Osteoarthritis occurs when the cartilage that gradually deteriorates as common aging-associated disease in humans and animals. There is no cure, but the treatments are available to manage to relieve pain through medication such as steroids. Growing interest has been focused on the role of cell-based therapies using mesenchymal stem cells (MSCs). In addition, mesenchymal stem cells can be isolated from almost adult tissues and known for their potential of becoming cartilage. Clinical and experimental studies indicate that the development of treatment using stem cells is double-edged sword involving a possibility such as tumorigenesis. This study focused on the electrical features during articular cartilage development and hypothesized that external electric fields promote pre-chondrogenic condensation without concern relating to genetic modification or exogenous factors. Here, it has been reported that exogenous direct electric fields drive pre-chondrogenic condensation which is the stage where cartilage formation begins by condensation of stem cells and cartilage cells in the microenvironment of the joint. Time-dependent observations also support the contribution of electrical stimulation (ES) to induce gradual aggregation of MSCs into highly compact structures within 3 days. Collectively, our findings provide the potential of electrical stimulation-driven chondrogenesis of mesenchymal stem cells in the absence of exogenous factors for repair of cartilage defects.

Mesenchymal stem cells (MSCs) have been considered an alternative source of neuronal lineage cells, which are difficult to isolate from brain and expand in vitro. Previous studies have reported that MSCs expressing Nestin (Nestin+ MSCs), a neuronal stem/progenitor cell marker, exhibit increased transcriptional levels of neural development-related genes, indicating that Nestin+ MSCs may exert potential with neurogenic differentiation. Accordingly, we investigated the effects of the presence of Nestin+ MSCs in bone-marrow-derived primary cells (BMPCs) on enhanced neurogenic differentiation of BMPCs by identifying the presence of Nestin+ MSCs in uncultured and cultured BMPCs. The percentage of Nestin+ MSCs in BMPCs was measured per passage by double staining with Nestin and CD90, an MSC marker. The efficiency of neurogenic differentiation was compared among passages, revealing the highest and lowest yields of Nestin+ MSCs. The presence of Nestin+ MSCs was identified in BMPCs before in vitro culture, and the highest and lowest percentages of Nestin+ MSCs in BMPCs was observed at the third (P3) and fifth passages (P5). Moreover, significantly the higher efficiency of differentiation into neurons, oligodendrocyte precursor cells and astrocytes was detected in BMPCs at P3, compared with P5. In conclusion, these results demonstrate that neurogenic differentiation can be enhanced by increasing the proportion of Nestin+ MSCs in cultured BMPCs.

Due to their anatomical, physiological and genetic similarities, pig is attractive animal model in biomedical research. In the recent stem cell research era, porcine derived stem cells also gain attention due to its use for the preclinical application of human. Mesenchymal stem cells (MSCs) have been studied by many researchers over decade, and their prospect for clinical application is recognized. Although porcine derived MSCs (pMSCs) have confirmed to be differentiated into various types of cells, such as osteocyte, chondrocyte, neuronal cell, cardiomyocyte and pancreatic β cell, few report has been studied regarding hepatocyte differentiation in vitro. The present study was therefore aimed for bone marrow MSCs derived from pig femur to differentiate into hepatocyte. The cells were confirmed as MSCs by characterizing their morphology, lineage differentiation capacity and surface phenotype. They showed spindle like morphology and adipocytic, osteoblastic, and chondrocytic differentiation potentials and displayed positive expression of mesenchymal markers CD29, CD44 and CD90 while lacked the expression of hematopoietic marker CD45. Under appropriate differentiation conditions, MSCs displayed hepatocyte-like morphology depending on duration of differentiation. The differentiated MSCs into hepatocyte expressed hepatocyte-specific genes including hepatocyte nuclear factor 4 (HNF4), albumin (ALB), alpha fetoprotein (AFP), alpha-1-anti trypsin (A1AT). They also showed hepatocyte-like function, glycogen storage which is identified by PAS staining. Taken together, it concluded that the bone marrow MSCs have the potential to differentiate into hepatocyte. Further studies are needed on additional hepatocytic functional assays, such as low density lipoprotein (LDL) uptake and urea synthesis of differentiated MSC.

Fucoidan has been extensively studied as medicinal materials due to its biological activities including osteoblastic differentiation effect. However, osteoblastic effect by fucoidan is unknown in alveolar bone marrow derived mesenchymal stem cells (ABM-MSCs). The present study was undertaken to evaluate the effect of fucoidan on Osteoblastic differentiation in ABM-MSCs and explore its mechanism. Cell proliferation was analyzed by crystal violet staining. Osteoblast differentiation was determined by alkaline phosphatase activity, calcium accumulation assay and gene expression of osteoblast markers. We found that fucoidan induced cell proliferation of ABM-MSCs. Furthermore, fucoidan increased the ALP activity, calcium accumulation, and osteoblast specific genes such as Runx2, type I collagen alpha 1. Moreover, fucoidan induces the expression of asporin and bone morphogenic protein (BMP)-2 and asporin. Based on these results, these finding indicate that fucoidan induces osteoblast differentiation in ABM-MSCs and partially enhanced the mRNA expression of BMP-2 and asporin.

Bioactive peptides function effectively with a minimal amount compared to proteins. Recently SPARC related modular calcium binding 1 (SMOC1) has been implicated in regulating osteoblast differentiation and limb and eye development. In this study we synthesized a peptide covering 16 amino acids derived from the extracellular calcium binding (EC) domain of SMOC1, and its effects on proliferation and osteoblast differentiation of human bone marrow mesenchymal stem cells were examined. Treatment of SMOC1 peptide did not modulate proliferation of BMSCs. However, mineralization of BMSCs was significantly increased with a dose dependent manner. Consistently expression of osteoblast differentiation marker genes including type 1 collagen and osteocalcin was also dose dependently increased. Taken together, these results suggest that peptide derived from the EC domain of SMOC1 recapitulates at least partially osteogenic function of SMOC1.

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.

Adult stem cell transplantation has been increased every year, because of the lack of organ donors for regenerative medicine. Therefore, development of reliable and safety cryopreservation and bio-baking method for stem cell therapy is urgently needed. The present study investigated safety of dimethyl sulfoxide (DMSO) such as common cryoprotectant on porcine bone marrow derived mesenchymal stem cells (pBM-MSCs) by evaluating the activation of Caspase-3 and -7, apoptosis related important signal pathway. pBM-MSCs used for the present study were isolated density gradient method by Ficoll-Paque Plus and cultured in A-DMEM supplemented 10% FBS at in 5% incubator. pBM-MSCs were cryopreserved in A-DMEM supplemented either with 5%, 10% or 20% DMSO by cooling rate at /min in a Kryo 360 (planner 300, Middlesex, UK) and kept into . Survival rate of cells after thawing did not differ between 5% and 10% DMSO but was lowest in 20% DMSO by 0.4% trypan blue exclusion. Activation of Caspase-3 and -7 by Vybrant FAM Caspase-3 and -7 Assay Assay Kit (Molecular probes, Inc.OR, USA) was analyzed with a flow cytometer. Both of cryopreserved and control groups (fresh pBM-MSCs) were observed after the activation of Caspase-3 and -7. The activation did not differ between 5% and 10% DMSO, but was observed highest in 20% DMSO. Therefore 5% DMSO can be possibly used for cell cryopreservation instead of 10% DMSO.

Human mesenchymal stem cell (hMSCs) isolated from human adult bone marrow have self-renewal capacity and can differentiate into multiple cell types in vitro and in vivo. A number of studies have now demonstrated that MSCs can differentiate into various neuronal populations. Due to their autologous characteristics, replacement therapy using MSCs is considered to be safe and does not involve immunological complications. The basic helix-loop-helix (bHLH) transcription factor Olig2 is necessary for the specification of both oligodendrocytes and motor neurons during vertebrate embryogenesis. To develop an efficient method for inducing neuronal differentiation from MSCs, we attempted to optimize the culture conditions and combination with Olig2 gene overexpression. We observed neuron-like morphological changes in the hMSCs under these induction conditions and examined neuronal marker expression in these cells by RTPCR and immunocytochemistry. Our data demonstrate that the combination of Olig2 overexpression and neuron-specific conditioned medium facilitates the neuronal differentiation of hMSCs in vitro. These results will advance the development of an efficient stem cell-mediated cell therapy for human neurodegenerative diseases.