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.

Enrofloxacin (EFX) is one of the 2nd generation quinolone antibiotics and is now widely used as a broad-spectrum antibiotic for industrial animals. Previous study showed that EFX reduces the cellular metabolic activity of spleen cells and modulates the inflammatory responses. However, little is known about its toxicity on bone marrow (BM) cells. In this study, BM cells were treated with EFX and cellular metabolic activity, cell death, the change of neutrophil (CD11b+Gr1+ cells) proportion, and antigen uptake ability of granulocytes were measured. Compared to the control, EFX-treated cells showed the decrease of cellular metabolic activity, the increase of cell death, and the decreased proportion of neutrophils. In contrast, the antigen uptake ability of granulocytes in BM cells was increased by EFX. These data suggest that EFX has only limited toxicity on BM cells. And also, EFX is safe on BM cells in a range of concentration, 6.25 – 25 μg/mL. This study can provide available data for the safety or toxicity of EFX.

Oriental medicine uses many herbs with biological activity. Among these, some have anti-inflammatory activities, but their action mechanisms have yet to be fully elucidated. Therefore, in this study, we harvested mouse bone marrow cells (BMs) and treated them with lipopolysaccharide (LPS), after which they were treated with the extracts of four medicinal herbs. The metabolic activity, cell death ratio, and tumor necrosis factor (TNF)-alpha production of the BMs was then evaluated. Among the four medicinal herbs, Caesalpinia sappan (CS) significantly decreased the metabolic activity, mitochondrial membrane potential of BMs, and production of TNF-alpha, especially in the presence of LPS. Moreover, CS increased the cell death of BMs stained with propidium iodide. Taken together, these findings indicate that CS inhibited the biological activities of LPS-treated BMs in multiple assays, confirming that CS has anti-inflammatory activity.

Developmental aspects of chicken embryos showed dramatic difference compared with those of mammals and consequently, such difference in various developmental events leads to different feasibility in both clinical and industrial application. We have concentrated on the studies for using of chicken bone marrow cells and currently we found number of unique cellular properties. Through this article, we reviewed characteristics and cell signaling of osteogenic cells during endochondral ossification in chicken long bone.

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.

Many kinds of medicinal herbs have been used to treat inflammation in Oriental medicine. However, there few studies have investigated the anti-inflammatory activity of medicinal herbs. In this study, we used mouse bone marrow cells (BMs) treated with lipopolysaccharide (LPS), a simulator of osteomyelitis, to screen medicinal herbs having anti-inflammatory activity. Specifically, we investigated the activity of an extract of Rhus chinensis (RC) using metabolic activity and cytokine production of the BMs treated with LPS and RC. The metabolic activity of BMs was measured using Cell Counting Kit-8® solution. RC decreased the metabolic activity of LPS-treated BMs. A viability assay using trypan blue solution demonstrated that RC marginally decreased the viability of LPS-treated BMs. Flow cytometry analysis revealed that RC decreased the mitochondrial membrane potential of BMs, regardless of LPS treatment. To investigate the anti-inflammatory activity of RC, we measured the production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 in BMs. LPS increased the production of both cytokines in BMs. Interestingly, RC induced a greater increase in IL-10 than TNF-alpha in LPS-treated BMs. Taken together, RC decreased metabolic activity and modulated the production of inflammation-related cytokines in LPS-treated BMs. These findings suggest that RC can be used as a medicinal herb with anti-inflammatory activity.

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.

Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of β-cells, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from α-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3∼4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in β-cell replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

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.

Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

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.

조혈 줄기 세포에의 효과적인 유전자 전달은 유전자 치료의 새로운 가능성을 제시할 수 있다. 레트로바이러스를 이용한 유전자 전달 기술은 많은 기초 연구와 임상 시도가 이루어진 대표적인 바이러스이다. 그러나 현재 사용되고 있는 in vitro에서의 조혈 줄기 세포에의 유전자 도입은 조혈 줄기 세포의 분화 유도, 자기 복제 능력과homing 능력의 저하 등 많은 문제점이 있다. 본 연구는 이러한 문제점을 극복하기 위한 방법으로서 마우스의 대퇴골에 직접 레트로바이러스를 이식하는 IBM (Intra-Bone Marrow) 방법을 이용하여 조혈 줄기 세포에의 효과적인 유전자 도입을 시도하였다. IBM 이식 2주 후 마우스의 각 조직을 분석한 결과, 골수뿐 아니라 림파절, 비장, 간장 세포 등에서 유전자가 안정적으로 발현하는 것을 관찰하였다. 또한, 6.4+-2.7%의 골수조직 존재 조혈줄기/전구세포에서 도입된 유전자가 안정적으로 발현하고 있는 사실을 확인하였다. 본 연구의 결과를 바탕으로 IBM 이식 방법을 이용한 생체 조직 내 레트로바이러스의 유전자 도입은 조혈 줄기 세포를 이용한 유전자 치료에 매우 효과적인 방법이라는 사실을 시사해주고 있다.