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Homma K.,Kyoto University | Homma K.,Keio University | Sone M.,Kyoto University | Taura D.,Kyoto University | And 12 more authors.
Atherosclerosis | Year: 2010

Objective: We previously succeeded in inducing and isolating vascular endothelial cells (ECs) from both human embryonic stem (ES) and induced pluripotent stem (iPS) cells. Here, we compared the functionality of human adult ECs (HAECs), human ES-derived ECs (ESECs) and human iPS-derived ECs (iPSECs). Methods and results: We compared the cell proliferative potential, potential for migration, and tolerance to oxidative stress. ESECs were significantly superior to HAECs in all of these cell functions. The cell functions of iPSECs were comparable to those of ESECSs and also superior to HAECs. We then analyzed the gene expressions of HAECs, ESECs and iPSECs, and observed that the expression level of Sirt1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, is higher in ESECs and iPSECs than in HAECs. The inhibition of Sirt1 with a Sirt1-specific inhibitor and siRNA antagonized these differences between the three types of cells. Conclusions: Sirt1 plays a key role in the high cellular function of ESECs and iPSECs. Although further in vivo investigations are required, this study initially demonstrated the potential of ESECs and iPSECs as the cell source for regenerative medicine, and also showed the potential of ES cells as a useful tool for elucidating the molecular mechanism of cell aging. © 2010. Source


Fujiwara M.,Kyoto University | Yan P.,Kyoto University | Otsuji T.G.,Stem Cell and Drug Discovery Institute | Otsuji T.G.,Kyoto University | And 18 more authors.
PLoS ONE | Year: 2011

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1+ common progenitor cells, and identified highly cardiogenic progenitors as Flk1+/CXCR4+/VE-cadherin- (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1+ cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1+ cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs. © 2011 Fujiwara et al. Source


Wada T.,Stem Cell and Drug Discovery Institute | Goparaju S.K.,Kyoto University | Tooi N.,Kyoto University | Inoue H.,Kyoto University | And 4 more authors.
Stem Cells Translational Medicine | Year: 2012

The generation of amyotrophic lateral sclerosis (ALS) disease models is an important subject for investigating disease mechanisms and pharmaceutical applications. In transgenic mice, expression of a mutant form of superoxide dismutase 1 (SOD1) can lead to the development of ALS that closely mimics the familial type of ALS (FALS). Although SOD1 mutant mice show phenotypes similar to FALS, dissimilar drug responses and size differences limit their usefulness to study the disease mechanism(s) and identify potential therapeutic compounds. Development of an in vitro model system for ALS is expected to help in obtaining novel insights into disease mechanisms and discovery of therapeutics. We report the establishment of an in vitro FALS model from human embryonic stem cells overexpressing either a wild-type (WT) or a mutant SOD1 (G93A) gene and the evaluation of the phenotypes and survival of the spinal motor neurons (sMNs), which are the neurons affected in ALS patients. The in vitro FALS model that we developed mimics the in vivo human ALS disease in terms of the following: (a) selective degeneration of sMNs expressing the G93A SOD1 but not those expressing the WT gene; (b) susceptibility of G93A SOD1-derived sMNs to form ubiquitinated inclusions; (c) astrocyte-derived factor(s) in the selective degeneration of G93A SOD1 sMNs; and (d) cell-autonomous, as well as non-cellautonomous, dependent sMN degeneration. Thus, this model is expected to help unravel the disease mechanisms involved in the development of FALS and also lead to potential drug discoveries based on the prevention of neurodegeneration. © AlphaMed Press. Source


Tatsumi R.,Stem Cell and Drug Discovery Institute | Suzuki Y.,Stem Cell and Drug Discovery Institute | Sumi T.,Niigata University | Sone M.,Kyoto University | And 2 more authors.
Cell Transplantation | Year: 2011

Endothelial cells derived from human embryonic stem cells (hESC-ECs) hold much promise as a valuable tool for basic vascular research and for medical application such as cell transplantation or regenerative medicine. Here we have developed an efficient approach for the production of hESC-ECs. Using a differentiation method consisting of a stepwise combination of treatment with glycogen synthase kinase-3β (GSK-3β) inhibitor and culturing in vascular endothelial growth factor (VEGF)-supplemented medium, hESC-ECs are induced in 5 days with about 20% efficiency. These cells express vascular endothelial cadherin (VEcadherin), VEGF receptor-2 (VEGFR-2), CD34, and platelet endothelial cell adhesion molecule-1 (PECAM-1). These hESC-ECs can then be isolated with 95% purity using a magnetic sorting system, and expanded to more than 100-fold within a month. The hESC-ECs thus produced exhibit the endothelial morphological characteristics and specific functions such as capillary tube formation and acetylated low-density lipoprotein uptake. We propose that our methodology is useful for efficient and large-scale production of hESC-ECs. © 2011 Cognizant Comm. Corp. Source


Murakami G.,Kyoto University | Inoue H.,Kyoto University | Inoue H.,Japan Science and Technology Agency | Tsukita K.,Kyoto University | And 9 more authors.
Journal of Biomolecular Screening | Year: 2011

Familial amyotrophic lateral sclerosis (fALS) accounts for 10% of ALS cases, and about 25% of fALS cases are due to mutations in superoxide dismutase 1 (SOD1). Mutant SOD1-mediated ALS is caused by a gain of toxic function of the mutant protein, and the SOD1 level in nonneuronal neighbors, including astrocytes, determines the progression of ALS (non-cell-autonomous toxicity). Therefore, the authors hypothesized that small molecules that reduce SOD1 protein levels in astrocytes might slow the progression of mutant SOD1-mediated ALS. They developed and optimized a cell-based, high-throughput assay to identify low molecular weight compounds that decrease SOD1 expression transcriptionally in human astrocyte-derived cells. Screening of a chemical library of 9600 compounds with the assay identified two hit compounds that selectively and partially downregulate SOD1 expression in a dose-dependent manner, without any detectable cellular toxicity. Western blot analysis showed that one hit compound significantly decreased the level of endogenous SOD1 protein in H4 cells, with no reduction in expression of ß-actin. The assay developed here provides a powerful strategy for discovering novel lead molecules for treating familial SOD1-mediated ALS. (Journal of Biomolecular Screening 2011;16:405-414) © 2011 Society for Laboratory Automation and Screening. Source

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