasts cultured in serum free media after 24, 48 and 72 h. The extraction of exosomes was performed from 10-20 ml of CM using total exosome isolation kit. The isolated exosomes were then subjected to western analysis for the identification of annexin-I, annexin-II, histone H3 and dysferlin proteins. Results demonstrated the expression of proteins in the conditioned media isolated from canine AT-MSCs reflecting their potential in reducing the extent of damage at cellular levels. In conclusion, the conditioned media derived from canine AT-MSCs can be helpful in restoring the normal structure of cells both in vivo and in vitro conditions.

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) serve as a unique source for cell therapy. We investigated whether exosomes from iMSCs promote the proliferation of human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs). iPSCs were established from human Wharton’s jelly MSCs and were allowed to differentiate into iMSCs. Exosomes were collected from the culture supernatant of MSCs (MSC-exo) and iMSCs (iMSC-exo), and their characteristics were investigated. Both exosome types possessed basic characteristics of exosomes and were taken up by skin cells in vitro and in vivo. A significant increase in HaCaT proliferation was observed with iMSC-exo, although both exosomes increased the viability and cell cycle progression in HaCaT and HDFs. No significant difference was observed in the closure of wound scratch and the expression of reparative genes between cells treated with the two exosome types. Both exosomes enhanced the secretion of collagen in HaCaT and HDFs; however, an increase in fibronectin level was observed only in HaCaT, and this effect was better with iMSC-exo treatment. Only iMSC-exo increased the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2. Our results indicate that iMSC-exo promote the proliferation of skin cells by stimulating ERK1/2 and highlight the application of iMSCs for producing exosomes.

Mesenchymal stem cells (MSCs), which are present in all tissues, can differentiate into cells with various specific functions. Recently, cell-based therapies using MSCs have been increasing in the veterinary research and related fields. In this study, we investigated the cellular morphology, proliferating capacities, expression of cell surface markers such as CD13, CD34, CD44, CD45, CD90, and CD105, mesodermal differentiation potentials, and expression of senescence-related markers of p53, p21, and telomerase reverse transcriptase in equine adipose tissue-derived MSCs (eAD-MSCs) after cryopreservation. The eAD-MSCs were analyzed immediately and after being frozen in liquid nitrogen for 1 year (< 1 year, G1) and more than 3 years (> 3 years, G2), respectively. After cryopreservation for 1 - 3 years, G2 eAD-MSCs showed similar cellular morphology, proliferating capacities, and expression of cell surface markers when compared with G1 eAD-MSCs. Moreover, cryopreservation did not affect the adipogenic, chondrogenic, or osteogenic differentiation potentials of G1 and G2 eAD-MSCs. Collectively, cryopreservation for (or over) 3 years maintained the stem cell phenotype and differentiation potentials of eAD-MSCs. These results will be an advantage that can be effectively used for future development of cell-based therapies.

The mesenchymal stem cells (MSC) has been investigated as a source of stem cell therapy to replace and treat damaged cells. Human endometrial epithelial and stromal cells was isolated from hysterectomy tissue and the direct evidence of stem/progenitor cells in the human endometrium was identified. Endometrium derived stem cells (EnMSCs) are known to have a high proliferative ability, genetic stability, lack of tumorigenicity and low immnunogenicity during long-term cultivation. Here, we aimed to identify MSC in canine endometrium and characterize its potential to differentiate into decidua cells. EnMSCs were isolated from thrown-away spayed uterus of adult canine depending on their estrus cycle, and identified by flow cytometry, immunocytochemistry and flow cytometry with MSC specific markers. We then characterized the ability of EnMSCs by the doubling-time analysis, colony-forming units and MSC differentiation assays. Isolated EnMSCs expressed stem cell specific genes (Sox2, Oct4, Nanog, MCAM, Endoglin, Susd2 and IGTB) and MSC surface markers (CD90, CD44 and CD117). EnMSCs are also differentiated into adipogenic, osteogenic and chondrogenic cells morphologically under modified conditions with the expression of lineage specific genetic markers. EnMSCs showed higher proliferation ability than canine amniotic fluid derived MSCs which were used as a positive control. EnMSCs were cultured at low density (10, 20, cells/cm2) and initiated to form small colonies of loosely-arranged cells and gradually formed large colonies of densely-packed cells which underwent self-renewal with high proliferative potential which is similar to the clonogenicity feature of human endometrium-derived stem cells. EnMSCs were then induced to differentiate into decidua cells with 0.5 mM dbcAMP. After 14 days, EnMSCs changed their morphology into the elongated and rounded shape. The induced decidual cells expressed PRL and IGFBP1 which are typically expressed in decidua cells. In conclusion, we successfully isolated and characterized MSC in the canine endometrium which differentiated into decidua cells. These results showed that endometrium may be a promising source of stem cells, and furthermore raise the possibility of canine EnMSCs as a novel hypothetical decidualisation model of infertility associated with decidualisation insufficiency and implantation failure.

Though hydrogen peroxide (H₂O₂) causes a deleterious effect to cells with its reactive oxygen species resulting in cell death, S-allyl cysteine (SAC, a bioactive organosulfur compound of aged garlic extract) has been known to have a cytoprotective effect. Few reported profiles of gene expression of H₂O₂ and SAC treated human cord blood derived mesenchymal stem cells (MSC). This study revealed changes in the profile of twenty-one genes grouped by oxidative stress, antioxidant, cell death, anti-apoptosis and anti-aging by quantitative real time PCR. A concentration of 100μM of SAC or 50μM of H₂O₂ was applied to MSC which show moderate growth and apoptosis pattern. H₂O₂ treatment enhanced expression of eleven genes out of twenty-one genes compared with that of control group, on the contrary SAC suppressed expression of eighteen genes out of twenty-one genes except C ros oncogene. SAC decreased expression of oxidative stress genes such as SOD1, CAT and GPX. These results seemed consistent with reports which elucidated over expression of NF-kB by H2O2, and suppression of it by SAC. This study will confer basic information for further experiments regarding the effects of SAC on gene levels.

Adipose tissue-derived mesenchymal stem cells (ASCs) are very interesting in several laboratory animals and humans because they are easy to harvest and expand to generate millions of cells from a small quantity of fat. ASCs are known as useful materials for clinical applications in human cell therapy and as a donor cell in somatic cell nuclear transfer (SCNT). Here, we investigated if 1) minipig ASCs can be isolated, self-renewed and differentiated into multiple tissue lineages, 2) ASCs can be a suitable donor cell type for generation of cloned pig. In order to isolate ASC, adipose tissues were collected from inguinal region of a 6-year-old female minipig. The ASCs were attached to the culture dish with a fibroblast-like morphology. They expressed cell-surface marker characteristics of stem cell, underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. To investigate its potential as donor cell for cloning, we respectively carried out SCNT using ASC, adult skin fibroblast (ASF) and fetal fibroblast (FF) derived from same minipig. The ratio of blastocysts to 2-cell embryos and total cell number of blastocysts were monitored as experimental parameters. In results, cleavage and developmental competence to blastocysts rate showed no significant difference among the three groups. On the other hand, total cell numbers of blastocysts derived from ASC and FF were significantly higher than in ASF (89±7.9 and 105±5.5 vs. 57.5±5.2, respectively). Our results demonstrated that ASC have potential compared to ASF and FF in terms of the in vitro development and blastocyst formation ability. In further study, we will investigate the in vivo developmental ability of ASC as donor cell for pig cloning. * This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), Institute for Veterinary Science, the BK21 program, TS Corporation and Optifarm Solution.

Bone marrow (BM) cell harvesting is a crucial element in the isolation of mesenchymal stem cells (MSCs). A simple method for harvesting cat BM cells is described. The results show that a large number of BM cells can rapidly be harvested from the cat by this simple procedure. MSCs prepared by density-gradient method were spindle-shaped morphology with bipolar or polygonal cell bodies and strongly positive for CD9 and CD44 and negative for CD18 and CD45-like. They were capable of differentiation to adipocytic and osteocytic phenotypes when exposed to appropriate induction media. The advantages of this method are its rapidity, simplicity, low invasiveness, and low donor attrition and good outcome.

Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal and differentiation into a variety of cell types. They represent an attractive source of cells for gene and cell therapy. The purpose of this study is to direct the specific expression of the DsRed reporter gene in Sca-1+ BMMSCs differentiated into a cardiomyogenic lineage. We constructed the prMLC-2v-DsRed vector expressing DsRed under the control of the 309 tp fragment of the rat MLC-2v 5'-flanking region. The specific expression of the DsRed reporter gene under the transcriptional control of the 309 bp fragment of the rat MLC-2v promoter was tested in 5-azacytidine healed-Sca-1+ BMMSCs over 2 weeks after the prMLC-2v-DsRed transfection. The prMLC-2v-DsRed was specifically expressed in the Sca-1+ BMMSCs with cardiomyogenic lineage differentiation and it demonstrates that the 309 bp sequences of the rat MLC-2v 5'-flanking region is sufficient to confer cardiac specific expression on a DsRed reporter gene. The cardiac-specific promoter-driven reporter vector provides an important tool for the study of stem cell differentiation and cell replacement therapy in ischemic cardiomyopathy.

Bisphosphonates have been widely used to treat metabolic bone diseases, although the . mechanism of bisphosphonate action on bone has not been fully understood. This study aimed to examine the direct action of pamidronate on cell proliferation and differentiation of cultured human mesenchymal stem cells(hMSC). Four experimental groups and two control groups were designed; Experimental groups included both osteogenic supplement(OS) and pamidronate-treated group, pamidronate-treated group after 1 week OS treatment, only pamidronate-treated group, OS-treated group after 1week pamidronate treatrnent. Control gr。니ps included DMEMtreated group and OS-treated group. Human MSCs were isolate from bone maπow ， and cultured for 7, 14, 21 days. For the detection of osteoblastic differentiation, AI.Pase activity was measured and the expression of type 1 collagen and osteocalcin were evaluated. Von Kossa’s silver stain was performed for the examination of calcification. As results, the proliferation rate of 바1SC was maintained to be more than 90% by 1uglml of pamidronate. AI.Pase activity showed the highest value at the concentration of 100nglml of pamidronate. In pamidronate-treated group, ALPase activity reached a peak at the third week and the expression of type 1 collagen mRNA and protein was enhanced compared to other experimental and control groups, whereas osteocalcin expression was found only in OStreated group. Calcification was decreased by a dose dependent manner followed by pamidronate treatment. This study su잃，est that pamidronate treatrnent may be able to enhance the osteoblastic differentiation of hMSC at the early stage. On the other hand, calcification appeared to be inhibited by pamidronate treatrnent.

The use of human mesenchymal stem cells (hMSCs) in cell-based therapy has attracted extensive interest in the field of regenerative medicine, and it shows applications to numerous incurable diseases. hMSCs show several superior properties for therapeutic use compared to other types of stem cells. Different cell types are discussed in terms of their advantages and disadvantages, with focus on the characteristics of hMSCs. hMSCs can proliferate readily and produce differentiated cells that can substitute for the targeted affected tissue. To maximize the therapeutic effects of hMSCs, a substantial number of these cells are essential, requiring extensive ex vivo cell expansion. However, hMSCs have a limited lifespan in an in vitro culture condition. The senescence of hMSCs is a double-edged sword from the viewpoint of clinical applications. Although their limited cell proliferation potency protects them from malignant transformation after transplantation, senescence can alter various cell functions including proliferation, differentiation, and migration, that are essential for their therapeutic efficacy. Numerous trials to overcome the limited lifespan of mesenchymal stem cells are discussed.

Transforming growth factor (TGF) family is well known to induce the chondrogenic differentiation of mesenchymal stem cells (MSC). However, the precise signal transduction pathways and underlying factors are not well known. Thus the present study aims to evaluate the possible role of C2 domain in the chondrogenic differentiation of human mesenchymal stem cells. To this end, 145 C2 domains in the adenovirus were individually transfected to hMSC, and morphological changes were examined. Among 145 C2 domains, C2 domain of protein kinase C eta (PKCη) was selected as a possible chondrogenic differentiation factor for hMSC. To confirm this possibility, we treated TGFβ3, a well known chondrogenic differentiation factor of hMSC, and examined the increased-expression of glycosaminoglycan (GAG), collagen type II (COL II) as well as PKCη using PT-PCR, immunocytochemistry and Western blot analysis. To further evaluation of C2 domain of PKCη, we examined morphological changes, expressions of GAG and COL II after transfection of PKCη -C2 domain in hMSC. Overexpression of PKCη-C2 domain induced morphological change and increased GAG and COL II expressions. The present results demonstrate that PKCη involves in the TGF-β3-induced chondrogenic differentiation of hMSC, and C2 domain of PKCη has important role in this process.

Mesenchymal stem cells (MSCs) are considered to be attractive approaching in gene or drug delivery for cancer therapeutic strategies. In this study, the ability and feasibility of human bone marrow derived MSCs expressing the cytosine deaminase (CD)/5-Fluorocytosin (5-FC) prodrug was evaluated to target human osteosarcoma cell line Cal-72. At first, the fibroblast-like cells were successfully obtained from human bone marrow and demonstrated that they contained full of stem characteristics by the ability of differentiation into adipocyte/osteocyte and expression of typical mesenchymal markers CD90, CD44, while negative for CD34 and CD133 markers. We established the stable CD-expressing MSCs cell line (CD-MSCs) by transfection of pEGFP-C3 containing cytosine deaminase::uracil phos-phoribosyltransferase (CD::UPRT) gene into MSCs, and confirmed that the manipulated MSCs still remained full characteristics of multipotent cells and shown migration toward human osteosarcoma cancer cells Cal-72 as high as origin MSCs. Based on bystander effect, the therapeutic CD-MSCs significantly augmented the cytotoxicity on cancer cell Cal72 in either direct co-culture or conditioned medium in the presence of 5-FC. Moreover, in osteosarcoma cancer- bearing mice, the therapeutic CD/5-FC MSCs showed the inhibition of tumor growth compared with control mice which was s.c injected with only Cal72. Our findings suggest that these therapeutic CD-MSCs may be suitable and viable cellular vehicles for targeting human osteosarcoma cancer.

One of the most effective and safe therapeutic methods for treating vitiligo, mixed autologous keratinocytes (KCs) and melanocytes (MCs) cultures have been used for autologous cell transplantation. However, the present transplantation method is faced with a problem that may require a large amount of skin tissue and keratinocytes have limited culture potency. We have found previously that human adipose derived stromal cells (hASCs) from aspirated fat tissue could be used in place of KCs and sufficient amounts of hASCs for transplantation could be obtained by small amount of aspirated fat tissue. The present investigation was determined the effect of ASCs on ex vivo expansion MCs for transplantation. In addition, we examined for a preservation conditions of MCs which have reported low recovery rates and a slowdown in growth after cryopreservation. Various conditions including ASCs ratio, incubation period, and additive materials for MCs cultivation was determined to improve the expansion ability of MCs. The growth rate of MCs colony was elevated 6.85 folds compared the previous conditions. These MCs showed a specific expression of immature melanocyte protein, Trp-2, but did not express the mature melanocyte proteins and markers (c-kit, CD133, and etc.) of mesenchymal stem cells that represents in ASCs feeder. Results in cryopreservation experiments were determined a preservation medium for MCs showing an increased recovery rates after thawing. The characteristics of MCs after cryopreservation using a designed medium were indicated consistent morphology and immunophenotype. In conclusion, ASCs as a feeder could be used in place of keratinocytes for ex vivo expansion of MCs. For clinical trial for vitiligo patients, efficiency experiments in preclinical state should be followed.

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. They are widely distributed in the human diet through crops, beans, fruits, vegetables and red wine. The specific health effects that anthocyanins might have in vivo are not known, although there are several possibilities related to obesity, cardiovascular disease, and cancer. In this study we used human subcutaneous adipose mesenchymal stem cells (hADSC) and mouse subcutaneous adipose mesenchymal stem cells (mADSC) to evaluate the effects of anthocyanins. And we examined the effect of cell activity and adipocyte differentiation by Cyanidin-3-O-glucoside (C3G), Delphinidin-3-ß-D-glucoside (D3G) that are among the anthocyanin family and black soybean extract. Using MTT assay method, we tested cellular metabolic activity. In mADSC, cell activity is significantly decreased by C3G and D3G (50 uM, 100 uM, and 200 uM), and black soybean extract (100 ug/ml and 200 ug/ml). In hADSC, cell activity is significantly decreased only by C3G (50 uM, 100 uM and 200 uM) unlike in mADSC. Cell activity is significantly increased of 100 uM D3G and black soybean extract (50 ug/ml, 100 ug/ml and 200 ug/ml). In mADSC, 50 uM C3G promoted differentiation into adipocyte but no effect in other concentration. D3G suppressed the differentiation of mADSC at 100 uM and 200 uM. 50 ug/ml black soybean extract promoted differentiation of mADSC, but 200 ug/ml black soybean extract suppressed differentiation. In hADSC, 50 uM, 100 uM and 200 uM C3G suppressed differentiation. 100 uM D3G promoted differentiation into adipocyte, but 200uM D3G suppressed it. Black soybean extract suppressed the differentiation into adipocyte at 50 ug/ml, 100 ug/ml and 200 ug/ml. These data showed that the responsibility to the C3G and D3G were different between hADSC and mADSC. Interestingly the responsibility to the black soybean extract was similar between hADSC and mADSC. Based on them, it is suggested that there are species-specificity to the cellular responsibility to the anthocyanins in subcutaneous ADSC.