Our previous research on sulfated polysaccharide purified from Ecklonia cava, a brown alga found in Jeju island, Korea, showed that sulfated polysaccharides modulate the apoptotic threshold of intestinal cells, thereby preventing intestinal damage caused by ionizing radiation. In this study, we investigated the ability of sulfated polysaccharide to augment restoration of small intestinal stem cells from γ-ray-induced damage. In our results, sulfated polysaccharide treatment increased the numbers of Ki-67-positive cells as well as inducible nitric oxide synthase (iNOS)-expressing cells in the small intestine compared with those of irradiated only mice. Meanwhile, exposure to irradiation increased the number of paneth cells, which are frequently associated with intestinal inflammation, whereas sulfated polysaccharide treatment reduced the number of paneth cells in the small intestinal crypt. Conclusively, our data suggest that reduction of iNOS-expressing cells and paneth cells in sulfated polysaccharide-treated mice contributes to the inhibition of radiation-induced intestinal inflammation.

Mesenchymal stem cell (MSC) based cell therapy has emerged as a promising therapeutic approach for treatment of several degenerative, infectious and non-infectious diseases. Numerous studies have demonstrated the remarkable immunosuppressive and antibacterial effects of MSCs both in vitro and in vivo, in animal models and in humans. However, the antibacterial effects of MSCs rely heavily on their paracrine factors rather than direct cell-to-cell contact and the effect is specific to disease and site of infection or injury. Furthermore, recent studies have demonstrated the double-edged sword effect of MSCs in bacterial infectious diseases. Despite their inherent potential for repair of damaged tissues, immunosuppression, and alleviation of various autoimmune as well as infectious diseases, MSCs also play a critical role in promoting persistent bacterial infection and disease progression. Therapeutic administration of MSCs successfully inhibited the bacterial growth and enhances survival by improved clearance of pathogenic bacteria in sepsis and pneumonic conditions. However, due to their abnormal transformation, they assist in long lasting survival and persistent infection of Mycobacterium tuberculosis (M. tuberculosis) and may also be responsible for progression of gastric cancer. This review focuses on recent advances that have broadened our understanding of MSC based therapy for bacterial diseases and provides new insight into the possible therapeutic targets of fatal bacterial diseases.

Objective. To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation–mimicking agent cobalt chloride (CoCl2) on the osteogenic differentiation of human mesenchy-mal stem cells (hMSCs) and elucidate the underlying mole-cular mechanisms. Study design. The dose and exposure periods for CoCl2 in hMSCs were optimized by cell viability assays. After confirmation of CoCl2-induced HIF-1α and vas-cular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with CoCl2 on hMSC osteogenic differentiation were evaluated by RT- PCR analysis of osteogenic gene expression, an alkaline phos-phatase (ALP) activity assay and by alizarin red S staining. Results. Variable CoCl2 dosages (up to 500 µM) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After CoCl2 treatment of hMSCs at 100 µM for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocal-cin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP acti-vity was increased in these treated cells in which an accele-rated osteogenic capacity was also verified by alizarin red S staining. Conclusions. The osteogenic differentiation poten-tial of hMSCs could be preserved and even enhanced by CoCl2 treatment.

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal α -1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from α-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% CO2 incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers (CD45－, 29+, 90+ and 105+) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited CD45－, 29+, 90+ and 105+ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at 1×103 cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs (15.0 ± 0.4 μm) had a little larger cell size than Wild BM-MSCs (13.5 ± 0.3 μm). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-34TM cell culture media at 37℃. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin α6 and β1, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.

Matrix metalloproteinases (MMPs) have been known to affect to cell migration, proliferation, morphogenesis and apoptosis by degrading the extracellular matrix. In the previous studies, undifferentiated mouse embryonic stem cells (ESCs) were successfully proliferated inside the extracellular matrix (ECM) analog-conjugated three-dimensional (3D) poly ethylene glycol (PEG)-based hydrogel. However, there is no report about MMP secretion in ESCs, which makes it difficult to understand and explain how ESCs enlarge space and proliferate inside 3D PEG-based hydrogel constructed by crosslinkers containing MMP-specific cleavage peptide sequence. Therefore, we investigated what types of MMPs are released from undifferentiated ESCs and how extracellular signals derived from various niche conditions affect MMP expression of ESCs at the transcriptional level. Results showed that undifferentiated ESCs expressed specifically MMP2 and MMP3 mRNAs. Transcriptional up-regulation of MMP2 was caused by the 3D scaffold, and activation of integrin inside the 3D scaffold upregulated MMP2 mRNAs synergistically. Moreover, mouse embryonic fibroblasts (MEFs) on 2D matrix and 3D scaffold induced upregulation of MMP3 mRNAs, and activation of integrins through conjugation of extracellular matrix (ECM) analogs with 3D scaffold upregulated MMP3 mRNAs synergistically. These results suggest that successful proliferation of ESCs inside the 3D PEG-based hydrogel may be caused by increase of MMP2 and MMP3 expression resulting from 3D scaffold itself as well as activation of integrins inside the 3D PEG-based scaffold.

A lot of works have been dedicated to clarify the reasons why the establishment of embryonic stem cells (ESCs) from pig is more difficult than that from mouse and human. Several concomitant factors such as culture condition including feeder layer, sensitivity of cell to cell contact, definitive markers of pluripotency for evaluation of the validity and optimal timing of derivation have been suggested as the disturbing factors in the establishment of porcine ESCs. Traditionally, attempts to derive stem cells from porcine embryos have depend on protocols established for mouse ESCs using inner cell mass (ICM) for the isolation and culture. And more recently, protocols used for primate ESCs were also applied. However, there is no report for the establishment of porcine ESCs. Indeed, ungulate species including pigs have crucial developmental differences unlike rodents and primates. Here we will review recent studies about issues for establishment of porcine ESCs and discuss the promise and strategies focusing on the timing for derivation and pluripotent state of porcine ESCs.

This study investigated the genes involved in the dif- ferentiation of odontoblasts derived from human dental pulp stem cells (hDPSCs). hDPSCs isolated from human tooth pulp were validated by fluorescence activated cell sor- ting (FACS). After odontogenic induction, hDPSCs were analyzed investigated by Alizaline red-S staining, ALP assay, ALP staining and RT-PCR. Differential display-poly- me rase chain reac tion (DD-PCR) was pe rformed to s c re en differentially expressed genes involved in the differentia- tion of hDPSCs. By FACS analysis, the stem cell markers CD24 and CD44 were found to be highly expressed in hDPSCs. When hDPSCs were treated with agents such as β- glycerophosphate (β-GP) and ascorbic acid (AA), nodule formation was exhibited within six weeks. The ALP activity of hDPSCs was found to elevate over time, with a detectable up-regulation at 14 days after odontogenic induc- tion. RT-PCR analysis revealed that dentin sialophospho- protein (DSPP) and osteocalcin (OC) expression had inc- reased in a time-dependent manner in the induction culture. Through the use of DD-PCR, several genes were diffe- rentially detected following the odontogenic induction. These results suggest that these genes may possibly be linked to a variety of cellular process during odontogenesis. Further-more, the characterization of these regulated genes during odontogenic induction will likely provide valuable new in- sights into the functions of odontoblasts.

Human umbilical cord is easy to obtain because it is discarded after birth, so that ethical issues can be avoided. Chondrogenesis studies using MSCs from bone marrow, cord blood, and adipose have indicated that TGFβ3 and BMP6 stimulate chondrogenesis. Therefore, we investigated chondrogenesis of hUC-MSCs on TGFβ3, BMP6, and combination of the two growth factors. We initiated chondrogenesis of cells by application of physical forces to form 3D cell clusters. After initiation, we designated four experimental groups for differentiation of cells, as follows: control, 10 ng/mL TGFβ3, 100 ng/mL BMP6, and the combination of 5 ng/mL TGFβ3 and 50 ng/mL BMP6. For analysis of chondrogenesis, GAG contents, mRNA expression, histological analysis and immunohistochemistry (IHC) were performed. For analysis of GAG contents, GAG assay was performed and RT-PCR was performed for determination of chondrogenic markers. Histological analysis was performed through safranin O, alcian blue, and IHC was performed using collagen type I and II. GAG contents were increased 184% by TGFβ3, 147% by BMP6, and 189% by the combination of TGFβ3 and BMP6, compared to control. The growth factors improved collagen II and aggrecan expression; in particular, TGFβ3 and BMP6 showed a synergistic effect, compared to only TGFβ3 or BMP6 treated. The results of histological and IHC analysis indicated that chondrogenic differentiation in TGFβ3 and the combination of TGFβ3 and BMP6 showed more cartilage deposition. In conclusion, TGFβ3 and BMP6 differentiated hUC-MSCs into chondrogenic clusters of the combination treatment of the two growth factors showed more efficient chondrogenic ability.

Embryonic stem cell classically cultured on feeder layer with FBS contained ES medium. Feeder-free mouse ES cell culture systems are essential to avoid the possible contamination of nonES cells. First we determined the difference between ES cell and MEF by Oct4 population. We demonstrate to culture and to induce differentiation on feeder free condition using a commercially available mouse ES cell lines.

Skin serves as an easily accessible source of multipotent stem cells with potential for cellular therapies. In pigs, stem cells from skin tissues of fetal and adult origins have been demonstrated as either floating spheres (cell aggregates) or adherent spindle-shaped mesenchymal stem cell (MSC)-like cells depending on culture conditions. The cells isolated from the epidermis and dermis of porcine skin showed plastic adherent growth in the presence of serum and positively expressed a range of surface and intracellular markers that are considered to be specific for MSCs. The properties of primitive stem cells have been observed with the expression of alkaline phosphatase and markers related to pluripotency. Further, studies have shown the ability of skin-derived MSCs to differentiate in vitro along mesodermal, neuronal and germ-line lineages. Moreover, preclinical studies have also been performed to assess their in vivo potential, and the findings appear to be effective in tissue regeneration at the defected site after transplantation. The present review describes the recent progress on the biological features of porcine skin-derived MSCs as adherent cells, and summarizes their potential in advancing stem cell based therapies.

Although embryonic stem cells (ESCs) or ES-like cells are reported from many mammalian species other than the mouse, the culture system for murine ESCs may not be suitable to the other species. Previously many other research groups have modified either human or mouse ESC culture systems for bovine ESC culture. In this study, we compared three different culture mediums consisting of DMEM, -MEM or KnockOut-DMEM (KO), which are modified from human or mouse ESC culture system, for the generation of bovine ESCs. In this study, some pre-requisite events which are important for establishment and long-term propagation of ESCs such as inner cell mass (ICM) attachment on feeder cells, primary colony formation and sustainability after passaging. Once the ICM clumps attached on feeder cells, this was designated as passage 0. In regards to the rate of ICM attachment, -MEM was superior to the other systems. For primary colony formation, there was no difference between DMEM and -MEM whereas KO showed lower formation rate than the other groups. For passaging, the colonies were split into 2~4 pieces and passed every 5~6 days. From passage 1 to passage 3, DMEM system seemed to be appropriate for maintaining putative bovine ESCs. On the other hand, -MEM tended to be more suitable after passage 6. Although -MEM support to maintain a ES-like cell progenies to passage 15, all three culture systems which are modified from human or mouse ESC culture media failed to retain the propagation and long-term culture of putative bovine ESCs. Our findings imply that more optimized alternative culture system is required for establishing bovine ESC lines.