Earticle

Hexanoic acid, six carbon and saturated fatty acid could be produced by bacteria like Clostridium kluyveri, Megsphaera elsdenii, and Clostridium sp. BS-1. In this study, we investigated optimization of hexanoic acid production by Clostridium sp. BS-1. To determine the effective factors for hexanoic acid production, fractional factorial design was used and concentration range of the factors was determined via steepest ascent method (SAM). Response surface methodology (RSM) was used for investigation of interactions between the factors and optimized hexaonoic acid production. The proposed solutions from RSM were verified experimentally. Yeast extract, FeSO4•7H2O, and butyric acid were determined as the effective factors.

A number of potential resources for biofuels and chemicals have been derived from marine algae. Sargassum fulvellum is a type of brown seaweed. Korea naturally grows 18 species of S. fulvellum. Among them, gulfweed and S. horneri are edible and others are used for fertilizers and seaweed powders. S. fulvellum only grows in certain regions and its production is declining due to over-gathering and pollution. Versatile biomass-derived platform compounds such as 5-hydroxymethylfurfural and levulinic acid can be used to form various chemicals. This study focused on optimized production of mono-sugars and chemicals such as 5-hydroxymethylfurfural and levulinic acid from Sargassum fulvellum using acid- and ionic liquid- catalysts with statistical approach. [This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (KRF-2008-313-D00303).]

Ionic liquids(ILs) have been known as a green solvent because they are stable solvents. However, it is not clearly known if ILs may possess potential toxic activities when released into the environment. In this study, we investigated the effects of long-term exposure to various ILs on Shewanella oneidensis MR-1, an environmental bacterium. MR-1 was acclimated through repeated exposure to ILs at sublethal concentrations. Among the resulting acclimated strains we selected an [Omim][BF4] acclimated strain for further study. The latter strain was named as SH-1 and compared with MR-1 in various aspects including morphology, growth, and cell surface hydrophobicity (CSH). When grown in a complex medium without IL, SH-1 showed slower growth compared to the MR-1. In the presence of IL growth of SH-1 was slower than that of SH-1 grown without IL. Cell morphology was observed by scanning electron microscopy(SEM) for each strains. Compared to the MR-1, SH-1 showed significantly elongated cell shape. Cell surface hydrophobicity(CSH) analysis was conducted to compare cell surface charges of the two strains. In the absence of ILs there was no significant difference between the two strains. However, upon exposure to ILs at sublethal concentrations, CSH of SH-1 was rapidly increased while that of MR-1 was not affected.

Biodegradation of laminaran, a main component of brown algae, was studied for reutilization of algae waste. Potential five bacteria were isolated newly, and their ability to degrade laminaran was carried out in a 100 ml flask with 20 ml of working volume. The inoculum size was 0.2% (w/v) and 10 g/L of laminaran was used. The reaction was executed at 37℃ and 180 rpm for 15 days. HPLC analyses indicated that few oligosaccharides other than glucose were also produced from laminaran. pH decreased up to 3.5 as laminaran was degraded. Cells reached a stationary phase after 9 days. At this time, 6.30 and 4.21 g/L of total reducing sugars and glucose were obtained, respectively. In ethanol fermentation using yeast, 1.88 g/L of ethanol was obtained from the 9-day sample.

Rice straw is a lignocellulosic biomass that is a renewable organic substance and alternative source of energy. For the first time, rice straw was pretreated in a novel manner using TiO2/UV system and the pretreated rice straw was hydrolyzed using Accellerase 1000™. Hydroxyl radicals (OH•) from TiO2/UV system could degrade lignin and carbohydrates. TiO2/UV pretreatment was essential step for conversion of hemicelluloses to xylose. The optimal conditions for TiO2/UV pretreatment were TiO2 concentration (0.1%, w/v), irradiation time (2 h), UV lamp type (UVC, 254 nm). Under these conditions and following enzymatic hydrolysis, the sugar yields were 59.6±0.7% for glucose (339±13 mg/g rice straw) and 50.3±2.8% for xylose (64±3 mg/g rice straw) of theoretical. The fermentation of enzymatic hydrolysates containing 10.5 g glucose/L and 3.2 g xylose/L with Pichia stipitis produced 3.9 g ethanol/L with corresponding yield of 0.39 g/g rice straw, which is 76.47% of the stoichiometric and fermentation efficiency yields. The TiO2/UV pretreatment method can be conducted at room temperature and atmospheric pressure. Also, TiO2/UV system has a price advantage.

With increasing environmental awareness and legislation, GS Caltex Corporation, one of the biggest petroleum and petrochemical companies in Korea, has extended its research to industrial biotechnology. In particular, the company is interested in the fermentative production of C4 alcohols, including n-butanol and 2,3-butanediol, because the both alcohols are industrially important chemicals and also can be used as fuel additives. We recently constructed a number of n-butanol over-producing recombinant Clostridium acetobutylicum strains by metabolic engineering. The heterogeneous expression of adh gene, encoding secondary alcohol dehydrogenase, in C. acetobutylicum successfully converted acetone into isopropanol, which is also well known a fuel additive. More recently, the company has started the research on 2,3-butanediol to be further converted into butadiene. Butadiene is mainly used as a raw material for the synthesis of synthetic rubber for manufacturing automotive tyre. We carried out a series of fermentations to evaluate the economic feasibility of the microbial production of 2,3-butanediol. The experimental results turn out that 2,3-butanediol can be produced at a productivity of more than 3.0 g/L/h with a yield of 0.45 g/g glucose. This work was supported by the Energy Efficiency & Resources of the Korea Institute of Energy Technology Evaluation and Planning(KETEP) grant funded by the Korea government Ministry of Knowledge Economy.

Alginates are a family of linear polysaccharides occurring as a structural component of the cell wall in marine brown algae and bacteria [1]. The monomers in alginate, β-D-mannuronic acid and its C-5 epimer α-L-guluronic acid, can be arranged in varying proportions and sequence in a chain bound by 1→4 linkages [2]. Alginates find considerable commercial application in food, pharmaceutical and industrial uses due to their solution properties [3]. In this poster section, we report the results of the alginate extraction yields from Laminaria japonica under the various extraction temperatures, concentrations of extraction solution, algae sizes, and stirring levels.

Due to fossil fuel depletion and climate change, anaerobic biomass digestion has been spotlighted as a cost-effective way to produce biogas. A two-stage anaerobic digestion system is preferred to independently produce volatile fatty acids (VFAs) and biogas in separate reactors and thus enhance the stability for biogas production because acidogenesis and methanogenesis requires different operating conditions. As a feedstock to anaerobic digestion, marine biomass has the significant potential to replace terrestrial biomass because of fast growth, the absence of lignin, and abundant carbohydrate. This study investigates the potential to produce methane from macro brown algae in a two-stage anaerobic digestion system. Laminaria japonica are fermented in two semi-continuous 7-L reactors, and the mass balance for the reactors is examined by analyzing methane production rate, VFA concentration, and chemical oxygen demand (COD). In the first stage, the total concentration and yield for VFAs are 2.31-2.94 g/L, 0.23-0.26 g TFA/g CODinput, respectively, at 35℃, pH 5.4-5.6, and hydraulic retention time (HRT) of 12day. In the second stage, the production rate and yield for methane are 6.71 L CH4/Lreactor/d and 0.88-0.96 L CH4/g COD, respectively, at 35℃, pH 7.5-7.7, and HRT of 30day. This study is on-going to determine the optimum design and operating conditions needed to maximize methane production.

Increased concentration of CO2 in the atmosphere has been considered to be one of the main causes of global warming problem. As a consequence of global warming many technologies are being developed for greenhouse gases mitigation. Chemical reaction-based CO2 mitigation approaches are energy-consuming and costly processes, and the only economical incentive is the CO2 credits to be generated under Kyoto Protocol. Biological CO2 fixation using photosynthetic organisms, especially microalgae, has been proposed as one of the possible methods for reducing CO2. The average temperature of winter is below 10 ℃ for 6 months in Korea. In agriculture field, Kerosene oil and butane gas were normally used as heating sources and consequently produced CO2 gas. This study aimed to investigate the potential of carbon dioxide fixation on microalgae for sequestration of generated flue gas after heating on green house at winter season. For this purpose, C. vulgaris was cultivated at the green house and CO2 sources were flue gases of Kerosene oil and butane gas. The results of FTIR analysis (Gas composition analysis) were indicated that flue gas contains SOX and NOX. Furthermore, the fresh cell weight under flue gas was 25% lower compared with pure CO2 condition. However the average lipid concentration (g/g) under flue gas cultivation was 12% higher than control.

Hyperglycemia can become severe and lead to serious complications, such as microvascular complications of diabetes and diabetic nephropathy (DN). Recent studies have focused on glomerular dysfunction found in DN. However, nephromegaly in the early stages of diabetes and the correlation of tubulointerstitial pathology rather than glomerular pathology with declining renal function in DN suggests the involvement of the tubulointerstitium. However, the etiology of the tubulointerstitial pathology in DN is not yet fully rnderstood. In this study, to understand the DN pathways, we planned to produce a 2-DE reference map for primitively cultured human proximal tubule (HK-2) cell under 5 mM and 25 mM glucose, which correspond to normoglycemia and hyperglycemia conditions, respectively. The differences in the expression of HK-2 cell cellular proteins under hyperglycemia condition were identified via ESI-Q-TOF MS/MS and confirmed by Western blotting; enolase 1 (up-regulated) and lactate dehydrogenase (down-regulated). The regulation of these proteins will contribute to understanding DN mechanism through the glycolysis metabolic pathways in high glucose stimulated HK-2 cells.

Alzheimer’s disease(AD) is the most common reason of dementia among people over the age of 65. AD is not yet fully understood about neurodegenerative disease pathology although there have been a lot of studies on AD. Therefore, proteome analysis is very important to understand the changed metabolism of AD brain. In this study, we compared proteome of transgenic (Tg) mice (6 mon Tg, n=16 / 12 mon Tg, n=9) expressing neuronspecific enolase (NSE)-controlled human wild-type tau (NSE-htau23) and control mice (6 mon non-Tg, n=9 / 12 mon non-Tg, n=5) by 2-dimensional electrophoresis (2-DE). As a result, the 27 differentially expressed proteins were identified by ESI-Q-TOF mass spectrometry and LC/MS mass spectrometry. Among the identified proteins, Proteins A, B and C were down-regulated with the progress of AD (p<0.05) and was Protein D up-regulated only in early-stage of AD (p<0.05) these proteins were confirmed by Western blotting. Conclusively, we suggests that these four proteins could help to understand the mechanism associated with neuronal degeneration in AD. And, they are expected to screening of a drug candidate to halt the cascade of disease-associated neurological damage.

The interface between neural electrodes and neural tissue plays an important role in chronic in vivo recording of neural signal. However, when these devices are implanted in to nerve tissue for recording of neural signal, they lose electrical connectivity due to inflammatory reaction, nonspecific protein adsorption and cell adhesion. In this study, to enhance the short-term and long-term biocompatibility of neural electrode, residual solvent extraction, anti-inflammatory drug loading and surface immobilization processes were carried out. Residual solvent in the neural electrode was reduced by solvent extraction and ultra-sonication processes. To reduce the post-inflammatory reaction, an anti-inflammatory drug embedded in a polyimide (PI) film. To modify the surface of the PI film for reducing protein absorption and cell adhesion, oligo (ethylene glycol) methacrylate (OEGMA) was grafted on the surface of PI films by surface-initiated atom transfer radical polymerization (SI-ATRP). The biocompatibility of the PI film was analyzed using the cytotoxicity, RT-PCR, and H&E staining. Neural electrode that is contained drug show better short term biocompatible characteristics over control neural electrode. These solvent reduction, drug containing, and surface immobilized PI film can be used to increase the biocompatibility of neural electrode.

Acellular bovine amniotic membrane(ABAM) is a biological product which has been used for medical applications including wound dressing. The application of ABAM as a wound dressing provides the prevention of heat and water loss from the wound surface and finally wound healing effect. In this study, the use of ABAM as a cell carrier has been evaluated in order to increase the wound healing efficacy. Keratinocyte was well attached to ABAM and grew on it. When ABAM containing keratinocyte was dressed on the wound induced on SD Rat, the wound healing was enhanced compared with dressing with ABAM only or graft with keratinocyte layer on the wound. In order to further enhance the wound healing effect, fibroblast layer was grafted on the wound, and the ABAM containing keratinocyte was dressed after 3days. The combination of fibroblast and ABAM containing keratinocyte reduced contraction, and enhanced epithelization, angiogenesis and the wound healing. Also ABAM was very bio-compatible and degraded well. Through this study, it was found that ABAM could be an efficient biological cell carrier for wound healing including burn and chronic ulcer. [This work was supported by MKE and KOTEF through the Human Resource Training Project for Strategic Technology.]

The methods currently used for treating alopecia have some limitations. The number of hair transplantation restricts less than 3 times because total transplantable hair number is no increase. To overcome these problems, researchers have attempted the in vitro culturing of hair follicle cells and implanting these cells in the treatment area. In the present study, culture expanded mesenchymal stem cells (MSCs) that do not possess aggregative activity were used to produce cell-aggregated spheroidal dermal papilla like tissues (DPLTs) with the aid of a special culture condition in vitro, and hair follicle inductive capacity pertinent to the aggregative activity was then evaluated. Isolated and cultivated MSCs from bone marrow and umbilical cord in vitro. After propagated MSCs underwent preconditioning in dermal papilla forming medium (DPFM), then subcultured MSCs formed self-aggregated DPLTs. We compared real human scalp dermal papilla cells (hDPCs) with DPLT employing DPCs, DPLT employing hBM-MSCs and DPLTs employing hUC-MSCs.The DPLTs have the same hair follicle inductive ability as natural dermal papilla (DP) tissue in vitro. As a result, MSCs from bone marrow and umbilical cord may be an applicable and novel cell source for the generation of human hair cell therapy.

Recent reports have suggested that tumors are organized in heterogeneous cell populations and that the capability to initiate malignancies resides in only a small subset of cancer cells called cancer stem cells. In the case of human colon cancer, cells expressing CD133 have been considered cancer stem cells. To determine specific mechanisms of colon cancer stem cells, colon cancer cell line (HT-29) was separated into CD133+ and CD133- cells by MACS, which was confirmed by RT-PCR. We observed that the sorted CD133+ cells possess high tumorigenecity and clonogenicity as compared to CD133- cells. Differentially expressed proteins between CD133+ cells and CD133- cells were detected by two-dimensional gel electrophoresis, and these proteins were identified by ESI-Q-TOF MS/MS. By the proteomic analysis, we obtained 10 proteins including acitn-related proteins. Notably the actin-related protein A and protein B were more expressed in CD133+ cells than CD133- cells, and these two proteins were verified by Western blot. The identification of specific protein markers for tumorigenic colon cells would provide the basis for detection, as well as clues for understanding the molecular mechanisms governing cancer progression.

The transplantation of pancreatic islet cells is a typical method for the treatment of type I diabetes. The treatment is limited by the shortage of islet cells, which can produce insulin in vivo. Various kinds of stem cells can be used for the preparation of insulin-producing cells (IPCs) such as adipocyte-derived mesenchymal stem cells (MSCs), bone marrow-derived MSCs and embryonic stem cells. In order to obtain IPCs from MSCs effectively, differentiation conditions should be optimized properly. In this study, periosteum-derived progenitor cells (PDPCs) from independent donors were used for the preparation of IPCs. PDPCs have been known to be another kind of MSCs and are expected to be used as a cell source to make IPCs. Homologous PDPCs was obtained by sorting from each donor’s periosteum-derived cells (PDCs) of cambium layer by fluorescent activated cell sorter (FACS) using surface markers of MSCs such as CD9, CD90, and CD166. For the differentiation into IPCs, PDPCs were cultured for 2 weeks in serum-free DMEM/F12 with several supplements containing activin A, exendin 4, nicotinamide, hepatocyte growth factor. It was confirmed that IPCs could be obtained efficiently by the differentiation of PDPCs using several analytical methods including PCR, real-time PCR, immunofluorescence, and ELISA.

Mesenchymal stem cells are multipotent cells capable of differentiating into various mesenchymal tissues, such as bone, cartilage, fat, tendon and muscle. In order to explore their usages in medical applications, the ex vivo expansion of MSCs to sufficient cell numbers is necessary. The expansion of MSCs strongly depends on the culture conditions like pH, temperature, oxygen, basal medium, serum and other supplements. In this study, a newly-formulated in-house medium introduced for effective culture of mesenchymal stem cells and culture environments such as pH, oxygen, carbon dioxide in gas phase have been investigated. The new in-house medium showed much better performances in cell expansion as well as differentiation potential of cultured cells when compared with commercial media including DMEM, KSFM, MSCGM and Mesencult. Carbon dioxide content in gas phase needs to be carefully considered particularly when the widely used DMEM is introduced as a basal medium, since there is strong possibility of increased pH value even in common carbon dioxide condition of 5%. Oxygen content in gas phase also has shown the influence in the culture of MSC.

Collagen and hyaluronic acid (HA) are the most widely used for implantable matrices, because of its biocompatibility and bioresorbability. In this study, we investigated the possibility of HA derivative particlescollagen (HADP-Col) gel as an injectable scaffold in cartilage tissue engineering. We extracted collagen from human umbilical cord matrix and HA were cross-liked by 1,4-butane-diol diglycidyl ether. Then they were mixed homogeneously with umbilical cord-mesenchymal stem cells to become a flowable gel. Consequently, the viability and growth of the cells was promoted in vitro. After chondrogenic induction, RT-PCR and histological analysis showed that the stem cells were successfully differentiated into chondrocytes. With the above results, the injectable HADP-Col gel can be used in the field of stem cell therapy.

Mesenchymal stem cells (MSCs) have are multipotent stem cells that can differentiate into a variety of cell types and contribute to regeneration of mesenchymal tissues. It is important for an organism to expand and maintain quantity and quality of MSCs with the advance of age. Therefore, it needs to address senescence-associated changes in metabolism of MSCs. In order to investigate cellular senescence, we used the cellular aging model of stress-induced premature senescence (SIPS) in human bone marrow-derived MSCs (hBM-MSCs) by treatment of sublethal dose of H2O2. Cellular senescence by oxidative stress was confirmed by various analysis, including WST-8 assay and cell cycle analysis in control and oxidative conditions (150 μM H2O2). In this study, proton (1H) nuclear magnetic resonance spectroscopy (NMR) was used to elucidate the difference of metabolites in control and hMSCs treated with H2O2. Among those metabolites, choline and leucine were identified as up-regulated metabolites and glycine and proline were identified as down-regulated metabolites by 1H-NMR. The results suggest that metabolomics approach provide senescence marker to study the effects of cellular senescence on cell metabolism.

Treatment of Type 1 diabetes mellitus (T1DM) has limited clinical relevance due to the shortage of pancreas donors. Generation of insulin-producing cells (IPCs) from mesenchymal stem cells (MSCs) is one of promising therapeutic options for future treatment of T1DM. Periosteum-derived progenitor cells (PDPCs) are a kind of MSCs. The cells can be isolated from periosteum using CD9/CD90/CD166 as positive surface markers and they have higher growth potency than the other tissue-derived MSCs. In this study, we investigated the effects of glucagon-like peptide-1 (GLP-1) differentiation of PDPCs into IPCs in vitro. GLP-1, an analog of exendin-4, could stimulate glucose-dependent insulin release from pancreatic islets. It relys on the activation of pancreas duodenum homeobox-1 (PDX-1) in IPCs. Using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), production level of insulin can be measured at gene and protein level, respectively. Our results showed the positive effects of the addition of GLP-1. Addition of 10 nM GLP-1 was found to be the optimum for the differentiation of PDPCs into IPCs.

From decades ago, pancreatic islet transplantation has received major spotlights from many researchers and practitioners to be the most favorable treatment for Type 1 Diabetes (T1D); however, a major obstacle of current islet transplantation procedure is generally recognized as an islet graft rejection by the recipient’s immune system. As studied and reviewed many times, immunosuppressive drugs are showing promising but still limited results on organ and cell transplantations. Also, in our previous studies, we have shown that PEGylation method along with Tacrolimus (FK506) could improve in certain extends, protection of transplanted islets from the rejection. Using Anti-CD154 monoclonal antibody (MR1 mAb) to block the costimulatory pathway of T-cell activation has been a successful method to prevent graft rejections. Though the importance and effectiveness of immunosuppressive drugs and mAb therapy were promising, their complications in high doses have interfered with their actual clinical uses. In this study, we have implemented the usage of MR1 to further suppress the host immune response in addition to PEGylation and FK506. After 300 Islets Equivalents (IEQ) were transplanted under left kidneys of C57BL/6 mice, MR1 was injected on 0, 2, 4, 6 days after Tx, while FK506 was given every day.

PCT15 Tissue engineering and regenerative medicine are new research areas investigating how to repair and regenerate impaired organs and tissues using the natural signaling pathway and components of the organism. In the case of joint cartilage, there are no blood vessels and the damaged tissues are not usually regenerated by themselves. For the treatment of damaged cartilage or initial symptom of osteoarthritis, transplant surgery should be carried out with scaffold beads to support the cells transplanted. In this study, the possibility of a manufactured HA-atelo scaffold bead as a support material for cartilage cell therapy was investigated. We compared the manufactured HA-atelo scaffold beads with two other types of commercial scaffold beads such as Cellgen and Cytodex3. The HA-atelo scaffold beads were manufactured through a five-step process including HA dilution, collagen mixture, bead formation, washing, and bead collection. Isolated chondrocytes are cultured and compared in three types of scaffold beads. Cell proliferation was determined by using MTT assay and secreted glycosaminoglycan (GAG) was analyzed quantitatively by using Blyscan GAG assay kit. In conclusion, the manufactured HA-atelo scaffold beads provided the best results for the cultivation of chondrocytes and they can be used in cartilage tissue engineering effectively.

Mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) represent encouraging candidates for the development of future cellular therapy strategies. MSCs have been found to have pluripotent which is able to differentiate into various tissues. One of the primary limitations in our study of the biology of human MSCs is the absence of prospective markers required for the monitoring of lineage-specific differentiation. hUCB-derived MSCs have been found to have significantly greater osteogenic potential. In this study, we focused on protein expression changes during osteogenic differentiation of hUCB-MSCs (n=3). And we analyzed the protein expression inherent to osteogenic differentiation by proteomic research including two-dimensional gel electrophoresis, ESI-Q-TOF, and Western blotting. Eleven differentially expressed proteins including β-actin, pyridoxal phosphatase, PGAM1, VBP1, Hsp27 and HSPC231 were confirmed between the two groups (before and after differentiation). These might also be proved as useful cytosolic biomarkers for osteogenesis, and might be employed in quality control of osteoblasts in cell-therapy applications.

Currently, even though the development of keratinocyte therapy products are actively carried out world wide, the research and development of test methods that are considered in quality evaluations require a lot of time because they vary for each product. Therefore, the purpose of this study was to suggest an optimal identity method of keratinocyte therapy products. we progressed immunofluoresence staining and Reverse Transcription-PCR(RT-PCR) by specific marker of keratinocyte. In addition, fluorescence activated cell sorting(FACS) test was conducted and morphologic identity test was carried out. CK14 was most prevalent while CK10 was not expressed at antibody-based immunofluorescence staining. With RT-PCR, it was possible to identity CK14, CK5, and E-cad as keratinocyte. FACS was not sufficient as a identity test because E-cad expressed in not only keratinocyte but also other cells, and the criterion is subjective. In addition, in the morphologic test, even though the layer differentiation is characteristic in keratinocyte, the test was not sufficient for keratinocyte therapy products, which has a short expiration date, because the test takes more than seven days.

The immunoisolation not only allows for successful transplantation of cells in the absence of immunosuppression but also for transplantation of cells from nonhuman origin. Microcapsules have been one of the most intensively studied extravascular devices because of the spherical shape and mechanical stability. However, it is reported that there may be some limitations for the microcapsules due to oxygen shortage and reduced diffusion capacity. Thus, this study was performed to evaluate the effects of exendin-4 on the viability and insulin secretion of microencapsulated islets. Pancreatic islets were isolated from male Sprague–Dawley rats and the exendin-4 was transduced into the islets. Microcapsules were prepared using an electrostatic bead generator. Microencapsulation of the untrasnduced islets was considered as control and the exendin-transduced one was regarded as test group. The mechanical stability of the microcapsules was determined using an osmotic pressure test and a rotational stress test. The viability and the insulin secretion stimulated by glucose were measured. After islet transplantation of the encapsulated islets, animals’ body weight and blood glucose levels of encapsulated islet recipients were monitored daily.It was shown that the exendin-4 transduced islets showed higher viability when compared with the control. Both groups were able to produce the insulin from the glucose challenge and the absolute amount of secreted insulin was higher in the test group. It may be suggested that the transduction of exendin-4 may be considered for the microencapsulation procedure to enhance the viability and the insulin secretion from the islets.

Mesenchymal stem cells (MSCs) have a great potential in cell-based therapies because of their multipotency and simple methods for in vitro manipulation. However, during in vitro manipulation, MSCs have aging and loss their multipotency and proliferation capability. Previous studies shown that calorie restriction (CR) induces proliferation of MSCs and diminishes apoptosis. Therefore, we observed the effect of low glucose in MSC in vitro. Proliferation level in the low glucose (LG, 1.375 mM) condition was compared with that in the normal glucose (NG, 5.5 mM) condition. We performed comparative studies of population doubling, β-galactosidase activity, ROS generation and differentiation capacity (osteocyte, adipocyte) in NG and LG conditions. We compared protein expressions between NG and LG conditions, several proteins were up-regulated or down-regulated in glucose restriction condition, LG condition. These results will be expected to provide a powerful tool for long-term and large scale culture of stem cells.

Adhesion of cells to surfaces is a basic and important requirement in cell culture and tissue engineering. Here, we designed artificial extracellular matrix (ECM) mimics for efficient cellular attachment, based on mussel adhesive protein (MAP) fusion with biofunctional peptides originating from ECM materials, including fibronectin, laminin, and collagen. Cellular behaviors, including attachment, proliferation, spreading, viability, and differentiation, were investigated with the artificial ECM material-coated surfaces, using three mammalian cell lines (pre-osteoblast, chondrocyte, and pre-adipocyte). All cell lines examined displayed superior attachment, proliferation, spreading, and survival properties on the MAP-based ECM mimics, compared to other commercially available cell adhesion materials, such as poly-L-lysine and the naturally extracted MAP mixture. Additionally, the degree of differentiation of pre-osteoblast cells on MAP-based ECM mimics was increased. These results collectively demonstrate that the artificial ECM mimics developed in the present work are effective cell adhesion materials. Moreover, we expect that the MAP peptide fusion approach can be extended to other functional tissue-specific motifs.

Stress induced premature senescence (SIPS) occurs after exposure to many different sublethal stresses including H2O2, hyperoxia, or tert-butylhydroperoxide. Human mesenchymal stem cells (hMSCs) exhibit limited proliferative potential in vitro, the so-called Hayflick limit. According to the free-radical theory, reactive oxygen species (ROS) might be the candidates responsible for senescence and age-related diseases. It may also be responsible for the production of high levels of ROS, in which the redox balance is disturbed and the cells shift into a state of oxidative stress, which subsequently leads to premature senescence with shortening telomeres. H2O2 has been the most commonly used inducer of SIPS, which shares features of replicative senescence (RS) including a similar morphology, senescenceassociated β-galactosidase activity, cell cycle regulation, etc. Therefore, in this study, the senescence of hMSC during SIPS was confirmed using a range of different analytical methods. In addition, we determined five differentially expressed spots in the 2-DE map, which were identified as Annexin A2 (ANXA2), myosin light chain 2 (MLC2), peroxisomal enoyl-CoA hydratase 1 (ECH1) and prosomal protein P30-33K (PSMA1) by ESI-Q-TOF MS/MS.

Type 1 diabetes is caused by the complete loss of pancreatic beta cells due to the body’s own immune system. Transplantation of beta cells is considered to be the best treatment for type 1 diabetes, however it is extremely limited by the shortage of human organ donors. Due to the shortage of donor pancreata, alternative sources of islets have been studied through the generation of the beta cells from stem cells and the expansion of the beta cells with various growth factors. However, differentiation of stem cells and expansion of insulin-producing cells (IPCs) have been limited. In this study, we used periosterium-derived progenitor cells (PDPCs) known as a kind of mesenchymal stem cells (MSCs) for the differentiation into IPCs. Even though IPCs can be obtained from PDPCs, maintenance of stability and expansion of IPCs was not easy. Therefore, effects of antioxidants on the stability and expansion of IPCs were investigated in this study. Enzyme-linked immunosorbent assay (ELISA) and MTT assay were used for the measurement of stability. In addition, real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect expression levels of insulin.

Podocytes are visceral epithelial cells in the kidney and play important roles in the glomerular filtration barrier. Podocyte injury is a key factor in the development of early diabetic nephropathy (DN). In this study, to understand the proteome changes in podocytes in DN, we constructed a 2-DE reference map for the primary cultured glomerular epithelial cells (GECs, podocytes) in the presence normal glucose (5 mM) and high glucose (30 mM), respectively. High glucose exposed GECs showed an increased production of ROS and more cellular hypertrophy compared with normal glucose exposed GECs. Differentially expressed GECs proteins between the normal and high glucose condition were identified using ESI-Q-TOF MS/MS. Poly (c) binding protein 1 was up-regulated and ATP synthase beta subunit, enolase 2, beta actin were downregulated in high glucose treated GECs. ATP synthase beta subunit expression was confirmed by Western blotting. The expression of vimentin (not significantly changed) and CLP36 (down-regulated), which are expected to be involved podocyte pathology in DN and then observed by Western blotting. These results will provide a better understanding of the development of early DN via glucose metabolism of GECs under hyperglycemic conditions.