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Imaizumi K.,RIKEN | Nishikawa S.-I.,RIKEN | Tarui H.,RIKEN | Akuta T.,RIKEN | Akuta T.,Kyokuto Pharmaceutical Industrial Co.
Protein Expression and Purification | Year: 2013

Leukemia inhibitor factor (LIF) is a three disulfide bridge-containing cytokine with numerous regulatory effects. In this report, we present the high level expression of a soluble recombinant human LIF (rhLIF) in Escherichia coli. A codon-optimized Profinity eXact™-tagged hLIF cDNA was cloned into pET3b vector, and transformed into E. coli OrigamiB(DE3) harboring the bacterial thioredoxin coexpression vector. By using an enzyme-based glucose release system (EnBase®) and high-aeration shake flask (Ultra Yield Flask™), the yield of soluble proteins was significantly improved in comparison to commonly-used 2 × YT media. The recombinant protein was purified via a single chromatographic step using an affinity tag-based protein purification system that processed by cleavage with sodium fluoride, resulting in the complete proteolytic removal the N-terminal tag. Soluble rhLIF yield was estimated to be approximately 1 mg/g of wet weight cells, with above 98% purity. The rhLIF protein specifically inhibited the proliferation of the murine myeloblastic leukemia M1 cell in a dose-dependent manner, and induced Stat3 phosphorylation in mouse ES cells. This novel expression and purification protocol for the production of recombinant hLIF is a simple, suitable, and effective method. © 2013 Elsevier Inc. All rights reserved.


Matsumoto A.,Japan National Institute of Materials Science | Matsumoto A.,University of Tsukuba | Matsumoto A.,Kyokuto Pharmaceutical Industrial Co. | Harada H.,Iwate Medical University | And 3 more authors.
In Vitro Cellular and Developmental Biology - Animal | Year: 2011

Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT- 7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions. © 2010 The Society for In Vitro Biology.


Todaka N.,Toyota Central R&D Labs. | Todaka N.,Kyokuto Pharmaceutical Industrial Co. | Nakamura R.,Toyota Central R&D Labs. | Moriya S.,RIKEN | And 5 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2011

For direct and efficient ethanol production from cellulosic materials, we screened optimal cellulases from symbiotic protists of termites through heterologous expression with Saccharomyces cerevisiae. 11 cellulases, belonging to glycoside hydrolase families 5, 7, and 45 endoglucanases (EGs), were confirmed to produce with S. cerevisiae for the first time. A recombinant yeast expressing SM2042B24 EG I was more efficient at degrading carboxylmethyl cellulose than was Trichoderma reesei EG I, a major EG with high cellulolytic activity.


Imaizumi K.,RIKEN | Imaizumi K.,Kyokuto Pharmaceutical Industrial Co. | Nishishita N.,RIKEN | Nishishita N.,Foundation for Biomedical Research and Innovation | And 10 more authors.
PLoS ONE | Year: 2014

Vitrification and slow-freezing methods have been used for the cryopreservation of human pluripotent stem cells (hPSCs). Vitrification requires considerable skill and post-thaw recovery is low. Furthermore, it is not suitable for cryopreservation of large numbers of hPSCs. While slow-freezing methods for hPSCs are easy to perform, they are usually preceded by a complicated cell dissociation process that yields poor post-thaw survival. To develop a robust and easy slow-freezing method for hPSCs, several different cryopreservation cocktails were prepared by modifying a commercially available freezing medium (CP-1™) containing hydroxyethyl starch (HES), and dimethyl sulfoxide (DMSO) in saline. The new freezing media were examined for their cryopreservation efficacy in combination with several different cell detachment methods. hPSCs in cryopreservation medium were slowly cooled in a conventional -80°C freezer and thawed rapidly. hPSC colonies were dissociated with several proteases. Ten percent of the colonies were passaged without cryopreservation and another 10% were cryopreserved, and then the recovery ratio was determined by comparing the number of Alkaline Phosphatase-positive colonies after thawing at day 5 with those passaged without cryopreservation at day 5. We found that cell detachment with Pronase/EDTA followed by cryopreservation using 6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline (termed CP-5E) achieved post-thaw recoveries over 80%. In summary, we have developed a new cryopreservation medium free of animal products for slow-freezing. This easy and robust cryopreservation method could be used widely for basic research and for clinical application. © 2014 Imaizumi et al.


Patent
Kyokuto Pharmaceutical Industrial Co. and Riken | Date: 2013-06-14

Provided are a stem cell preservation medium, stem cell preservation method and stem cell preservation system which can be used in a slow-freezing method that is used in place of vitrification, have a high cell survival rate and are convenient and highly efficient. The stem cell preservation medium comprises hydroxyethyl starch (HES), dimethyl sulfoxide (DMSO), and ethylene glycol (EG). The stem cell preservation method comprises: a dissociation step in which stem cells are dissociated using a pronase solution; and a freezing step in which the dissociated stem cells are slow-frozen in the stem cell preservation medium. The stem cell preservation system comprises: the pronase solution which is a dissociation means for dissociating stem cells; the stem cell preservation medium according to the present invention; and a slow-freezing means with which the dissociated stem cells are slow-frozen in the stem cell preservation medium.

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