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Nishigaki T.,Kyoto University | Teramura Y.,Kyoto University | Suemori H.,Institute for Frontier Medical science | Iwata H.,Kyoto University
Cryobiology | Year: 2010

Human embryonic stem (hES) cells are expected to be useful in the fields of regenerative medicine and tissue engineering due to their pluripotency. Therefore, it is necessary to establish highly efficient and reliable methods for the cryopreservation of hES cells. We have cryopreserved cynomolgus and human ES cells by the vitrification method, using a chemically-defined dimethyl sulfoxide (Me2SO)-free and serum-free medium composed of Euro-Collins solution as a base medium and 40% (v/v) ethylene glycol (EG) and 10% (w/v) polyethylene glycol (PEG) as cryoprotectants. When the vitrification and the cryoprotectants were combined, the recovery ratio of hES cells was 22.9 ± 7.7%, compared to 0.4 ± 0.2% when the conventional slow-freezing method was used. After the cryopreservation and thawing cycle, hES cells were easily cultured and expressed undifferentiated cell markers such as Nanog, Oct-4, SSEA-4, and alkaline phosphatase activity after several subculturing steps. We also found that the pluripotency of hES cells was maintained, as demonstrated by teratoma formation of ES cells transplanted into severe combined immunodeficient (SCID) mice. Thus, we conclude that we have successfully cryopreserved primate ES cells with high efficiency using a Me2SO-free, chemically-defined medium. © 2009 Elsevier Inc. All rights reserved. Source

Toguchida J.,Institute for Frontier Medical science
Clinical calcium | Year: 2014

The application of iPS cells for diseases in the locomotive system includes the cell therapy using bone or cartilage cells and the understanding of pathogenesis and drug discovery for intractable diseases using patients' derived iPS cells. To promote these researches, the proper knowledge concerning the process from iPS cells to multipotential mesenchymal stromal cell (MSC) and terminally differentiated cells is inevitable. In addition, because there are several different ways to induce terminally differentiation, the biological quality of cells induced by each method should be compared in future. Source

Fujiwara H.,Tohoku University | Saiki Y.,Tohoku University | Sato M.,Tohoku University | Sakamoto N.,Tohoku University | And 3 more authors.
Journal of Vascular Surgery | Year: 2010

Objectives: We investigated the outcomes of reinforcing anastomotic sites using (1) nonbiodegradable polytetrafluoroethylene (PTFE) felt, (2) biodegradable polyglycolic acid (PGA) felt, and (3) PGA felt with basic fibroblast growth factor (bFGF) in a canine descending thoracic aortic replacement model. Methods: Thirty-seven beagles underwent descending thoracic aorta replacement using a prosthetic graft with one of the above-mentioned reinforcements or no reinforcement for controls. Histologic evaluations were carried out 1 month and 3 months after surgery. The biomechanical strength of the anastomosis was assessed along the longitudinal axis of the aortic segments using a tensile tester. Local compliance at the anastomotic site was also evaluated in the circumferential direction. Results: The media was significantly thinner in the PTFE group than in the control group (65.8% ± 5.1% vs 95.0% ± 9.3% of normal thickness; P < .05). Relative to the control group, the adventitial layer was significantly thinner in the PTFE group (42.3% ± 8.2% of control; P < .05) but significantly thicker in the PGA and the PGA + bFGF groups (117.2% ± 11.3% and 134.1% ± 14.2% of control, respectively; P < .05). There were more vessels in the adventitial layer in the PGA + bFGF group than in the control, PTFE, and PGA groups (29.2 ± 2.1/mm2 vs 13.8 ± 0.8, 5.4 ± 0.7, 17.0 ± 1.3/mm2, respectively; P < .01). There were no significant differences between the four groups in the failure force at anastomotic sites. Local compliance at the anastomotic site was higher in the PGA group than that in the PTFE group (11.6 ± 1.6 10-6 m2/N vs 5.6 ± 1.9 10-6 m2/N; P < .05). Conclusion: Reinforcement of the experimental aortic wall with PTFE felt resulted in thinning of the media and adventitia and fewer vessels at the anastomotic site. These histologic changes were not observed when biodegradable felt was used. The bFGF failed to augment the modification of the aortic wall with the exception of increased adventitial vessel number. Biomechanical strength of the anastomosis along the longitudinal axis was comparable in all four groups; however, local vascular compliance was better in the biodegradable PGA felt group. © 2010 Society for Vascular Surgery. Source

Nagae G.,Research Center for Advanced Science and Technology | Isagawa T.,Research Center for Advanced Science and Technology | Shiraki N.,Kumamoto University | Fujita T.,Research Center for Advanced Science and Technology | And 15 more authors.
Human Molecular Genetics | Year: 2011

Epigenetic regulation is essential in determining cellular phenotypes during differentiation. Although tissuespecific DNA methylation has been studied, the significance of methylation variance for tissue phenotypes remains unresolved, especially for CpG-poor promoters. Here, we comprehensively studied methylation levels of 27 578 CpG sites among 21 human normal tissues from 12 anatomically different regions using an epigenotyping beadarray system. Remarkable changes in tissue-specific DNA methylation were observed within CpG-poor promoters but not CpG-rich promoters. Of note, tissue-specific hypomethylation is accompanied by an increase in gene expression, which gives rise to specialized cellular functions. The hypomethylated regions were significantly enriched with recognition motifs for transcription factors that regulate cell-type-specific differentiation. To investigate the dynamics of hypomethylation events, we analyzed methylation levels of the entire APOA1 gene locus during in vitro differentiation of embryonic stem cells toward the hepatic lineage. A decrease in methylation was observed after day 13, coinciding with alpha-feto- protein detection, in the vicinity of its transcription start sites (TSSs), and extends up to ~200 bp region encompassing the TSS at day 21, equivalent to the hepatoblastic stage. This decrease is even more pro- nounced in the adult liver, where the entire APOA1 gene locus is hypomethylated. Furthermore, when we compared the methylation status of induced pluripotent stem (iPS) cells with their parental cell, IMR-90, we found that fibroblast-specific hypomethylation is restored to a fully methylated state in iPS cells after reprogramming. These results illuminate tissue-specific methylation dynamics in CpG-poor promoters and provide more comprehensive views on spatiotemporal gene regulation in terminal differentiation. © The Author 2011. Published by Oxford University Press. All rights reserved. Source

Nishimura H.,Institute for Integrated Cell Material science | Nishimura H.,Institute for Frontier Medical science | Ritchie K.,Purdue University | Kasai R.S.,Institute for Integrated Cell Material science | And 10 more authors.
Journal of Cell Biology | Year: 2013

Fluorescence microscopy is used extensively in cellbiological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for =10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1-2 μm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed. ©2013 Nishimura et al. Source

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