• Journal of Embryo Transfer
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• v.28 no.3
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• pp.281-287
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• 2013

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal ${\alpha}$-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 ${\alpha}$-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% $CO_2$ 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 $1{\times}10^3$ 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($15.0{\pm}0.4{\mu}m$) had a little larger cell size than Wild BM-MSCs ($13.5{\pm}0.3{\mu}m$). 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.

• Journal of Periodontal and Implant Science
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• v.26 no.4
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• pp.949-965
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• 1996

The main goal of periodontal therapy is to restore the lost periodontal tissue and establish the attachment appratus. Current acceptable therapeutic techniques are included : removal of diseased soft tissue, demineralization of exposed root surface, using the barrier membrane for preventing the downgrowth of gingival epithelial cell, insertion of graft materials as a scaffolding action, and biological mediators for promoting the cell activity. The latest concept one among them has been studied which based on the knowledge of cellular biology of destructed tissue. Platelet-derived growth factor(PDGF) is one of the polypeptide growth factor which have been reported as a biological mediator to regulate activities of wound healing progress including cell proliferation, migration, and metabolism. The purposes of this study is to evaluate the influences of the PDGF as biological mediator to periodontal ligament and bone marrow cell. Both right and left maxillary first molar were extracted from rat which had treated with 0.4% ${\beta}-Aminopropionitril$ for 5 days, and feeded until designed date to sacrifice under anesthesisa. Periodontal ligament were removed from the extracted socket of the rat, and cultured with Dulbecco's Modified Essential Medium(DMEM) contained with 10% Fetal Bovine Serum, 100U/ml penicillin, $100{\mu}g/ml$ streptomycin, $0.5{\mu}g/ml$ amphotericin-B. Bone marrow cell were culture from bone marrow suspension with which washed out from femur with same medium. The study was performed to evaluate the effect of PDGF to periodontal ligament and bone cell, cell proliferation rate, total protein synthesis, and alkaline phosphatase activity of rat periodontal ligament(PDL) cell and bone stromal(RBS) cell in vitro. The effects of growth factors on both cells were measured at 3, 5th day after cell culture with (control group) or without growth factors(experimental group). The results were as follows: 1. The tendency of cell proliferation under the influence of PDGF showed more rapid proliferation pattern than control at 3 and 5 days after inoculation. 2. The activity of Alkaline phosphatase revealed 14, 80% increased respectively at 3, 5 days culture than control group. Measurements of ALPase levels indicated that PDL cells had significantly higher activity when compared with that of co-culture groups and GF only(P<0.05). And, ALPase activity in 10 days was higher than that of 7 days(P<0.05). 3. The tendency of formation of the mineralized nodule were observed dose-depend pattern of PDL cells. There was statistically significant difference among group l(PDL 100%), 2(PDL 70%:GF 30%), and 3(PDL 50%:GF 50%)(P<0.01). But, there was no difference among group 3, 4(PDL 30%:GF 70%), and 5(GF 100%). 4. Also, the number of nodule was greater in co-culture of PDL 70% and GF 30% than in culture of PDL 70%(P<0.05). From the above results, it is assumed that the PDGF on PDL cells and RMB cell culture. GF stimulates the cell growth, which is not that of PDL cells but GF. And, the activity of ALPase depends on the ratio of PDL cells, and ALPase may relate to the initial phase of nodule formation. Also, it is thought that the calcified nodule formation principally depends on PDL cells, is inhibited by GF, and affected by cell density. In conclusion, platelet-derived growth factor can promote rapid osteogenesis during early stage of periodontal tissue regeneration.

• Archives of Plastic Surgery
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• v.38 no.4
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• pp.438-444
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• 2011

Purpose: Adipose-derived stromal cells (ASCs) are readily harvested from lipoaspirated tissue or subcutaneous adipose tissue fragments. The stromal vascular fraction (SVF) is a heterogeneous set of cell populations that surround and support adipose tissue, which includes the stromal cells, ASCs, that have the ability to differentiate into cells of several lineages and contains cells from the microvasculature. The mechanisms that drive the ASCs into the osteoblast lineage are still not clear, but the process has been more extensively studied in bone marrow stromal cells. The purpose of this study was to investigate the osteogenic capacity of adipose derived SVF cells and evaluate bone formation following implantation of SVF cells into the bone defect of human phalanx. Methods: Case 1 a 43-year-old male was wounded while using a press machine. After first operation, segmental bone defects of the left 3rd and 4th middle phalanx occurred. At first we injected the SVF cells combined with demineralized bone matrix (DBM) to defected 4th middle phalangeal bone lesion. We used P (L/DL)LA [Poly (70L-lactide-co-30DL-lactide) Co Polymer P (L/DL)LA] as a scaffold. Next, we implanted the SVF cells combined with DBM to repair left 3rd middle phalangeal bone defect in sequence. Case 2 was a 25-year-old man with crushing hand injury. Three months after the previous surgery, we implanted the SVF cells combined with DBM to restore right 3rd middle phalangeal bone defect by syringe injection. Radiographic images were taken at follow-up hospital visits and evaluated radiographically by means of computerized analysis of digital images. Results: The phalangeal bone defect was treated with autologous SVF cells isolated and applied in a single operative procedure in combination with DBM. The SVF cells were supported in place with mechanical fixation with a resorbable macroporous sheets acting as a soft tissue barrier. The radiographic appearance of the defect revealed a restoration to average bone density and stable position of pharyngeal bone. Densitometric evaluations for digital X-ray revealed improved bone densities in two cases with pharyngeal bone defects, that is, 65.2% for 4th finger of the case 1, 60.5% for 3rd finger of the case 1 and 60.1% for the case 2. Conclusion: This study demonstrated that adipose derived stromal vascular fraction cells have osteogenic potential in two clinical case studies. Thus, these reports show that cells from the SVF cells have potential in many areas of clinical cell therapy and regenerative medicine, albeit a lot of work is yet to be done.

• Development and Reproduction
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• v.13 no.4
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• pp.227-237
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• 2009

Recently, adipose mesenchymal stem cells (AdMSC) that are similar to bone marrow MSC and blood derived MSC are thought to be another source for stem cell therapy. However, the diseases that can be applied for stem cells therapy are age-dependent degenerative diseases. Accordingly, the present study investigated the growth and differentiation potential to neural cells of human AdMSC (hAdMSC) obtained from aged thirty, forty and fifty. The growth of cells and cell viability were measured by passage and neural differentiation of hAdMSC was induced in neural differentiation condition for 10 days. Our results demonstrated that cell number, viability and morphology were not different from hAdMSC by age and passage. Immunofluorescence analysis of neural cell marker (TuJ1, NSE, Sox2, GFAP or MAP2) demonstrated no significant differences in neural cell differentiation by age and passage. As the number of passage was increased, the mRNA level of MAP2 and Sox2 was decreased in hAdMSC from age of 50 compared to hAdMSC from age of 30. In conclusion, the present study demonstrated that ability of neural cell differentiation of hAdMSC was maintained with ages, suggesting that autologous stem cells from aged people can be applied for stem cell therapy with age-dependent neural disease with the same stem cell quality and ability as stem cell derived from young age.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.95-97
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• 2003

Despite recent advances in the treatment of acute myocardial infarction, the ability to repair extensive myocardial damage is limited. To develop a new therapy for myocardial infarction, we examined the possibility of regenerating myocardium by implanting bone marrow-derived mononuclear cells(BM-MNC) . Histological and immunohistochemical examination showed myocardium regeneration and angiogenesis in the cell transplantation site. Isolated perfused (Langendorff) heart experiments revealed enhanced functions of heart. These results suggest that BM-MNC transplantation induce cardiac muscle regeneration and that this approach could be applied as a possible treatment for myocardial infarction.

• Biomolecules & Therapeutics
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• v.21 no.6
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• pp.447-453
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• 2013

Chondroitin sulfate proteoglycan (CSPG) inhibits neurite outgrowth of various neuronal cell types, and CSPG-associated inhibition of neurite outgrowth is mediated by the Rho/ROCK pathway. Mesenchymal stromal/stem cells (MSCs) have the potential to differentiate into neuron-like cells under specific conditions and have been shown to differentiate into neuron-like cells by co-treatment with the ROCK inhibitor Y27632 and the hypoxia condition mimicking agent $CoCl_2$. In this study, we addressed the hypothesis that a ROCK inhibitor might be beneficial to regenerate neurons during stem cell therapy by preventing transplanted MSCs from inhibition by CSPG in damaged tissues. Indeed, dose-dependent inhibition by CSPG pretreatment was observed during morphological changes of Wharton's jelly-derived MSCs (WJ-MSCs) induced by Y27632 alone. The formation of neurite-like structures was significantly inhibited when WJ-MSCs were pre-treated with CSPG before induction under Y27632 plus $CoCl_2$ conditions, and pretreatment with a protein kinase C inhibitor reversed such inhibition. However, CSPG treatment resulted in no significant inhibition of the WJ-MSC morphological changes into neuron-like cells after initiating induction by Y27632 plus $CoCl_2$. No marked changes were detected in expression levels of neuronal markers induced by Y27632 plus $CoCl_2$ upon CSPG treatment. CSPG also blocked the morphological changes of human bone marrow-derived MSCs into neuron-like cells under other neuronal induction condition without the ROCK inhibitor, and Y27632 pre-treatment blocked the inhibitory effect of CSPG. These results suggest that a ROCK inhibitor can be efficiently used in stem cell therapy for neuronal induction by avoiding hindrance from CSPG.

• Molecules and Cells
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• v.28 no.6
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• pp.515-520
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• 2009

Applications of bone marrow-derived mesenchymal stem cells in gene therapy have been hampered by the low efficiency of gene transfer to these cells. In current transduction protocols, retrovirus particles with foreign genes make only limited contact with their target cells by passive diffusion and have short life spans, thereby limiting the chances of viral infection. We theorized that mechanically agitating the virus-containing cell suspensions would increase the movement of viruses and target cells, resulting in increase of contact between them. Application of our mechanical agitation for transduction process has increased the absorption of retrovirus particles more than five times compared to the previous static method without changing cell growth rate and viability. The addition of a mechanical agitation step increased transduction efficiency to 42%, higher than that of any other previously-known static transduction protocol.

• Biomolecules & Therapeutics
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• v.19 no.2
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• pp.206-210
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• 2011

Natural Killer (NK) cells are the lymphocytes that are derived from hematopoietic stem cells, developed in the bone marrow from hematopoietic stem cells (HSC) by sequential acquisition of functional surface receptors, and express the repertoire of inhibitory and activating receptors. Recently, Osteopontin (OPN) has been identified as a critical factor for differentiation of natural killer cells. However, the detailed mechanism of OPN-induced NK differentiation has been still to be elucidated. Here, we determined the signaling pathway and possible receptor for OPN in NK differentiation. OPN induced expression of Bcl-2 and activation of Erk kinase. Inhibition of Erk pathway decreased the effect of OPN on NK differentiation. In addition, the expression of integrin ${\alpha}9$ was significantly increased by OPN during NK differentiation, suggesting the possible role of a major signaling molecule for OPN- induced NK differentiation.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.249-255
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• 2015

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 ${\beta}-cells$, 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 ${\alpha}$-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 ${\beta}-cell$ replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.3
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• pp.167-174
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• 2012

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-$kit^+$ bone marrow cells (c-$kit^+$ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-$kit^+$ BM cells that give rise to $CD2^+CD8^+$ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-$kit^+$ BM cells than those of controls. And, we compared the ability of the cytotoxicity of $CD2^+CD8^+$ NK cells differentiated by cytokines from c-$kit^+$ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100 : 1 for 4 h once a week. In results, $CD2^+CD8^+$ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-$kit^+$ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-$kit^+$ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.