Takeshi Teramura

Induction of Mesenchymal Progenitor Cells with Chondrogenic Property from Mouse-Induced Pluripotent Stem Cells

Despite recent cell-engineering advances, treatment and repair of cartilage remains challenging. Although stem cell transplantation therapy using mesenchymal stem cells (MSCs) is considered a prominent strategy, the major problem of limited proliferative capacity of autologous cells has been unsolved. Recently, an induced pluripotent stem (iPS) cell line was suggested as an alternative way to cure various human diseases due to their potential proliferating infinitely while possessing the capacity to form all types of cells. However, the method to induce lineage-restricted differentiation has not been well examined or established. Here, we suggest a simple method to induce mesenchymal progenitors possessing chondrogenic property from mouse iPS cells. The MSC-like cells produced in our study expressed some MSC markers, and could also differentiate to osteoblast and adipocyte. The present study demonstrates the property of iPS cells as an alternative candidate for treatment of articular disorders, and suggests an effective approach for preparing chondrocyte from iPS cells.

Induction of hypertrophic chondrocyte-like phenotypes by oxidized LDL in cultured bovine articular chondrocytes through increase in oxidative stress

OBJECTIVE It has been reported that the lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor 1 (LOX-1) is expressed by chondrocytes in osteoarthritis (OA) cartilage and that Ox-LDL binding to LOX-1 increases intracellular oxidative stress in cultured bovine articular chondrocytes (BACs). It was recently demonstrated that reactive oxygen species (ROS) induce hypertrophic differentiation of chondrocytes in the growth plate. It has also been shown that activated chondrocytes in OA have hypertrophic chondrocyte-like phenotypes. The purpose of this study was to determine whether Ox-LDL induces hypertrophic chondrocyte-like phenotypes in BACs. DESIGN Changes in type X collagen (COL10) and runt-related transcription factor 2 (Runx2) mRNA expression in BACs after Ox-LDL stimulation were investigated using real-time polymerase chain reaction (PCR). Western blotting and immunofluorescent cell staining were used to investigate changes in protein level. The antioxidant N-acetyl cysteine (NAC) was used to ascertain whether oxidative stress is involved in COL10 and Runx2 expression. We induced LOX-1 knockdown cells using small interfering RNA (siRNA) to examine the receptor specificity of Ox-LDL. RESULTS COL10 expression was upregulated by Ox-LDL in a time- and dose-dependent manner. Immunofluorescent staining showed that Ox-LDL increased COL10 production in the extracellular matrix. Ox-LDL-induced upregulation of COL10 was suppressed by pretreatment with NAC and siRNA. Expression of Runx2 was upregulated by Ox-LDL and H(2)O(2), and these effects were suppressed by NAC pretreatment. CONCLUSION Ox-LDL binding to LOX-1 induces a hypertrophic chondrocyte-like phenotype through oxidative stress, indicating that Ox-LDL plays a role in the degeneration of cartilage.

Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells.

Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

Reactive oxygen species induce Cox-2 expression via TAK1 activation in synovial fibroblast cells

Oxidative stress within the arthritis joint has been indicated to be involved in generating mediators for tissue degeneration and inflammation. COX‐2 is a mediator in inflammatory action, pain and some catabolic reactions in inflamed tissues. Here, we demonstrated a direct relationship between oxidative stress and Cox‐2 expression in the bovine synovial fibroblasts. Furthermore, we elucidated a novel mechanism, in which oxidative stress induced phosphorylation of MAPKs and NF‐κB through TAK1 activation and resulted in increased Cox‐2 and prostaglandin E2 expression. Finally, we demonstrated that ROS‐induced Cox‐2 expression was inhibited by supplementation of an antioxidant such as N‐acetyl cysteamine and hyaluronic acidin vitro andin vivo. From these results, we conclude that oxidative stress is an important factor for generation of Cox‐2 in synovial fibroblasts and thus its neutralization may be an effective strategy in palliative therapy for chronic joint diseases.

Rho-associated kinase inhibitor Y-27632 promotes survival of cynomolgus monkey embryonic stem cells

Non-human primates are suitable models for preclinical research aimed at cell-replacement therapies. Recently, it has been reported that Rho-associated kinase inhibitor Y-27632 markedly reduced dissociation-induced apoptosis of human embryonic stem (hES) cells, and is expected as a novel supplement for hES cell maintenance or differentiation inductions; however, the effects of the chemical are still to be determined in model animals. Here, we demonstrated the effect of Y-27632 on cynomolgus monkey ES (cyES) cells. Also, in cyES cells, Y-27632 treatment dramatically improved the efficiency of colony formation from single cells without affecting the pluripotent state and karyotype. Y-27632 supplementation was also effective for feeder-free culture and differentiation induction. Neural stem cells directly induced from cyES cells could give rise to neurons, astrocytes and dopamine producing cells. The present result not only suggests that the chemical was effective for improving the culture system of primate ES cells, but also the similarity between cyES and hES cells regarding the reactions to the chemical, which might be further evidence that cyES cells are superior models for hES cells.

Isolation and characterization of side population stem cells in articular synovial tissue

BackgroundAutologous chondrocyte implantation is an established technique for the repair of degenerated articular cartilage. Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 (Ho) fluorescence dye, has attracted attention as a method of stem cell isolation. Although SP cells from synovial tissue were expected to be an excellent source for this tissue engineering, their precise character in the synovial tissue has not been determined.MethodsSynovial tissues from bovine metacarpophalangeal joints were used as a stem cell source. For efficient collection of stem cells, we first prepared a preculture before sorting in medium containing FBS at variable concentrations for 4 days. Using a cell sorter and the Ho-dye, a poorly stained population enriched with stem cells was then isolated. To determine the characteristics of the stem cells, specific marker genes such as CD34, Flk-1, c-Kit, Abcg-2 were identified by real-time PCR. Sorted SP cells were cultured in a stem cell medium supplemented with bFGF, SCF and fibronectin, and evaluated for their differentiation potentials into chondrocytes, osteocytes and myocytes.ResultsSP cells of synovium tissue were increased from 2% of the total cell population to approximately 10% of the total cells by preculture in the 1%FBS contained medium. Sorted SP cells expressed CD34, Flk-1, c-Kit, Abcg-2 and Mdr-1 -all are important marker genes for stem cell characteristics. The SP cells could be further expanded ex vivo while maintaining stem cell potentials such as marker gene expression, Ho-dye efflux potential and multiple differentiation potentials into chondrocyte, osteocyte and myocyte.ConclusionIn the present study, we demonstrated that the cells with outstanding stem cell properties were efficiently collected as a SP fraction from bovine synovial membrane. Furthermore, we have described an efficient isolation method and the culture conditions for ex vivo expansion that maintains their important characteristics. Our results suggest that the SP cells of synovium tissue might be important candidates as sources for cell transplantation.

Oxidized LDL binding to LOX-1 enhances MCP-1 expression in cultured human articular chondrocytes

OBJECTIVE It has been suggested that oxidized low-density lipoprotein (ox-LDL) has some roles in progression of osteoarthritis. The purpose of this study is to investigate whether ox-LDL binding to lectin-like ox-LDL receptor 1 (LOX-1) enhances monocyte chemoattractant protein 1 (MCP-1) expression in cultured human articular chondrocytes (HACs). METHOD The time course and dose response of MCP-1 mRNA expression and MCP-1 protein release into medium following ox-LDL stimulation were investigated using quantitative Real time PCR (delta-delta Ct method) and enzyme-linked immunosorbent assay (ELISA), respectively. To examine the receptor specificity of ox-LDL action, HACs were preincubated with anti-human LOX-1 monoclonal antibody (TS92). RESULTS A time-course study revealed that MCP-1 mRNA expression increased 5.09+/-0.86 fold 12h after ox-LDL stimulation compared to time-0. ox-LDL stimulation increased MCP-1 protein level in conditioned medium in a time-dependent manner. Increased MCP-1 level was evident 6h after stimulation, reaching 830+/-91 pg/ml at 24h (33+/-8 pg/ml at time-0). Dose responses of MCP-1 expression were also evident in mRNA and protein levels. Pretreatment with TS92 markedly suppressed these stimulating effects of ox-LDL, although that with non-specific IgG did not. Native LDL did not affect MCP-1 expression. CONCLUSION Our results suggest that ox-LDL enhances MCP-1 expression in HACs and supports the hypothesis that ox-LDL is involved in cartilage degeneration.

Hyaluronan reversed proteoglycan synthesis inhibited by mechanical stress: possible involvement of antioxidant effect

IntroductionAbnormal mechanical stress loaded on the cartilage leads to the osteoarthritis (OA). Although intraarticular hyaluronan (HA) injection is an effective treatment for OA, the underlying mechanism has not been made clear.MethodsMechanical compression was loaded on the bovine cartilage using the Biopress system. Proteoglycan (PG) and reactive oxygen species (ROS) synthesis were measured with [35S] incorporation and fluorescent dye, respectively. Accumulation of peroxynitrite was determined with western blotting using nitrotyrosine antibody.ResultsMechanical compression inhibited PG synthesis and enhanced ROS. Externally added HA reversed stress-inhibited PG synthesis and attenuated ROS synthesis. HA also significantly decreased the generation of nitrotyrosine.ConclusionsHA neutralized stress-enhanced ROS synthesis and resulted in the reversing of PG synthesis. These data suggest that HA plays an anabolic effect as an antioxidant.

Leukemia Inhibitory Factor (LIF) Enhances Germ Cell Differentiation from Primate Embryonic Stem Cells

Recently, several research groups have shown that germ cells can be produced in vitro from pluripotent embryonic stem cells (ESCs). In the mouse, live births of offspring using germ cells induced from ESCs in vitro have been reported. Furthermore, some efficient methods for inducing the useful number of germ cells from ESCs have also been developed. On the other hand, in primates, despite the appearances of germ cell-like cells including meiotic cells were observed by spontaneous differentiation or introducing transgenes, it has not been determined whether fully functional germ cells can be derived from ESCs. To elucidate the property for the germ cells induced from primate ESCs, specification of the promoting factors for the germ cell development and improving the efficiency of germ cell derivation are essential. Leukemia inhibitory factor (LIF) has been reported as one of the important factors for mouse primordial germ cell (PGC) survival in vitro. However, the effects of LIF on germ cell formation from pluripotent cells of primates have not been examined. The aim of this study is to determine whether LIF addition can improve in vitro germ cell production from cynomolgus monkey ESCs (cyESCs). After 8 days of differentiation, LIF added culture induced dome-shaped germ cell colonies as indicated by the intense expression of alkaline phosphatase activity (ALP). These cells also demonstrate high-level expression of the germ cell-marker VASA, OCT-4, and BLIMP-1, and show SSEA-1 expression that supports their early stage germ cell identity. Finally, we observed that adding LIF to differentiating cultures inhibited meiotic gene expressions and increased the percentage of ALP-positive cells, and demonstrate that the addition of LIF to differentiation media increases differentiation of early germ cells from the cyESCs.

Reduced oxygen concentration enhances conversion of embryonic stem cells to epiblast stem cells.

Recently, an additional type of pluripotent stem cell-line derived from mouse embryos has been established and termed epiblast stem cell (EpiSC), and is expected to be an important tool for studying the mechanisms of maintenance of pluripotency since they depend on basic fibroblast growth factor-MAPK and Activin A-Smad2/3 signaling to maintain pluripotency, unlike mouse embryonic stem cells (ESCs). Further, because of the similarities between mouse EpiSCs and human ESCs, EpiSCs are expected to be effective experimental models for human stem cell therapy. Recently, study for conversion from ESC state to EpiSC state or reversion from EpiSC state to ESC state has attracted interest since these techniques may lead to increasing the potential of pluripotent stem cells and our knowledge about their developmental status. In the present study, we find that a low oxygen concentration in culture environment accelerated, improved, and stabilized the EpiSC state of the converted cells from the ESC state using Oct4ΔPE-GFP transgenic ESCs. Induced EpiSCs (iEpiSCs) in hypoxia possess closer gene expression patterns to native EpiSCs, and bisulfite sequences for the promoter regions of Stella and Oct4 genes have elucidated that the iEpiSC gain EpiSC-specific methylation patterns in hypoxia. Our data provide evidence that oxygen concentration is an important factor for establishment of the EpiSC-specific state.