九州大学 研究者情報
論文一覧
河邉 佳典(かわべ よしのり) データ更新日:2021.11.02

准教授 /  工学研究院 化学工学部門 分子・生物システム工学講座


原著論文
1. Akitsu Hotta, Masamichi Kamihira, Kanako Itoh, Mahboob Morshed, Kawabe Yoshinori, Ken Ichiro Ono, Hiroyuki Matsumoto, Ken Ichi Nishijima, Shinji Iijima, Production of anti-CD2 chimeric antibody by recombinant animal cells, Journal of Bioscience and Bioengineering, 10.1263/jbb.98.298, 98, 4, 298-303, 2004.01, [URL], Expression vectors for chimeric anti-CD2 antibody were constructed in order to clarify the importance of the expression ratio of heavy (H-) and light (L-) chains of antibody to antibody production in animal cells. The antibody genes were introduced into cells using plasmid DNA vectors or replication-defective retroviral vectors. Productivity was maximal when the expression ratio of H- and L-chains was 1:1, and decreased when the ratio was not equal. We also examined the expression of antibody using one-packed vectors in which the bicistronic expression of H- and L-chain genes was mediated by an internal ribosomal entry site (IRES) sequence derived from encephalomyocarditis virus (EMCV). The translation efficiency was unbalanced between 5′Cap-and IRES-dependent genes. Using the retroviral vectors, it was estimated that the IRES-dependent translation efficiency was 5-fold lower than the 5′Cap-dependent translation efficiency. The cells exhibiting an unbalanced expression of H- and L-chains tended to accumulate H-chain protein..
2. Hotta A, Kamihira M, Itoh K, Morshed M, Kawabe Y, Ono K, Matsumoto H, Nishijima K, Iijima S, Production of anti-CD2 chimeric antibody by recombinant animal cells., J Biosci Bioeng , doi:10.1016/S1389-1723(04)00285-3, 98, 4, 298-303, 2004.10.
3. Hotta A, Saito Y, Kyogoku K, Kawabe Y, Nishijima K, Kamihira M, Iijima S, Characterization of transient expression system for the retroviral vector production, J Biosci Bioeng, doi:10.1263/jbb.101.361, 101, 4, 361-368, 2006.04.
4. Akitsu Hotta, Yoshikazu Saito, Kenji Kyogoku, Kawabe Yoshinori, Ken ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Characterization of transient expression system for retroviral vector production, Journal of Bioscience and Bioengineering, 10.1263/jbb.101.361, 101, 4, 361-368, 2006.04, [URL], The production of retroviral vectors using a transient expression system has been improved to obtain a high-titer virus preparation that is difficult to produce using packaging cell lines due to the cytotoxic or cytostatic effect of transgenes. Here, we used one such production method, the so-called Q-vector system, and examined its potential for virus production. The Q-vector system could produce a similar level of viral vectors compared with the packaging cell system but the production seemed to depend on the size and nature of transgenes. In the process of investigation of the quantitative difference in viral components between the transient expression system and the packaging cell system, we found that the Q-vector system could express higher amounts of viral RNA and proteins compared with the packaging cell system. However, this did not lead to a higher virus titer compared with that produced by the packaging cell system. This suggests that retroviral RNA transcribed from the plasmid in the transient system seemed to be used mainly for translation and only some of the RNA molecules were packaged in viral particles..
5. Akitsu Hotta, Yoshikazu Saito, Kenji Kyogoku, Yoshinori Kawabe, Ken-ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Characterization of transient expression system for retroviral vector production., Journal of bioscience and bioengineering, 10.1263/jbb.101.361, 101, 4, 361-8, 2006.04, The production of retroviral vectors using a transient expression system has been improved to obtain a high-titer virus preparation that is difficult to produce using packaging cell lines due to the cytotoxic or cytostatic effect of transgenes. Here, we used one such production method, the so-called Q-vector system, and examined its potential for virus production. The Q-vector system could produce a similar level of viral vectors compared with the packaging cell system but the production seemed to depend on the size and nature of transgenes. In the process of investigation of the quantitative difference in viral components between the transient expression system and the packaging cell system, we found that the Q-vector system could express higher amounts of viral RNA and proteins compared with the packaging cell system. However, this did not lead to a higher virus titer compared with that produced by the packaging cell system. This suggests that retroviral RNA transcribed from the plasmid in the transient system seemed to be used mainly for translation and only some of the RNA molecules were packaged in viral particles..
6. Yoshinori Kawabe, Akitsu Hotta, Ken-ichiro Ono, Kazuhisa Esaka, Ken-ichi Nishijima, Masamichi Kamihira, and Shinji Iijima, Production of chimeric antibodies by transgenic chicken bioreactor, Animal Cell Technology: Basic & Applied Aspects, 14,307-314, 2006.05.
7. Kawabe Y, Kamihira M, Ono K, Kyogoku K, Nishijima K, Iijima S, Production of scFv-Fc Fusion Protein by Genetically Manipulated Quails, J Biosci Bioeng , doi:10.1263/jbb.102.297, 102, 4, 297-303, 2006.10.
8. Kawabe Yoshinori, Masamichi Kamihira, Ken ichiro Ono, Kenji Kyogoku, Ken ichi Nishijima, Shinji Iijima, Production of scFv-Fc fusion protein using genetically manipulated quails, Journal of Bioscience and Bioengineering, 10.1263/jbb.102.297, 102, 4, 297-303, 2006.10, [URL], The use of transgenic avian species as a transgenic bioreactor for the production of recombinant proteins has been proposed. In recent years, although various procedures for generating transgenic chickens have been reported, the expression of a useful protein at a commercially feasible level has rarely been attained. In this study, we injected a concentrated retroviral vector into quail embryos to generate genetically manipulated quails that produce recombinant proteins. We found that transgene expression in the whole body at a high level was observed for viral injection into the heart of the developing embryos after a 48-h incubation. For the practical production of a useful protein, a retroviral vector encoding an anti-prion scFv-Fc gene under the control of the β-actin promoter was injected into quail embryos. The quails that hatched stably produced scFv-Fc at a high level in their serum and egg white. The production of scFv-Fc was maintained throughout the breeding period. scFv-Fc purified from the egg white retained the antigen-binding activity. This system exhibited the potential of transgenic quails for the commercial production of recombinant proteins..
9. Yoshinori Kawabe, Masamichi Kamihira, Ken-ichiro Ono, Kenji Kyogoku, Ken-ichi Nishijima, Shinji Iijima, Production of scFv-Fc fusion protein using genetically manipulated quails., Journal of bioscience and bioengineering, 10.1263/jbb.102.297, 102, 4, 297-303, 2006.10, The use of transgenic avian species as a transgenic bioreactor for the production of recombinant proteins has been proposed. In recent years, although various procedures for generating transgenic chickens have been reported, the expression of a useful protein at a commercially feasible level has rarely been attained. In this study, we injected a concentrated retroviral vector into quail embryos to generate genetically manipulated quails that produce recombinant proteins. We found that transgene expression in the whole body at a high level was observed for viral injection into the heart of the developing embryos after a 48-h incubation. For the practical production of a useful protein, a retroviral vector encoding an anti-prion scFv-Fc gene under the control of the beta-actin promoter was injected into quail embryos. The quails that hatched stably produced scFv-Fc at a high level in their serum and egg white. The production of scFv-Fc was maintained throughout the breeding period. scFv-Fc purified from the egg white retained the antigen-binding activity. This system exhibited the potential of transgenic quails for the commercial production of recombinant proteins..
10. Kawabe Y, Naka T, Ando-Noumi N, Matsumoto H, Ono K, Nishijima K, Kamihira M, Iijima S, Transport of human Immunoglobulin G and Fc-Fusion Proteins to Chicken Egg Yolk, J Biosci Bioen, doi:10.1263/jbb.102.518, 102, 6, 518-523, 2006.12.
11. Kawabe Yoshinori, Tsutomu Naka, Naoko Ando-Noumi, Hiroyuki Matsumoto, Ken ichiro Ono, Ken ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Transport of human immunoglobulin G and Fc-fusion proteins to chicken egg yolk, Journal of Bioscience and Bioengineering, 10.1263/jbb.102.518, 102, 6, 518-523, 2006.12, [URL], We examined the transport of human immunoglobulin G (IgG) subclasses and fusion proteins with the Fc region of human IgG to the egg yolk, after the proteins were injected into a vein of hens. Human IgGs were efficiently transported and accumulated into the yolk, whereas the proteins were not detected in the egg white. Among human IgG subclasses, IgG2 was transported most efficiently. Fc-fusion proteins injected were also transported into the yolk. A fusion protein with the Fc region derived from human IgG2 was more efficiently transported into the yolk than the counterpart fusion with the Fc region from human IgG1. This study shows that the recovery of recombinant antibodies and Fc-fusion proteins from the yolk is an effective method in transgenic chicken bioreactors..
12. Yoshinori Kawabe, Tsutomu Naka, Naoko Ando-Noumi, Hiroyuki Matsumoto, Ken-Ichiro Ono, Ken-Ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Transport of human immunoglobulin G and Fc-fusion proteins to chicken egg yolk., Journal of bioscience and bioengineering, 10.1263/jbb.102.518, 102, 6, 518-23, 2006.12, We examined the transport of human immunoglobulin G (IgG) subclasses and fusion proteins with the Fc region of human IgG to the egg yolk, after the proteins were injected into a vein of hens. Human IgGs were efficiently transported and accumulated into the yolk, whereas the proteins were not detected in the egg white. Among human IgG subclasses, IgG2 was transported most efficiently. Fc-fusion proteins injected were also transported into the yolk. A fusion protein with the Fc region derived from human IgG2 was more efficiently transported into the yolk than the counterpart fusion with the Fc region from human IgG1. This study shows that the recovery of recombinant antibodies and Fc-fusion proteins from the yolk is an effective method in transgenic chicken bioreactors..
13. Ito A, Akiyama H, Kawabe Y, Kamihira M., Magnetic force-based cell patterning using Arg-Gly-Asp (RGD) peptide-conjugated magnetite cationic liposomes., J Biosci Bioeng. , 2007 Oct;104(4):288-93., 2007.10.
14. Ito A, Jitsunobu H, Kawabe Y, Kamihira M., Construction of heterotypic cell sheets by magnetic force-based 3-D coculture of HepG2 and NIH3T3 cells., J Biosci Bioeng. , 2007 Nov;104(5):371-8., 2007.10.
15. Akira Ito, Hirokazu Akiyama, Kawabe Yoshinori, Masamichi Kamihira, Magnetic force-based cell patterning using Arg-Gly-Asp (RGD) peptide-conjugated magnetite cationic liposomes, Journal of Bioscience and Bioengineering, 10.1263/jbb.104.288, 104, 4, 288-293, 2007.10, [URL], Micropatterning of target cells is highly desired for tissue engineering and cell biology. Although recent progress in surface chemistry has enabled the spatial control of cell adhesion onto substrates, conventional methods usually require specialized devices and time-consuming processes to fabricate the substrate. In this study, we demonstrate a simple and rapid cell-patterning procedure using magnetite nanoparticles and magnetic force. To label the target cells magnetically, magnetite nanoparticles were encapsulated in cationic liposomes (magnetite cationic liposomes; MCLs). To promote cell attachment, an Arg-Gly-Asp (RGD)-motif-containing peptide was coupled to the phospholipid of MCLs (RGD-MCLs). A human keratinocyte cell line, HaCaT, which has a high anchorage dependency, was used as a model. The RGD-MCLs were added to an ultralow-attachment plate, whose culture surface is modified with a covalently bound hydrogel layer that is hydrophilic and neutrally charged, and then HaCaT cells were seeded to the plates. The RGD-MCLs induced cell adhesion, spreading, cytoskeletal organization, and fibronectin expression. When steel plates with a 200 μm width placed on a magnet were set under a culture surface, magnetically labeled cells aligned on the surface where the steel plate was positioned, resulting in cell patterning. Furthermore, various cell patterns using a computer-aided design were successfully fabricated. These results suggest that cell patterning using RGD-MCLs is a promising approach to tissue engineering and studies in cell biology..
16. Akira Ito, Hirokazu Akiyama, Yoshinori Kawabe, Masamichi Kamihira, Magnetic force-based cell patterning using Arg-Gly-Asp (RGD) peptide-conjugated magnetite cationic liposomes., Journal of bioscience and bioengineering, 10.1263/jbb.104.288, 104, 4, 288-93, 2007.10, Micropatterning of target cells is highly desired for tissue engineering and cell biology. Although recent progress in surface chemistry has enabled the spatial control of cell adhesion onto substrates, conventional methods usually require specialized devices and time-consuming processes to fabricate the substrate. In this study, we demonstrate a simple and rapid cell-patterning procedure using magnetite nanoparticles and magnetic force. To label the target cells magnetically, magnetite nanoparticles were encapsulated in cationic liposomes (magnetite cationic liposomes; MCLs). To promote cell attachment, an Arg-Gly-Asp (RGD)-motif-containing peptide was coupled to the phospholipid of MCLs (RGD-MCLs). A human keratinocyte cell line, HaCaT, which has a high anchorage dependency, was used as a model. The RGD-MCLs were added to an ultralow-attachment plate, whose culture surface is modified with a covalently bound hydrogel layer that is hydrophilic and neutrally charged, and then HaCaT cells were seeded to the plates. The RGD-MCLs induced cell adhesion, spreading, cytoskeletal organization, and fibronectin expression. When steel plates with a 200 microm width placed on a magnet were set under a culture surface, magnetically labeled cells aligned on the surface where the steel plate was positioned, resulting in cell patterning. Furthermore, various cell patterns using a computer-aided design were successfully fabricated. These results suggest that cell patterning using RGD-MCLs is a promising approach to tissue engineering and studies in cell biology..
17. Akira Ito, Hideaki Jitsunobu, Kawabe Yoshinori, Masamichi Kamihira, Construction of Heterotypic Cell Sheets by Magnetic Force-Based 3-D Coculture of HepG2 and NIH3T3 Cells, Journal of Bioscience and Bioengineering, 10.1263/jbb.104.371, 104, 5, 371-378, 2007.11, [URL], Heterotypic 3-D coculture is essential to mimic tissues and organs, because cell-cell interaction between various types of cells is believed to be important for the activation of cellular functions. In this study, magnetic force was applied to construct a 3-D coculture system of HepG2 and NIH3T3 cells as a model of hepatocytes and mesenchymal cells. Magnetite cationic liposomes (MCLs) were used to label target cells. NIH3T3 cells labeled with MCLs were seeded onto ultralow-attachment plates, whose surface is composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. When a magnet was placed under the plate, cells accumulated on the bottom of the well. After a 24-h incubation period, the cells formed a multilayered cell sheet, which contained the major mesenchymal extracellular matrix (ECM) components (fibronectin and type I collagen), suggesting that the use of stromal NIH3T3 cells gave sufficient strength to cell sheets. Both NIH3T3 and HepG2 cells were labeled with MCLs, and cocultured by two methods: NIH3T3 cell sheets were constructed and HepG2 cells were subsequently seeded onto NIH3T3 cell sheets, and then allowed to form layered cell sheets by applying magnetic force; or NIH3T3 and HepG2 cells were mixed and then allowed to form mixed cell sheets by applying magnetic force. These heterotypic multilayered cell sheets were successfully constructed and an enhanced albumin secretion by HepG2 cells was observed. These results suggest that the new tissue engineering technique using magnetite nanoparticles and magnetic force, to which we refer to as magnetic force-based tissue engineering (Mag-TE), is a promising approach to construct multilayered cell sheets consisting of heterotypic cocultured cells..
18. Akira Ito, Hideaki Jitsunobu, Yoshinori Kawabe, Masamichi Kamihira, Construction of heterotypic cell sheets by magnetic force-based 3-D coculture of HepG2 and NIH3T3 cells., Journal of bioscience and bioengineering, 10.1263/jbb.104.371, 104, 5, 371-8, 2007.11, Heterotypic 3-D coculture is essential to mimic tissues and organs, because cell-cell interaction between various types of cells is believed to be important for the activation of cellular functions. In this study, magnetic force was applied to construct a 3-D coculture system of HepG2 and NIH3T3 cells as a model of hepatocytes and mesenchymal cells. Magnetite cationic liposomes (MCLs) were used to label target cells. NIH3T3 cells labeled with MCLs were seeded onto ultralow-attachment plates, whose surface is composed of a covalently bound hydrogel layer that is hydrophilic and neutrally charged. When a magnet was placed under the plate, cells accumulated on the bottom of the well. After a 24-h incubation period, the cells formed a multilayered cell sheet, which contained the major mesenchymal extracellular matrix (ECM) components (fibronectin and type I collagen), suggesting that the use of stromal NIH3T3 cells gave sufficient strength to cell sheets. Both NIH3T3 and HepG2 cells were labeled with MCLs, and cocultured by two methods: NIH3T3 cell sheets were constructed and HepG2 cells were subsequently seeded onto NIH3T3 cell sheets, and then allowed to form layered cell sheets by applying magnetic force; or NIH3T3 and HepG2 cells were mixed and then allowed to form mixed cell sheets by applying magnetic force. These heterotypic multilayered cell sheets were successfully constructed and an enhanced albumin secretion by HepG2 cells was observed. These results suggest that the new tissue engineering technique using magnetite nanoparticles and magnetic force, to which we refer to as magnetic force-based tissue engineering (Mag-TE), is a promising approach to construct multilayered cell sheets consisting of heterotypic cocultured cells..
19. Akira Ito, Tetsuya Takahashi, Yujiro Kameyama, Kawabe Yoshinori, Masamichi Kamihira, Magnetic manipulation of a retroviral vector using magnetite cationic liposomes, 2008 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2008, with Symposium on "COE for Education and Research of Micro-Nano Mechatronics", Symposium on "System Cell Engineering by Multi-scale Manipulation"
2008 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2008, with Symposium on "COE for Education and Research of Micro-Nano Mechatronics"
, 10.1109/MHS.2008.4752479, 367-371, 2008, [URL], In the present study, we used magnetite nanoparticles and magnetic force to concentrate the retroviral vectors in order to enhance the transduction efficiency and to enable their magnetic manipulation. Magnetite nanoparticles modified with Cationic liposomes were added to a solution containing a retroviral vector. The magnetic particles which captured the viral vectors were collected by a magnetic force, and seeded into target cells. The viral titer increased up to 55-fold and 3 x 108 IU/mL. Additionally, the magnetically labeled retroviral vectors can be directed to the desired regions for infection by applying magnetic fields, and micro-patterns of gene-transduced cell regions could be created on a cellular monolayer using micro-patterned magnetic concentrators. These results suggest that this new technique provides a promising approach to capture and concentrate viral vectors, thus achieving high transduction efficiency and the ability to deliver genes to a specifically injured site by applying a magnetic field..
20. Daisuke Kodama, Daisuke Nishimiya, Ken ichi Iwata, Kazuhisa Yamaguchi, Kazuhiro Yoshida, Kawabe Yoshinori, Makoto Motono, Hiroyuki Watanabe, Takashi Yamashita, Ken ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Production of human erythropoietin by chimeric chickens, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2008.01.020, 367, 4, 834-839, 2008.03, [URL], The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells..
21. Daisuke Kodama, Daisuke Nishimiya, Ken-Ichi Iwata, Kazuhisa Yamaguchi, Kazuhiro Yoshida, Yoshinori Kawabe, Makoto Motono, Hiroyuki Watanabe, Takashi Yamashita, Ken-Ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Production of human erythropoietin by chimeric chickens., Biochemical and biophysical research communications, 10.1016/j.bbrc.2008.01.020, 367, 4, 834-9, 2008.03, The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells..
22. Kodama D, Nishimiya D, Iwata K, Yamaguchi K, Yoshida K, Kawabe Y, Motono M, Watanabe H, Yamashita T, Nishijima K, Kamihira M, Iijima S., Production of human erythropoietin by chimeric chickens., Biochem Biophys Res Commun. , 2008 Mar 21;367(4):834-9. Epub 2008 Jan 15., 2008.05.
23. Kyogoku K, Yoshida K, Watanabe H, Yamashita T, Kawabe Y, Motono M, Nishijima K, Kamihira M, Iijima S, Production of recombinant TNFR/Fc fusion protein by genetically manipulated chickens, Journal of Bioscience and Bioengineering, doi:10.1263/jbb.105.454, 105, 5, 454-459, 2008.05.
24. Kenji Kyogoku, Kazuhiro Yoshida, Hiroyuki Watanabe, Takashi Yamashita, Kawabe Yoshinori, Makoto Motono, Ken Ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens, Journal of Bioscience and Bioengineering, 10.1263/jbb.105.454, 105, 5, 454-459, 2008.05, [URL], We previously reported the production of recombinant proteins using genetically manipulated chickens and quails. In this study, we constructed a retroviral vector encoding an expression cassette for a fusion protein of the extracellular domain of the human tumor necrosis factor (TNF) receptor 2 and Fc region of human IgG1 (TNFR/Fc), which is expected as an effective drug for inflammatory diseases such as rheumatoid arthritis. The concentrated viral vector was injected into developing chicken embryos. The chickens that hatched stably produced TNFR/Fc in the serum and egg yolk for six months. It appears that the fused protein is transported and accumulated into yolk from the serum, which is mediated by the Fc receptor. The protein purified from the yolk and serum inhibited the cytotoxic activity of TNF-α toward L929 cells, indicating that the protein produced by the chickens is biologically active. These results indicate the effectiveness of the recovery of Fc-fused proteins from the yolk of genetically manipulated chickens..
25. Kenji Kyogoku, Kazuhiro Yoshida, Hiroyuki Watanabe, Takashi Yamashita, Yoshinori Kawabe, Makoto Motono, Ken-Ichi Nishijima, Masamichi Kamihira, Shinji Iijima, Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens., Journal of bioscience and bioengineering, 10.1263/jbb.105.454, 105, 5, 454-9, 2008.05, We previously reported the production of recombinant proteins using genetically manipulated chickens and quails. In this study, we constructed a retroviral vector encoding an expression cassette for a fusion protein of the extracellular domain of the human tumor necrosis factor (TNF) receptor 2 and Fc region of human IgG1 (TNFR/Fc), which is expected as an effective drug for inflammatory diseases such as rheumatoid arthritis. The concentrated viral vector was injected into developing chicken embryos. The chickens that hatched stably produced TNFR/Fc in the serum and egg yolk for six months. It appears that the fused protein is transported and accumulated into yolk from the serum, which is mediated by the Fc receptor. The protein purified from the yolk and serum inhibited the cytotoxic activity of TNF-* toward L929 cells, indicating that the protein produced by the chickens is biologically active. These results indicate the effectiveness of the recovery of Fc-fused proteins from the yolk of genetically manipulated chickens..
26. Ito A, Kiyohara T, Kawabe Y, Ijima H, Kamihira M, Enhancement of cell function through heterotypic cell-cell interactions using E-cadherin-expressing NIH3T3 cells., J Biosci Bioeng, doi:10.1263/jbb.105.679 , 105, 6, 679-682, 2008.06.
27. Kameyama Y, Kawabe Y, Ito A, Kamihira M, Antibody-dependent gene transduction using gammaretroviral and lentiviral vectors pseudotyped with chimeric vesicular stomatitis virus glycoprotein, J Virol Methods, doi:10.1016/j.jviromet.2008.06.013, 153, 1, 49-54, 2008.06.
28. Akira Ito, Takehiko Kiyohara, Kawabe Yoshinori, Hiroyuki Ijima, Masamichi Kamihira, Enhancement of cell function through heterotypic cell-cell interactions using E-cadherin-expressing NIH3T3 cells, Journal of Bioscience and Bioengineering, 10.1263/jbb.105.679, 105, 6, 679-682, 2008.06, [URL], An epithelial cell adhesion molecule, E-cadherin was expressed in NIH3T3 fibroblasts, and cell-cell interactions between keratinocytes and fibroblasts, or between hepatocytes and fibroblasts were artificially engineered. When the E-cadherin-expressing NIH3T3 cells were co-cultured with rat hepatocytes, the cell-cell contacts were formed with high frequency, and enhanced albumin secretion was observed..
29. Akira Ito, Takehiko Kiyohara, Yoshinori Kawabe, Hiroyuki Ijima, Masamichi Kamihira, Enhancement of cell function through heterotypic cell-cell interactions using E-cadherin-expressing NIH3T3 cells., Journal of bioscience and bioengineering, 10.1263/jbb.105.679, 105, 6, 679-82, 2008.06, An epithelial cell adhesion molecule, E-cadherin was expressed in NIH3T3 fibroblasts, and cell-cell interactions between keratinocytes and fibroblasts, or between hepatocytes and fibroblasts were artificially engineered. When the E-cadherin-expressing NIH3T3 cells were co-cultured with rat hepatocytes, the cell-cell contacts were formed with high frequency, and enhanced albumin secretion was observed..
30. Yujiro Kameyama, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Antibody-dependent gene transduction using gammaretroviral and lentiviral vectors pseudotyped with chimeric vesicular stomatitis virus glycoprotein, Journal of Virological Methods, 10.1016/j.jviromet.2008.06.013, 153, 1, 49-54, 2008.10, [URL], Gammaretroviral and lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) have been used for stable gene transfer because of their broad host range and high mechanical strength. In the present study, an expression plasmid for chimeric VSV-G, consisting of a ZZ fragment derived from Staphylococcal protein A fused to the N-terminus of VSV-G (ZZ-VSV-G), was constructed to produce viral vectors capable of antibody-dependent gene transduction. Gammaretroviral (based on mouse stem cell virus, MSCV) and lentiviral (based on human immunodeficiency virus type 1, HIV-1) vectors pseudotyped with ZZ-VSV-G were produced without the loss of antibody-binding activity. The production of infectious viral particles was promoted by the addition of an expression plasmid for native VSV-G and antibody-dependent gene transduction was achieved using plates coated with antibodies. This system may be useful for the genetic transduction of cells expressing specific proteins on their surface, and for screening of antibodies specific for cell surface receptors..
31. Yujiro Kameyama, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Antibody-dependent gene transduction using gammaretroviral and lentiviral vectors pseudotyped with chimeric vesicular stomatitis virus glycoprotein., Journal of virological methods, 10.1016/j.jviromet.2008.06.013, 153, 1, 49-54, 2008.10, Gammaretroviral and lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) have been used for stable gene transfer because of their broad host range and high mechanical strength. In the present study, an expression plasmid for chimeric VSV-G, consisting of a ZZ fragment derived from Staphylococcal protein A fused to the N-terminus of VSV-G (ZZ-VSV-G), was constructed to produce viral vectors capable of antibody-dependent gene transduction. Gammaretroviral (based on mouse stem cell virus, MSCV) and lentiviral (based on human immunodeficiency virus type 1, HIV-1) vectors pseudotyped with ZZ-VSV-G were produced without the loss of antibody-binding activity. The production of infectious viral particles was promoted by the addition of an expression plasmid for native VSV-G and antibody-dependent gene transduction was achieved using plates coated with antibodies. This system may be useful for the genetic transduction of cells expressing specific proteins on their surface, and for screening of antibodies specific for cell surface receptors..
32. Kawabe Y, Naka T, Komatsu H, Nishijima K, Iijima S, Kamihira M., Retroviral gene transduction into chicken embryo gonads through blood circulation, J Biosci Bioeng, 106(6):598-601, 2008.12.
33. Kawabe Yoshinori, Tsutomu Naka, Hiroyuki Komatsu, Ken Ichi Nishijima, Shinji Iijima, Masamichi Kamihira, Retroviral Gene Transduction into Chicken Embryo Gonads through Blood Circulation, Journal of Bioscience and Bioengineering, 10.1263/jbb.106.598, 106, 6, 598-601, 2008.12, [URL], A retroviral vector was injected into the heart of developing chicken embryos to determine the best timing for effective gene transduction into the gonads. Transduction efficiency and transgene expression were both highest when the viral solution was injected into embryos at stages 14-15 (Hamburger-Hamilton stages), in which primordial germ cells (PGCs) can be found in the blood circulation. This study shows that PGCs in the blood are important targets for gene transfer into the gonads using retroviral vectors..
34. Yoshinori Kawabe, Tsutomu Naka, Hiroyuki Komatsu, Ken-ichi Nishijima, Shinji Iijima, Masamichi Kamihira, Retroviral gene transduction into chicken embryo gonads through blood circulation., Journal of bioscience and bioengineering, 10.1263/jbb.106.598, 106, 6, 598-601, 2008.12, A retroviral vector was injected into the heart of developing chicken embryos to determine the best timing for effective gene transduction into the gonads. Transduction efficiency and transgene expression were both highest when the viral solution was injected into embryos at stages 14-15 (Hamburger-Hamilton stages), in which primordial germ cells (PGCs) can be found in the blood circulation. This study shows that PGCs in the blood are important targets for gene transfer into the gonads using retroviral vectors..
35. Hirokazu Akiyama, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Fabrication of complex three-dimensional tissue architectures using a magnetic force-based cell patterning technique, Biomedical Microdevices, 10.1007/s10544-009-9284-x, 11, 4, 713-721, 2009, [URL], We describe the fabrication of three-dimensional tissue constructs using a magnetic force-based tissue engineering technique, in which cellular organization is controlled by magnetic force. Target cells were labeled with magnetite cationic liposomes (MCLs) so that the MCL-labeled cells could be manipulated by applying a magnetic field. Line patterning of human umbilical vein endothelial cells (HUVECs) labeled with MCLs was successfully created on monolayer cells or skin tissues using a magnetic concentrator device. Multilayered cell sheets were also inducible on a culture surface by accumulating MCL-labeled cells under a uniform magnetic force. Based on these results, we attempted to construct a complex multilayered myoblast C2C12 cell sheet. Here, patterned HUVECs were embedded by alternating the processes of magnetic accumulation of C2C12 cells for cell layer formation and magnetic patterning of HUVECs on the cell layers. This technique may be applicable for the fabrication of complex tissue architectures required in tissue engineering..
36. Akira Ito, Hirokazu Akiyama, Yasunori Yamamoto, Kawabe Yoshinori, Masamichi Kamihira, Skeletal muscle tissue engineering using functional magnetite nanoparticles, 20th Anniversary MHS 2009 and Micro-Nano Global COE - 2009 International Symposium on Micro-NanoMechatronics and Human Science
20th Anniversary MHS 2009 and Micro-Nano Global COE - 2009 International Symposium on Micro-NanoMechatronics and Human Science
, 10.1109/MHS.2009.5351986, 379-382, 2009, [URL], Skeletal muscular tissues were constructed using magnetic force-based tissue engineering (Mag-TE) techniques. Mouse myoblast C2C12 cells labeled with magnetite cationic liposomes (MCLs) were seeded into a well of 24-well ultra-low cell attachment culture plates. When a magnet was positioned underneath the well, cells accumulated evenly onto the culture surface and formed a multilayered cell sheet. Furthermore, because an angiogenic potential of transplants is considered to be important for the long-term maintenance of cell survival and tissue functions, a vascular endothelial growth factor (VEGF) gene-modified C2C12 (C2C12/VEGF) cell sheets were also fabricated by the Mag-TE technique. The secretion level of C2C12/VEGF sheets was 3.0 ng/day, indicating that VEGF gene-expressing cell sheets were successfully fabricated. Since the shape of artificial tissue constructs can be controlled by magnetic force, a cellular string-like assembly was formed by placing a linear-shaped magnetic field concentrator with a magnet. These cellular sheets and strings shrank and did not maintain their shapes for an additional in vitro culture period during myogenic differentiation. On the other hand, when a silicone plug was positioned at the center of well during the fabrication of cell sheets, the cell sheets shrank and formed a ring-like assembly around the plug. After 6-d cultivation of cell rings in differentiation medium, the C2C12 cells differentiated to form multinucleated myotubes. Thus, these procedures can provide a novel strategy for skeletal muscular tissue engineering..
37. Akira Ito, Tetsuya Takahashi, Yujiro Kameyama, Kawabe Yoshinori, Masamichi Kamihira, Magnetic concentration of a retroviral vector using magnetite cationic liposomes, Tissue Engineering - Part C: Methods, 10.1089/ten.tec.2008.0275, 15, 1, 57-64, 2009.03, [URL], For tissue engineering purposes, retroviral vectors represent an efficient method of delivering exogenous genes such as growth factors to injured tissues because gene-transduced cells can produce stable and constant levels of the gene product. However, retroviral vector technology suffers from low yields. In the present study, we used magnetite nanoparticles and magnetic force to concentrate the retroviral vectors to enhance the transduction efficiency and to enable their magnetic manipulation. Magnetite nanoparticles modified with cationic liposomes were added to a solution containing a retroviral vector pseudotyped with vesicular stomatitis virus glycoprotein. The magnetic particles that captured the viral vectors were collected using a magnetic force and seeded into mouse neuroblastoma Neuro2a cells. The viral titer was up to 55 times greater (up to 3 × 108 infectious units/mL). Additionally, the magnetically labeled retroviral vectors can be directed to the desired regions for infection by applying magnetic fields, and micro-patterns of gene-transduced cell regions could be created on a cellular monolayer using micro-patterned magnetic concentrators. These results suggest that this technique provides a promising approach to capturing and concentrating viral vectors, thus achieving high transduction efficiency and the ability to deliver genes to a specific injured site by applying a magnetic field..
38. Akira Ito, Tetsuya Takahashi, Yujiro Kameyama, Yoshinori Kawabe, Masamichi Kamihira, Magnetic concentration of a retroviral vector using magnetite cationic liposomes., Tissue engineering. Part C, Methods, 10.1089/ten.tec.2008.0275, 15, 1, 57-64, 2009.03, For tissue engineering purposes, retroviral vectors represent an efficient method of delivering exogenous genes such as growth factors to injured tissues because gene-transduced cells can produce stable and constant levels of the gene product. However, retroviral vector technology suffers from low yields. In the present study, we used magnetite nanoparticles and magnetic force to concentrate the retroviral vectors to enhance the transduction efficiency and to enable their magnetic manipulation. Magnetite nanoparticles modified with cationic liposomes were added to a solution containing a retroviral vector pseudotyped with vesicular stomatitis virus glycoprotein. The magnetic particles that captured the viral vectors were collected using a magnetic force and seeded into mouse neuroblastoma Neuro2a cells. The viral titer was up to 55 times greater (up to 3 x 10(8) infectious units/mL). Additionally, the magnetically labeled retroviral vectors can be directed to the desired regions for infection by applying magnetic fields, and micro-patterns of gene-transduced cell regions could be created on a cellular monolayer using micro-patterned magnetic concentrators. These results suggest that this technique provides a promising approach to capturing and concentrating viral vectors, thus achieving high transduction efficiency and the ability to deliver genes to a specific injured site by applying a magnetic field..
39. Kamihira M, Kawabe Y, Shindo T, Ono K, Esaka K, Yamashita T, Nishijima K, Iijima S, Production of chimeric monoclonal antibodies by genetically manipulated chickens, J Biotechnol. , 141(1-2):18-25., 2009.04.
40. Masamichi Kamihira, Kawabe Yoshinori, Takuya Shindo, Ken ichiro Ono, Kazuhisa Esaka, Takashi Yamashita, Ken ichi Nishijima, Shinji Iijima, Production of chimeric monoclonal antibodies by genetically manipulated chickens, Journal of Biotechnology, 10.1016/j.jbiotec.2009.02.022, 141, 1-2, 18-25, 2009.04, [URL], Genetically manipulated chickens producing chimeric monoclonal antibodies were generated by injecting retroviral vectors encoding genes for the heavy and light chains of antibodies into developing embryos. The transgene was detected in all chickens that hatched, and they stably produced the chimeric antibodies in their serum. After sexual maturation, the antibodies were also produced in eggs laid by the manipulated hens. The stable antibody production was observed both in egg white and yolk throughout the breeding period. The chimeric antibodies produced by the chickens were properly assembled and exhibited antigen-binding activities. Furthermore, we characterized the structures of the N-linked oligosaccharide chains added to the Fc-region of the recombinant antibodies produced in the serum, egg white and yolk of the chickens..
41. Masamichi Kamihira, Yoshinori Kawabe, Takuya Shindo, Ken-ichiro Ono, Kazuhisa Esaka, Takashi Yamashita, Ken-ichi Nishijima, Shinji Iijima, Production of chimeric monoclonal antibodies by genetically manipulated chickens., Journal of biotechnology, 10.1016/j.jbiotec.2009.02.022, 141, 1-2, 18-25, 2009.04, Genetically manipulated chickens producing chimeric monoclonal antibodies were generated by injecting retroviral vectors encoding genes for the heavy and light chains of antibodies into developing embryos. The transgene was detected in all chickens that hatched, and they stably produced the chimeric antibodies in their serum. After sexual maturation, the antibodies were also produced in eggs laid by the manipulated hens. The stable antibody production was observed both in egg white and yolk throughout the breeding period. The chimeric antibodies produced by the chickens were properly assembled and exhibited antigen-binding activities. Furthermore, we characterized the structures of the N-linked oligosaccharide chains added to the Fc-region of the recombinant antibodies produced in the serum, egg white and yolk of the chickens..
42. Ito A, Takahashi T, Kameyama Y, Kawabe Y, Kamihira M., Magnetic concentration of a retroviral vector using magnetite cationic liposomes., Tissue Eng Part C Methods. , 15(1):57-64., 2009.05.
43. Akiyama H, Ito A, Kawabe Y, Kamihira M., Fabrication of complex three-dimensional tissue architectures using a magnetic force-based cell patterning technique, Biomed Microdevices. , 11(4):713-21., 2009.08.
44. Hirokazu Akiyama, Akira Ito, Yoshinori Kawabe, Masamichi Kamihira, Fabrication of complex three-dimensional tissue architectures using a magnetic force-based cell patterning technique., Biomedical microdevices, 10.1007/s10544-009-9284-x, 11, 4, 713-21, 2009.08, We describe the fabrication of three-dimensional tissue constructs using a magnetic force-based tissue engineering technique, in which cellular organization is controlled by magnetic force. Target cells were labeled with magnetite cationic liposomes (MCLs) so that the MCL-labeled cells could be manipulated by applying a magnetic field. Line patterning of human umbilical vein endothelial cells (HUVECs) labeled with MCLs was successfully created on monolayer cells or skin tissues using a magnetic concentrator device. Multilayered cell sheets were also inducible on a culture surface by accumulating MCL-labeled cells under a uniform magnetic force. Based on these results, we attempted to construct a complex multilayered myoblast C2C12 cell sheet. Here, patterned HUVECs were embedded by alternating the processes of magnetic accumulation of C2C12 cells for cell layer formation and magnetic patterning of HUVECs on the cell layers. This technique may be applicable for the fabrication of complex tissue architectures required in tissue engineering..
45. Ito A, Jitsunobu H, Kawabe Y, Ijima H, Kamihira M., Magnetic separation of cells in coculture systems using magnetite cationic liposomes, Tissue Eng Part C Methods., 15(3):413-23., 2009.09.
46. Ito A, Takahashi T, Kawabe Y, Kamihira M., Human beta defensin-3 engineered keratinocyte sheets constructed by a magnetic force-based tissue engineering technique., J Biosci Bioeng. , 108(3):244-7., 2009.09.
47. Akira Ito, Tetsuya Takahashi, Kawabe Yoshinori, Masamichi Kamihira, Human beta defensin-3 engineered keratinocyte sheets constructed by a magnetic force-based tissue engineering technique, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2009.04.004, 108, 3, 244-247, 2009.09, [URL], A multilayered keratinocyte sheet overexpressing human beta defensin-3 (HBD-3) gene was prepared by the magnetic force-based tissue engineering technique. The cell sheet demonstrated significant antimicrobial activity, indicating that the therapeutic introduction of HBD-3 gene into cell sheets may provide a new gene therapy strategy for infectious diseases..
48. Akira Ito, Hideaki Jitsunobu, Kawabe Yoshinori, Hiroyuki Ijima, Masamichi Kamihira, Magnetic separation of cells in coculture systems using magnetite cationic liposomes., Tissue Engineering - Part C: Methods, 10.1089/ten.tec.2008.0496, 15, 3, 413-423, 2009.09, [URL], In tissue engineering, coculture systems have been employed for two major purposes: (1) construction of tissue and organ substitutes (e.g., coculture of parenchymal and nonparenchymal cells in liver tissue engineering) and (2) maintenance of cellular functions (e.g., coculture of embryonic stem cells with embryonic fibroblasts as the feeder cells). For the characterization and recovery of specific cell types, however, target cells have to be isolated from other cells. We report here a novel magnetic separation method to isolate target cells in coculture systems. In this method, target cells were cocultured with nontarget cells labeled with magnetite cationic liposomes (MCLs). Thus, when necessary, the MCL-labeled nontarget cells were magnetically removed from the coculture, resulting in negative isolation of the target cells. As the separation models, three deferent types of coculture systems were examined: rat hepatocytes with various MCL-labeled cells (mouse NIH3T3, STO, or human umbilical vein endothelial cells), human keratinocyte HaCaT cells with MCL-labeled NIH3T3 cells, and mouse embryonic stem cells with MCL-labeled STO cells. In these cocultures, target cells were separated with 94% purity and 98%recovery yield on average. This technique provides a promising approach to isolate and recover target cells for further analysis and application..
49. Akira Ito, Tetsuya Takahashi, Yoshinori Kawabe, Masamichi Kamihira, Human beta defensin-3 engineered keratinocyte sheets constructed by a magnetic force-based tissue engineering technique., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2009.04.004, 108, 3, 244-7, 2009.09, A multilayered keratinocyte sheet overexpressing human beta defensin-3 (HBD-3) gene was prepared by the magnetic force-based tissue engineering technique. The cell sheet demonstrated significant antimicrobial activity, indicating that the therapeutic introduction of HBD-3 gene into cell sheets may provide a new gene therapy strategy for infectious diseases..
50. Akira Ito, Hideaki Jitsunobu, Yoshinori Kawabe, Hiroyuki Ijima, Masamichi Kamihira, Magnetic separation of cells in coculture systems using magnetite cationic liposomes., Tissue engineering. Part C, Methods, 10.1089/ten.tec.2008.0496, 15, 3, 413-23, 2009.09, In tissue engineering, coculture systems have been employed for two major purposes: (1) construction of tissue and organ substitutes (e.g., coculture of parenchymal and nonparenchymal cells in liver tissue engineering) and (2) maintenance of cellular functions (e.g., coculture of embryonic stem cells with embryonic fibroblasts as the feeder cells). For the characterization and recovery of specific cell types, however, target cells have to be isolated from other cells. We report here a novel magnetic separation method to isolate target cells in coculture systems. In this method, target cells were cocultured with nontarget cells labeled with magnetite cationic liposomes (MCLs). Thus, when necessary, the MCL-labeled nontarget cells were magnetically removed from the coculture, resulting in negative isolation of the target cells. As the separation models, three deferent types of coculture systems were examined: rat hepatocytes with various MCL-labeled cells (mouse NIH3T3, STO, or human umbilical vein endothelial cells), human keratinocyte HaCaT cells with MCL-labeled NIH3T3 cells, and mouse embryonic stem cells with MCL-labeled STO cells. In these cocultures, target cells were separated with 94% purity and 98%recovery yield on average. This technique provides a promising approach to isolate and recover target cells for further analysis and application..
51. Ken-ichi Nishijima, Makoto Motono, Daisuke Kodama, Yoshinori Kawabe, Masamichi Kamihira, Shinji Iijima, Retroviral gene transfer to chickens for the production of pharmaceutical proteins into eggs, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 10.1016/j.jbiosc.2009.08.109, 108, S25-S25, 2009.11.
52. Yamamoto Y, Ito A, Kato M, Kawabe Y, Shimizu K, Fujita H, Nagamori E, Kamihira M., Preparation of artificial skeletal muscle tissues by a magnetic force-based tissue engineering technique., J Biosci Bioeng. , 108(6):538-43., 2009.12.
53. Yasunori Yamamoto, Akira Ito, Masahiro Kato, Kawabe Yoshinori, Kazunori Shimizu, Hideaki Fujita, Eiji Nagamori, Masamichi Kamihira, Preparation of artificial skeletal muscle tissues by a magnetic force-based tissue engineering technique, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2009.05.019, 108, 6, 538-543, 2009.12, [URL], Artificial muscle tissues composed of mouse myoblast C2C12 cells were prepared using a magnetic force-based tissue engineering technique. C2C12 cells labeled with magnetite nanoparticles were seeded into the wells of 24-well ultralow-attachment culture plates. When a magnet was positioned underneath each plate, the cells accumulated evenly on the culture surface and formed multilayered cell sheets. Since the shapes of artificial tissue constructs can be controlled by magnetic force, cellular string-like assemblies were formed by using a linear magnetic field concentrator with a magnet. However, the resulting cellular sheets and strings shrank considerably and did not retain their shapes during additional culture periods for myogenic differentiation. On the other hand, when a silicone plug was positioned at the center of the well during the fabrication of a cell sheet, the cell sheet shrank drastically and formed a ring-like assembly around the plug. A histological examination revealed that the cells in the cellular ring were highly oriented in the direction of the circumference by the tension generated within the structure. Individual cellular rings were hooked around two pins separated by 10 mm, and successfully cultured for 6 d without breakage. After a 6-d culture in differentiation medium, the C2C12 cells differentiated to form myogenin-positive multinucleated myotubes. Highly dense and oriented skeletal muscle tissues were obtained using this technique, suggesting that this procedure may represent a novel strategy for muscle tissue engineering..
54. Yasunori Yamamoto, Akira Ito, Masahiro Kato, Yoshinori Kawabe, Kazunori Shimizu, Hideaki Fujita, Eiji Nagamori, Masamichi Kamihira, Preparation of artificial skeletal muscle tissues by a magnetic force-based tissue engineering technique., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2009.05.019, 108, 6, 538-43, 2009.12, Artificial muscle tissues composed of mouse myoblast C2C12 cells were prepared using a magnetic force-based tissue engineering technique. C2C12 cells labeled with magnetite nanoparticles were seeded into the wells of 24-well ultralow-attachment culture plates. When a magnet was positioned underneath each plate, the cells accumulated evenly on the culture surface and formed multilayered cell sheets. Since the shapes of artificial tissue constructs can be controlled by magnetic force, cellular string-like assemblies were formed by using a linear magnetic field concentrator with a magnet. However, the resulting cellular sheets and strings shrank considerably and did not retain their shapes during additional culture periods for myogenic differentiation. On the other hand, when a silicone plug was positioned at the center of the well during the fabrication of a cell sheet, the cell sheet shrank drastically and formed a ring-like assembly around the plug. A histological examination revealed that the cells in the cellular ring were highly oriented in the direction of the circumference by the tension generated within the structure. Individual cellular rings were hooked around two pins separated by 10 mm, and successfully cultured for 6 d without breakage. After a 6-d culture in differentiation medium, the C2C12 cells differentiated to form myogenin-positive multinucleated myotubes. Highly dense and oriented skeletal muscle tissues were obtained using this technique, suggesting that this procedure may represent a novel strategy for muscle tissue engineering..
55. Akiyama H, Ito A, Kawabe Y, Kamihira M., Genetically engineered angiogenic cell sheets using magnetic force-based gene delivery and tissue fabrication techniques., Biomaterials. , 31(6):1251-9., 2010.02.
56. Hirokazu Akiyama, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Genetically engineered angiogenic cell sheets using magnetic force-based gene delivery and tissue fabrication techniques, Biomaterials, 10.1016/j.biomaterials.2009.11.017, 31, 6, 1251-1259, 2010.02, [URL], A major limitation in tissue engineering is the insufficient formation of blood vessels in implanted tissues, resulting in reduced cell density and graft size. We report here the fabrication of angiogenic cell sheets using a combination of two magnetic force-based techniques which use magnetite cationic liposomes (MCLs), magnetofection and magnetic cell accumulation. A retroviral vector encoding an expression cassette of vascular endothelial growth factor (VEGF) was labeled with MCLs, to magnetically attract the particles onto a monolayer of mouse myoblast C2C12 cells, for gene delivery. MCL-mediated infection increased transduction efficiency by 6.7-fold compared with the conventional method. During the fabrication of the tissue constructs, MCL-labeled cells were accumulated in the presence of a magnetic field to promote the spontaneous formation of a multilayered cell sheet. VEGF gene-engineered C2C12 (C2C12/VEGF) cell sheets, constructed using both magnetic force-based techniques, were subcutaneously transplanted into nude mice. Histological analyses revealed that on day 14 the C2C12/VEGF cell sheet grafts had produced thick tissues, with a high-cell density, and promoted vascularization. This suggests that the method described here represents a powerful strategy in tissue engineering..
57. Hirokazu Akiyama, Akira Ito, Yoshinori Kawabe, Masamichi Kamihira, Genetically engineered angiogenic cell sheets using magnetic force-based gene delivery and tissue fabrication techniques., Biomaterials, 10.1016/j.biomaterials.2009.11.017, 31, 6, 1251-9, 2010.02, A major limitation in tissue engineering is the insufficient formation of blood vessels in implanted tissues, resulting in reduced cell density and graft size. We report here the fabrication of angiogenic cell sheets using a combination of two magnetic force-based techniques which use magnetite cationic liposomes (MCLs), magnetofection and magnetic cell accumulation. A retroviral vector encoding an expression cassette of vascular endothelial growth factor (VEGF) was labeled with MCLs, to magnetically attract the particles onto a monolayer of mouse myoblast C2C12 cells, for gene delivery. MCL-mediated infection increased transduction efficiency by 6.7-fold compared with the conventional method. During the fabrication of the tissue constructs, MCL-labeled cells were accumulated in the presence of a magnetic field to promote the spontaneous formation of a multilayered cell sheet. VEGF gene-engineered C2C12 (C2C12/VEGF) cell sheets, constructed using both magnetic force-based techniques, were subcutaneously transplanted into nude mice. Histological analyses revealed that on day 14 the C2C12/VEGF cell sheet grafts had produced thick tissues, with a high-cell density, and promoted vascularization. This suggests that the method described here represents a powerful strategy in tissue engineering..
58. Hirokazu Akiyama, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Cell-patterning using poly (ethylene glycol)-modified magnetite nanoparticles, Journal of Biomedical Materials Research - Part A, 10.1002/jbm.a.32313, 92, 3, 1123-1130, 2010.03, [URL], Development of cell-patterning techniques is a major challenge for the construction of functional tissues and organs in tissue engineering. Recent progress in surface chemistry has enabled spatial control of cell adhesion onto cultural substrates by varying hydrophilicity, for example, by using poly (ethylene glycol) (PEG). In the present study, we developed a novel cell-patterning procedure using PEG-modified magnetite particles (PEG-Mags) and magnetic force. Using an array-patterned magnet, PEG-Mags were magnetically patterned on the surface of a tissue culture dish. The resultant substrate surface consisted of two regions: the PEG-Mag surface that acts as a cell-resistant region and the native substrate surface that promotes cell adhesion. When human keratinocyte HaCaT cells were seeded onto the PEG-Mag-patterned surface, cells adhered only to the native substrate surface, resulting in cell-patterning on the tissue culture dish. The patterned PEG-Mags were then washed away to expose the native substrate surface, and thereafter, when mouse myoblast C2C12 cells were seeded to the dish, cells adhered to the exposed substrate surface, resulting in a patterned coculture of heterotypic cells. Moreover, it is worth noting that the magnetic force-based cell-patterning procedure is not limited by the property of cultural substrate surfaces, and that cell-patterning of mouse fibroblast NIH3T3 cells on a monolayer of HaCaT cells was successfully achieved using PEG-Mags and magnetic force. These results indicate that this procedure provides a novel concept for cell-patterning and may be useful for tissue engineering and cell biology..
59. Hirokazu Akiyama, Akira Ito, Yoshinori Kawabe, Masamichi Kamihira, Cell-patterning using poly (ethylene glycol)-modified magnetite nanoparticles., Journal of biomedical materials research. Part A, 10.1002/jbm.a.32313, 92, 3, 1123-30, 2010.03, Development of cell-patterning techniques is a major challenge for the construction of functional tissues and organs in tissue engineering. Recent progress in surface chemistry has enabled spatial control of cell adhesion onto cultural substrates by varying hydrophilicity, for example, by using poly (ethylene glycol) (PEG). In the present study, we developed a novel cell-patterning procedure using PEG-modified magnetite particles (PEG-Mags) and magnetic force. Using an array-patterned magnet, PEG-Mags were magnetically patterned on the surface of a tissue culture dish. The resultant substrate surface consisted of two regions: the PEG-Mag surface that acts as a cell-resistant region and the native substrate surface that promotes cell adhesion. When human keratinocyte HaCaT cells were seeded onto the PEG-Mag-patterned surface, cells adhered only to the native substrate surface, resulting in cell-patterning on the tissue culture dish. The patterned PEG-Mags were then washed away to expose the native substrate surface, and thereafter, when mouse myoblast C2C12 cells were seeded to the dish, cells adhered to the exposed substrate surface, resulting in a patterned coculture of heterotypic cells. Moreover, it is worth noting that the magnetic force-based cell-patterning procedure is not limited by the property of cultural substrate surfaces, and that cell-patterning of mouse fibroblast NIH3T3 cells on a monolayer of HaCaT cells was successfully achieved using PEG-Mags and magnetic force. These results indicate that this procedure provides a novel concept for cell-patterning and may be useful for tissue engineering and cell biology..
60. Penno CA, Kawabe Y, Ito A, Kamihira M., Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens, Transgenic Res. , 19(2):187-95., 2010.04.
61. Kameyama Y, Kawabe Y, Ito A, Kamihira M., An accumulative site-specific gene integration system using Cre recombinase-mediated cassette exchange., Biotechnol Bioeng. , 105(6):1106-14., 2010.04.
62. Yujiro Kameyama, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, An accumulative site-specific gene integration system using Cre recombinase-mediated cassette exchange, Biotechnology and Bioengineering, 10.1002/bit.22619, 105, 6, 1106-1114, 2010.04, [URL], The Cre-loxP system is frequently used for sitespecific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site-specific gene integration system using Cre recombinase and mutated loxPs in which the Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms..
63. Carlos Alberto Penno, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens, Transgenic Research, 10.1007/s11248-009-9310-z, 19, 2, 187-195, 2010.04, [URL], We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors..
64. Yujiro Kameyama, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, An accumulative site-specific gene integration system using Cre recombinase-mediated cassette exchange., Biotechnology and bioengineering, 10.1002/bit.22619, 105, 6, 1106-14, 2010.04, The Cre-loxP system is frequently used for site-specific recombination in animal cells. The equilibrium and specificity of the recombination reaction can be controlled using mutated loxPs. In the present study, we designed an accumulative site-specific gene integration system using Cre recombinase and mutated loxPs in which the Cre-mediated cassette exchange reaction is infinitely repeatable for target gene integration into loxP target sites. To evaluate the feasibility and usefulness of this system, a series of integration reactions were repeated and confirmed in vitro using Cre recombinase protein and plasmids. Accumulative gene integration was also performed on the genome of Chinese hamster ovary (CHO) cells. The results indicated that the system was applicable for repeated gene integration of multiple genes to the target sites on both plasmids and CHO cell genomes. This gene integration system provides a novel strategy for gene amplification and for biological analyses of gene function through the genetic modification of cells and organisms..
65. Carlos Alberto Penno, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens., Transgenic research, 10.1007/s11248-009-9310-z, 19, 2, 187-95, 2010.04, We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors..
66. Akiyama H, Ito A, Kawabe Y, Kamihira M., Cell-patterning using poly (ethylene glycol)-modified magnetite nanoparticles, J Biomed Mater Res A. , 92(3):1123-30., 2010.05.
67. Akiyama H, Ito A, Sato M, Kawabe Y, Kamihira M, Construction of cardiac tissue rings using a magnetic tissue fabrication technique, Int J Mol Sci , 11:2910-20, 2010.08.
68. Hirokazu Akiyama, Akira Ito, Masanori Sato, Kawabe Yoshinori, Masamichi Kamihira, Construction of cardiac tissue rings using a magnetic tissue fabrication technique, International Journal of Molecular Sciences, 10.3390/ijms11082910, 11, 8, 2910-2920, 2010.08, [URL], Here we applied a magnetic force-based tissue engineering technique to cardiac tissue fabrication. A mixture of extracellular matrix precursor and cardiomyocytes labeled with magnetic nanoparticles was added into a well containing a central polycarbonate cylinder. With the use of a magnet, the cells were attracted to the bottom of the well and allowed to form a cell layer. During cultivation, the cell layer shrank towards the cylinder, leading to the formation of a ring-shaped tissue that possessed a multilayered cell structure and contractile properties. These results indicate that magnetic tissue fabrication is a promising approach for cardiac tissue engineering..
69. Hirokazu Akiyama, Akira Ito, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, Construction of Cardiac Tissue Rings Using a Magnetic Tissue Fabrication Technique, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 10.3390/ijms11082910, 11, 8, 2910-2920, 2010.08, Here we applied a magnetic force-based tissue engineering technique to cardiac tissue fabrication. A mixture of extracellular matrix precursor and cardiomyocytes labeled with magnetic nanoparticles was added into a well containing a central polycarbonate cylinder. With the use of a magnet, the cells were attracted to the bottom of the well and allowed to form a cell layer. During cultivation, the cell layer shrank towards the cylinder, leading to the formation of a ring-shaped tissue that possessed a multilayered cell structure and contractile properties. These results indicate that magnetic tissue fabrication is a promising approach for cardiac tissue engineering..
70. Horie M, Ito A, Kiyohara T, Kawabe Y, Kamihira M, E-cadherin gene-engineered feeder systems for supporting undifferentiated growth of mouse embryonic stem cells, J Biosci Bioeng, 110:582-7, 2010.11.
71. Huang S, Kawabe Y, Ito A, Kamihira M , Cre recombinase-mediated site-specific modification of a celluar genome using an integrase-defective retroviral vector, Biotechnol Bioeng, 107: 717-29, 2010.11.
72. Shuohao Huang, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector, Biotechnology and Bioengineering, 10.1002/bit.22863, 107, 4, 717-729, 2010.11, [URL], Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors. Biotechnol. Bioeng. 2010;107:717-729..
73. Masanobu Horie, Akira Ito, Takehiko Kiyohara, Kawabe Yoshinori, Masamichi Kamihira, E-cadherin gene-engineered feeder systems for supporting undifferentiated growth of mouse embryonic stem cells, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2010.06.002, 110, 5, 582-587, 2010.11, [URL], Conventionally, embryonic stem (ES) cells are cultured on a cell layer of mouse embryonic fibroblasts (MEFs) as feeder cells to support undifferentiated growth of ES cells. In this study, cell-cell interactions between mouse ES and feeder cells were artificially engineered via an epithelial cell adhesion molecule, E-cadherin, whose expression is considerable in ES cells. Mouse mesenchymal STO and NIH3T3 cells that were genetically engineered to express E-cadherin were used in ES cell cultures as feeder cells. ES cells cultured on the E-cadherin-expressing feeder cells maintained the expression of stem cell markers, alkaline phosphatase (AP), Oct3/4, Nanog and Sox2, and the efficiency of AP-positive colony formation was comparable to MEFs, and much better than parental STO and NIH3T3 cells. Furthermore, ES cells maintained on the E-cadherin-expressing feeder cells possessed the ability to differentiate into the three germ layers both in vitro and in vivo. The results indicated that E-cadherin expression in feeder cells could improve the performance of feeder cells, which may be further applicable to create new artificial feeder cell lines..
74. Shuohao Huang, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Cre recombinase-mediated site-specific modification of a cellular genome using an integrase-defective retroviral vector., Biotechnology and bioengineering, 10.1002/bit.22863, 107, 4, 717-29, 2010.11, Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors..
75. Masanobu Horie, Akira Ito, Takehiko Kiyohara, Yoshinori Kawabe, Masamichi Kamihira, E-cadherin gene-engineered feeder systems for supporting undifferentiated growth of mouse embryonic stem cells., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2010.06.002, 110, 5, 582-7, 2010.11, Conventionally, embryonic stem (ES) cells are cultured on a cell layer of mouse embryonic fibroblasts (MEFs) as feeder cells to support undifferentiated growth of ES cells. In this study, cell-cell interactions between mouse ES and feeder cells were artificially engineered via an epithelial cell adhesion molecule, E-cadherin, whose expression is considerable in ES cells. Mouse mesenchymal STO and NIH3T3 cells that were genetically engineered to express E-cadherin were used in ES cell cultures as feeder cells. ES cells cultured on the E-cadherin-expressing feeder cells maintained the expression of stem cell markers, alkaline phosphatase (AP), Oct3/4, Nanog and Sox2, and the efficiency of AP-positive colony formation was comparable to MEFs, and much better than parental STO and NIH3T3 cells. Furthermore, ES cells maintained on the E-cadherin-expressing feeder cells possessed the ability to differentiate into the three germ layers both in vitro and in vivo. The results indicated that E-cadherin expression in feeder cells could improve the performance of feeder cells, which may be further applicable to create new artificial feeder cell lines..
76. Yamamoto Y, Ito A, Fujita H, Nagamori E, Kawabe Y, Kamihira M, Functional evaluation of artificial skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique., Tissue Eng Part A., 17(1-2):107-14., 2011.01.
77. Yasunori Yamamoto, Akira Ito, Hideaki Fujita, Eiji Nagamori, Kawabe Yoshinori, Masamichi Kamihira, Functional evaluation of artificial skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique, Tissue Engineering - Part A., 10.1089/ten.tea.2010.0312, 17, 1-2, 107-114, 2011.01, [URL], Skeletal muscle tissue engineering is currently applied in a variety of research fields, including regenerative medicine, drug screening, and bioactuator development, all of which require the fabrication of biomimic and functional skeletal muscle tissues. In the present study, magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs. Histological studies revealed that elongated and multinucleated myotubes were observed within the tissue. Expression of muscle-specific markers, such as myogenin, myosin heavy chain and tropomyosin, were detected in the tissue constructs by western blot analysis. Further, creatine kinase activity increased during differentiation. In response to electric pulses, the artificial tissue constructs contracted to generate a physical force (the maximum twitch force, 33.2μN [1.06mN/mm2]). Rheobase and chronaxie of the tissue were determined as 4.45V and 0.72ms, respectively. These results indicate that the artificial skeletal muscle tissue constructs fabricated in this study were physiologically functional and the data obtained for the evaluation of their functional properties may provide useful information for future skeletal muscle tissue engineering studies..
78. Yasunori Yamamoto, Akira Ito, Hideaki Fujita, Eiji Nagamori, Yoshinori Kawabe, Masamichi Kamihira, Functional Evaluation of Artificial Skeletal Muscle Tissue Constructs Fabricated by a Magnetic Force-Based Tissue Engineering Technique, TISSUE ENGINEERING PART A, 10.1089/ten.tea.2010.0312, 17, 1-2, 107-114, 2011.01, Skeletal muscle tissue engineering is currently applied in a variety of research fields, including regenerative medicine, drug screening, and bioactuator development, all of which require the fabrication of biomimic and functional skeletal muscle tissues. In the present study, magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs. Histological studies revealed that elongated and multinucleated myotubes were observed within the tissue. Expression of muscle-specific markers, such as myogenin, myosin heavy chain and tropomyosin, were detected in the tissue constructs by western blot analysis. Further, creatine kinase activity increased during differentiation. In response to electric pulses, the artificial tissue constructs contracted to generate a physical force (the maximum twitch force, 33.2 mu N [1.06 mN/mm(2)]). Rheobase and chronaxie of the tissue were determined as 4.45V and 0.72 ms, respectively. These results indicate that the artificial skeletal muscle tissue constructs fabricated in this study were physiologically functional and the data obtained for the evaluation of their functional properties may provide useful information for future skeletal muscle tissue engineering studies..
79. Yasunori Yamamoto, Akira Ito, Hideaki Fujita, Eiji Nagamori, Yoshinori Kawabe, Masamichi Kamihira, Functional evaluation of artificial skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique., Tissue engineering. Part A, 10.1089/ten.TEA.2010.0312, 17, 1-2, 107-14, 2011.01, Skeletal muscle tissue engineering is currently applied in a variety of research fields, including regenerative medicine, drug screening, and bioactuator development, all of which require the fabrication of biomimic and functional skeletal muscle tissues. In the present study, magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs. Histological studies revealed that elongated and multinucleated myotubes were observed within the tissue. Expression of muscle-specific markers, such as myogenin, myosin heavy chain and tropomyosin, were detected in the tissue constructs by western blot analysis. Further, creatine kinase activity increased during differentiation. In response to electric pulses, the artificial tissue constructs contracted to generate a physical force (the maximum twitch force, 33.2 μN [1.06 mN/mm2]). Rheobase and chronaxie of the tissue were determined as 4.45 V and 0.72 ms, respectively. These results indicate that the artificial skeletal muscle tissue constructs fabricated in this study were physiologically functional and the data obtained for the evaluation of their functional properties may provide useful information for future skeletal muscle tissue engineering studies..
80. Masanobu Horie, Akira Ito, Takeshi Maki, Kawabe Yoshinori, Masamichi Kamihira, Magnetic separation of cells from developing embryoid bodies using magnetite cationic liposomes, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.04.011, 112, 2, 184-187, 2011.08, [URL], Embryoid bodies resemble post-implantation egg-cylinder stage embryos and are used to differentiate embryonic stem cells in vitro. In this study, we enriched mouse vasa homolog-positive germ cells from embryoid bodies after 8. d of differentiation using a magnetic separation method with magnetite cationic liposomes..
81. Masanobu Horie, Akira Ito, Takeshi Maki, Yoshinori Kawabe, Masamichi Kamihira, Magnetic separation of cells from developing embryoid bodies using magnetite cationic liposomes., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2011.04.011, 112, 2, 184-7, 2011.08, Embryoid bodies resemble post-implantation egg-cylinder stage embryos and are used to differentiate embryonic stem cells in vitro. In this study, we enriched mouse vasa homolog-positive germ cells from embryoid bodies after 8d of differentiation using a magnetic separation method with magnetite cationic liposomes..
82. Sato M, Ito A, Kawabe Y, Kamihira M, Enhanced contractile force generation by artificial skeletal muscle tissues using IGF-I gene-engineered myoblast cells., J Biosci Bioeng, doi:10.1016/j.jbiosc.2011.05.007, 112, 3, 273-278, 2011.09.
83. Horie M, Ito A, Kawabe Y, Kamihira M, A genetically engineered STO feeder system expressing E-cadherin and leukemia inhibitory factor for mouse pluripotent stem cell culture., J Bioproces Biotechniq, doi:10.4172/2155-9821.S3-001, S3, 001, 2011.09.
84. Masanori Sato, Akira Ito, Kawabe Yoshinori, Eiji Nagamori, Masamichi Kamihira, Enhanced contractile force generation by artificial skeletal muscle tissues using IGF-I gene-engineered myoblast cells, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.05.007, 112, 3, 273-278, 2011.09, [URL], The aim of this study was to investigate whether insulin-like growth factor (IGF)-I gene delivery to myoblast cells promotes the contractile force generated by hydrogel-based tissue-engineered skeletal muscles in vitro. Two retroviral vectors allowing doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into mouse myoblast C2C12 cells to evaluate the effects of IGF-I gene expression on these cells. IGF-I gene expression stimulated the proliferation of C2C12 cells, and a significant increase in the growth rate was observed for IGF-I-transduced C2C12 cells with Dox addition, designated C2C12/IGF (Dox+) cells. Quantitative morphometric analyses showed that the myotubes induced from C2C12/IGF (Dox+) cells had a larger area and a greater width than control myotubes induced from normal C2C12 cells. Artificial skeletal muscle tissues were prepared from the respective cells using hydrogels composed of type I collagen and Matrigel. Western blot analyses revealed that the C2C12/IGF (Dox+) tissue constructs showed activation of a skeletal muscle hypertrophy marker (Akt) and enhanced expression of muscle-specific markers (myogenin, myosin heavy chain and tropomyosin). Moreover, the creatine kinase activity was increased in the C2C12/IGF (Dox+) tissue constructs. The C2C12/IGF (Dox+) tissue constructs contracted in response to electrical pulses, and generated a significantly higher physical force than the control C2C12 tissue constructs. These findings indicate that IGF-I gene transfer has the potential to yield functional skeletal muscle substitutes that are capable of in vivo restoration of the load-bearing function of injured muscle or acting as in vitro electrically-controlled bio-actuators..
85. Masanori Sato, Akira Ito, Yoshinori Kawabe, Eiji Nagamori, Masamichi Kamihira, Enhanced contractile force generation by artificial skeletal muscle tissues using IGF-I gene-engineered myoblast cells., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2011.05.007, 112, 3, 273-8, 2011.09, The aim of this study was to investigate whether insulin-like growth factor (IGF)-I gene delivery to myoblast cells promotes the contractile force generated by hydrogel-based tissue-engineered skeletal muscles in vitro. Two retroviral vectors allowing doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into mouse myoblast C2C12 cells to evaluate the effects of IGF-I gene expression on these cells. IGF-I gene expression stimulated the proliferation of C2C12 cells, and a significant increase in the growth rate was observed for IGF-I-transduced C2C12 cells with Dox addition, designated C2C12/IGF (Dox+) cells. Quantitative morphometric analyses showed that the myotubes induced from C2C12/IGF (Dox+) cells had a larger area and a greater width than control myotubes induced from normal C2C12 cells. Artificial skeletal muscle tissues were prepared from the respective cells using hydrogels composed of type I collagen and Matrigel. Western blot analyses revealed that the C2C12/IGF (Dox+) tissue constructs showed activation of a skeletal muscle hypertrophy marker (Akt) and enhanced expression of muscle-specific markers (myogenin, myosin heavy chain and tropomyosin). Moreover, the creatine kinase activity was increased in the C2C12/IGF (Dox+) tissue constructs. The C2C12/IGF (Dox+) tissue constructs contracted in response to electrical pulses, and generated a significantly higher physical force than the control C2C12 tissue constructs. These findings indicate that IGF-I gene transfer has the potential to yield functional skeletal muscle substitutes that are capable of in vivo restoration of the load-bearing function of injured muscle or acting as in vitro electrically-controlled bio-actuators..
86. Yamamoto H, Kawabe Y, Ito A, Kamihira M, Enhanced liver functions in mouse hepatoma cells by induced overexpression of liver-enriched transcription factors., Biochem Eng J, doi:10.1016/j.bej.2011.10.004, 60, 15, 67-73, 2012.01.
87. Huang S, Kawabe Y, Ito A, Kamihira M, Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19., Biochem Biophys Res Commun, doi:10.1016/j.bbrc.2011.11.059, 417, 1, 78-83, 2012.01.
88. Shuohao Huang, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2011.11.059, 417, 1, 78-83, 2012.01, [URL], Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site..
89. Hideaki Yamamoto, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Enhanced liver functions in mouse hepatoma cells by induced overexpression of liver-enriched transcription factors, Biochemical Engineering Journal, 10.1016/j.bej.2011.10.004, 60, 67-73, 2012.01, [URL], Hepatoma cells, which are derived from liver carcinoma, are able to proliferate infinitely under culture conditions. However, the liver functions of hepatoma cells are generally low compared with those of hepatocytes in a liver. Here, we attempted to create genetically engineered hepatoma cells with enhanced liver functions by overexpression of liver-enriched transcription factors (LETFs), which are associated with the transcription of liver-specific genes and hepatic differentiation. For this purpose, genes for eight LETFs, hepatocyte nuclear factor (HNF)-1α, HNF-1β, HNF-3β, HNF-4α, HNF-6, CCAAT/enhancer binding protein (C/EBP)-α, C/EBP-β and C/EBP-γ, were obtained from the mouse liver. Mouse hepatoma Hepa1-6 cells were transduced with retroviral vectors, in which inducible expression cassettes for the LETF genes were introduced. Cell clones with inducible expression of high liver functions were established. Upon overexpression of the LETF genes, cell proliferation ceased and the cells exhibited an epithelial morphology, indicating hepatic maturation of hepatoma cells. This approach for genetic modification of hepatoma cells may be promising for the construction of cells for use in bioartificial liver support systems..
90. Yasunori Yamamoto, Akira Ito, Hideaki Jitsunobu, Katsuya Yamaguchi, Kawabe Yoshinori, Hiroshi Mizumoto, Masamichi Kamihira, Hollow fiber bioreactor perfusion culture system for magnetic force-based skeletal muscle tissue engineering, Journal of Chemical Engineering of Japan, 10.1252/jcej.11we237, 45, 5, 348-354, 2012.01, [URL], Large-scale skeletal muscle tissue cultures are often limited by nutrient supplementation and oxygen diffusion. In the present study, we used a hollow-fiber bioreactor system to supply nutrients and oxygen for the cultivation of high cell-density skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique. C2C12 cells, magnetically-labeled with magnetite cationic liposomes (MCLs), were mixed with a type I collagen solution and seeded into the cell culture space of the hollow-fiber bioreactor. A magnet was then placed underneath the bioreactor to accumulate MCL-labeled cells in the space between the hollow fibers by magnetic force. Perfusion culture was performed using a myogenic differentiation medium for 7 d. Histological observation revealed that high cell-dense and viable tissue constructs containing myotubes were successfully formed. Furthermore, muscle-specific proteins, such as myosin heavy chain and tropomyosin, were detected by western blot, indicating that C2C12 cells underwent myogenic differentiation. These findings indicate that the hollow-fiber bioreactor system is an effective approach for the in vitro culture of large skeletal muscle tissue constructs, fabricated by magnetic force-based tissue engineering. © 2012 The Society of Chemical Engineer, Japan..
91. Shuohao Huang, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19., Biochemical and biophysical research communications, 10.1016/j.bbrc.2011.11.059, 417, 1, 78-83, 2012.01, Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site..
92. Hideaki Yamamoto, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Enhanced liver functions in mouse hepatoma cells by induced overexpression of liver-enriched transcription factors, BIOCHEMICAL ENGINEERING JOURNAL, 10.1016/j.bej.2011.10.004, 60, 67-73, 2012.01, Hepatoma cells, which are derived from liver carcinoma, are able to proliferate infinitely under culture conditions. However, the liver functions of hepatoma cells are generally low compared with those of hepatocytes in a liver. Here, we attempted to create genetically engineered hepatoma cells with enhanced liver functions by overexpression of liver-enriched transcription factors (LETFs), which are associated with the transcription of liver-specific genes and hepatic differentiation. For this purpose, genes for eight LETFs, hepatocyte nuclear factor (HNF)-1 alpha, HNF-1 beta, HNF-3 beta, HNF-4 alpha, HNF-6, CCAAT/enhancer binding protein (C/EBP)-alpha, C/EBP-beta and C/EBP-gamma, were obtained from the mouse liver. Mouse hepatoma Hepa1-6 cells were transduced with retroviral vectors, in which inducible expression cassettes for the LETF genes were introduced. Cell clones with inducible expression of high liver functions were established. Upon overexpression of the LETF genes, cell proliferation ceased and the cells exhibited an epithelial morphology, indicating hepatic maturation of hepatoma cells. This approach for genetic modification of hepatoma cells may be promising for the construction of cells for use in bioartificial liver support systems. (C) 2011 Elsevier B.V. All rights reserved..
93. Kodama D, Nishimiya D, Nishijima K, Okino Y, Inayoshi Y, Ono K, Motono M, Miyake K, Kawabe Y, Kyogoku K, Yamashita T, Kamihira M, Iijima S, Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system., J Biosci Bioeng, doi:10.1016/j.jbiosc.2011.10.006, 113, 2, 146-153, 2012.02.
94. Daisuke Kodama, Daisuke Nishimiya, Ken ichi Nishijima, Yuuki Okino, Yujin Inayoshi, Yasuhiro Kojima, Ken ichiro Ono, Makoto Motono, Katsuhide Miyake, Kawabe Yoshinori, Kenji Kyogoku, Takashi Yamashita, Masamichi Kamihira, Shinji Iijima, Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.10.006, 113, 2, 146-153, 2012.02, [URL], We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to - 3861. bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning - 3861 to - 1569. bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately - 1800. bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (- 2112 to - 1569. bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene..
95. Daisuke Kodama, Daisuke Nishimiya, Ken-Ichi Nishijima, Yuuki Okino, Yujin Inayoshi, Yasuhiro Kojima, Ken-Ichiro Ono, Makoto Motono, Katsuhide Miyake, Yoshinori Kawabe, Kenji Kyogoku, Takashi Yamashita, Masamichi Kamihira, Shinji Iijima, Chicken oviduct-specific expression of transgene by a hybrid ovalbumin enhancer and the Tet expression system., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2011.10.006, 113, 2, 146-53, 2012.02, We generated genetically manipulated chickens and quail by infecting them with a retroviral vector expressing the human growth hormone under the control of chicken ovalbumin promoter/enhancer up to -3861 bp from the transcriptional start site. The growth hormone was expressed in an oviduct-specific manner and was found in egg white, although its level was low. The DNA sequence of the integrated form of the viral vector in the packaging cells was shown to be truncated and contained only the sequence spanning -3861 to -1569 bp. This represented only the DNase I hypersensitive site (DHS) III of the 4 DHSs and lacked the proximal promoter of the ovalbumin control region. We found several TATA-like and other promoter motifs of approximately -1800 bp and considered that these promoter motifs and DHS III may cause weak but oviduct-specific expression of the growth hormone. To prove this hypothesis and apply this system to oviduct-specific expression of the transgene, the truncated regulatory sequence was fused to an artificial transactivator-promoter system. In this system, initial weak but oviduct-specific expression of the Tet activator from the promoter element in the ovalbumin control sequence triggered a self-amplifying cycle of expression. DsRed was specifically expressed in oviduct cells of genetically manipulated chickens using this system. Furthermore, deletion of a short region possibly containing the promoter elements (-2112 to -1569 bp) completely abrogated oviduct-specific expression. Taken together, these results suggest that weak expression of this putative promoter causes oviduct-specific expression of the transgene..
96. Hirokazu Obayashi, Kawabe Yoshinori, Hirokatsu Makitsubo, Ryoko Watanabe, Yujiro Kameyama, Shuohao Huang, Yuta Takenouchi, Akira Ito, Masamichi Kamihira, Accumulative gene integration into a pre-determined site using Cre/loxP, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.10.027, 113, 3, 381-388, 2012.03, [URL], Site-specific gene recombination systems, such as Cre/. loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes..
97. Hirokazu Obayashi, Yoshinori Kawabe, Hirokatsu Makitsubo, Ryoko Watanabe, Yujiro Kameyama, Shuohao Huang, Yuta Takenouchi, Akira Ito, Masamichi Kamihira, Accumulative gene integration into a pre-determined site using Cre/loxP., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2011.10.027, 113, 3, 381-8, 2012.03, Site-specific gene recombination systems, such as Cre/loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes..
98. Yamamoto Y, Ito A, Jitsunobu H, Yamaguchi K, Kawabe Y, Mizumoto H, Kamihira M, Hollow fiber bioreactor perfusion culture system for magnetic force-based skeletal muscle tissue engineering., J Chem Eng Japan, doi: 10.1252/jcej.11we237, 45, 5, 1-7, 2012.05.
99. Obayashi H, Kawabe Y, Makitsubo H, Watanabe R, Kameyama Y, Huang S, Takenouchi Y, Ito A, Kamihira M, Accumulative gene integration into a pre-determined site using Cre/loxP., J Biosci Bioeng, doi:10.1016/j.jbiosc.2011.10.027, 113, 3, 381-388, 2012.05.
100. Kawabe Yoshinori, Makitsubo H, Kameyama Y, Huang S, Akira Ito, Masamichi Kamihira, Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system., Cytotechnology, DOI: 10.1007/s10616-011-9397-y, 64(3)、267-279, 2012.05.
101. Kawabe Yoshinori, Hirokatsu Makitsubo, Yujiro Kameyama, Shuohao Huang, Akira Ito, Masamichi Kamihira, Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system, Cytotechnology, 10.1007/s10616-011-9397-y, 64, 3, 267-279, 2012.05, [URL], We previously reported an accumulative site-specific gene integration system using Cre recombinase and mutated loxP sites, where a recombinasemediated cassette exchange (RMCE) reaction is repeatable. This gene integration system was applied for antibody production using recombinant Chinese hamster ovary (CHO) cells. We introduced an exchange cassette flanked by wild-type and mutated loxP sites into the chromosome of CHO cells for the establishment of recipient founder cells. Then, the donor plasmids including an expression cassette for an antibody gene flanked by a compatible pair of loxP sites were prepared. The donor plasmid and a Cre expression vector were co-transfected into the founder CHO cells to give rise to RMCE in the CHO genome, resulting in site-specific integration of the antibody gene. The RMCE procedure was repeated to increase the copy numbers of the integrated gene. Southern blot and genomic PCR analyses for the established cells revealed that the transgenes were integrated into the target site. Antibody production determined by ELISA and western blotting was increased corresponding to the number of transgenes. These results indicate that the accumulative site-specific gene integration system could provide a useful tool for increasing the productivity of recombinant proteins..
102. Yoshinori Kawabe, Hirokatsu Makitsubo, Yujiro Kameyama, Shuohao Huang, Akira Ito, Masamichi Kamihira, Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system., Cytotechnology, 10.1007/s10616-011-9397-y, 64, 3, 267-79, 2012.05, We previously reported an accumulative site-specific gene integration system using Cre recombinase and mutated loxP sites, where a recombinase-mediated cassette exchange (RMCE) reaction is repeatable. This gene integration system was applied for antibody production using recombinant Chinese hamster ovary (CHO) cells. We introduced an exchange cassette flanked by wild-type and mutated loxP sites into the chromosome of CHO cells for the establishment of recipient founder cells. Then, the donor plasmids including an expression cassette for an antibody gene flanked by a compatible pair of loxP sites were prepared. The donor plasmid and a Cre expression vector were co-transfected into the founder CHO cells to give rise to RMCE in the CHO genome, resulting in site-specific integration of the antibody gene. The RMCE procedure was repeated to increase the copy numbers of the integrated gene. Southern blot and genomic PCR analyses for the established cells revealed that the transgenes were integrated into the target site. Antibody production determined by ELISA and western blotting was increased corresponding to the number of transgenes. These results indicate that the accumulative site-specific gene integration system could provide a useful tool for increasing the productivity of recombinant proteins..
103. Akira Ito, Okamoto Noriaki, Yamaguchi Masaki, Kawabe Yoshinori, Masamichi Kamihira, Heat-inducible transgene expression with transcriptional amplification mediated by a transactivator, Int J Hyperthermia, doi: 10.3109/02656736.2012.738847, 28(8):788-98, 2012.08.
104. Yamaguchi M, Ito A, Okamoto N, Kawabe Y, Kamihira M, Heat-inducible transgene expression system incorporating a positive feedback loop of transcriptional amplification for hyperthermia-induced gene therapy, J Biosci Bioeng, http://dx.doi.org/10.1016/j.jbiosc.2012.05.006, 114, 4, 460-465, 2012.10.
105. Ito A, Yamaguchi M, Okamoto N, Sanematsu Y, Kawabe Y, Wakamatsu K, Ito S, Honda H, Kobayashi T, Nakayama E, Tamura Y, Okura M, Yamashita T, Jimbow K, Kamihira M, T-cell receptor repertoires of tumor-infiltrating lymphocytes after hyperthermia using functionalized magnetite nanoparticles, Nanomedicine (Lond), doi: 10.2217/nnm.12.142, 2012.10.
106. Kawabe Y, Hayashida Y, Numata K, Harada S, Hayashida Y, Ito A, Kamihira M , Oral immunotherapy for pollen allergy using T-cell epitope-containing egg white derived from genetically manipulated chickens, PLOS ONE, doi:10.1371/journal.pone.0048512, 7, e48512, 2012.10.
107. Masaki Yamaguchi, Akira Ito, Noriaki Okamoto, Kawabe Yoshinori, Masamichi Kamihira, Heat-inducible transgene expression system incorporating a positive feedback loop of transcriptional amplification for hyperthermia-induced gene therapy, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.05.006, 114, 4, 460-465, 2012.10, [URL], One of the major goals of gene therapy is to regulate the expression of therapeutic genes in desired cells or tissues. For this purpose, heat-inducible vectors have been exploited for cancer gene therapy combined with hyperthermia, which can result in considerable improvement of therapeutic effects. In the present study, we constructed a novel heat-inducible gene expression system incorporating a transactivation system with a positive feedback loop of transcriptional amplification. The target gene expression mediated by the transactivator under the control of a heat shock protein 70B' promoter is enhanced by self-promoted transactivator gene expression. This expression system showed tight control of target gene expression together with high-level expression; enhanced expression of the reporter gene was observed in transfected cells upon heat treatment, while negligible gene expression was detected in non-heated cells. When a therapeutic gene was used as the target gene, a considerable cytotoxic effect was observed after heat treatment of cancer cells transfected with the plasmids. The heat-induced transgene expression system is a promising new approach for the development of both a safe and effective vector for hyperthermia-based cancer gene therapy..
108. Masaki Yamaguchi, Akira Ito, Noriaki Okamoto, Yoshinori Kawabe, Masamichi Kamihira, Heat-inducible transgene expression system incorporating a positive feedback loop of transcriptional amplification for hyperthermia-induced gene therapy., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2012.05.006, 114, 4, 460-5, 2012.10, One of the major goals of gene therapy is to regulate the expression of therapeutic genes in desired cells or tissues. For this purpose, heat-inducible vectors have been exploited for cancer gene therapy combined with hyperthermia, which can result in considerable improvement of therapeutic effects. In the present study, we constructed a novel heat-inducible gene expression system incorporating a transactivation system with a positive feedback loop of transcriptional amplification. The target gene expression mediated by the transactivator under the control of a heat shock protein 70B' promoter is enhanced by self-promoted transactivator gene expression. This expression system showed tight control of target gene expression together with high-level expression; enhanced expression of the reporter gene was observed in transfected cells upon heat treatment, while negligible gene expression was detected in non-heated cells. When a therapeutic gene was used as the target gene, a considerable cytotoxic effect was observed after heat treatment of cancer cells transfected with the plasmids. The heat-induced transgene expression system is a promising new approach for the development of both a safe and effective vector for hyperthermia-based cancer gene therapy..
109. Akira Ito, Noriaki Okamoto, Masaki Yamaguchi, Kawabe Yoshinori, Masamichi Kamihira, Heat-inducible transgene expression with transcriptional amplification mediated by a transactivator, International Journal of Hyperthermia, 10.3109/02656736.2012.738847, 28, 8, 788-798, 2012.12, [URL], Purpose: Control of therapeutic gene expression in tumours is a major goal of gene therapy research, as it can restrict cytotoxic gene expression in cancer cells. In addition, the combination of hyperthermia with gene therapy through the application of heat-inducible vectors can result in considerable improvements in therapeutic efficiency. In this study, to combine heat-inducibility with high-level transgene expression, we developed a heat-inducible transgene expression system with transcriptional amplification mediated by a tetracycline-responsive transactivator. Materials and methods: A hybrid promoter was generated by placing the heat shock protein (HSP) 70B′ promoter under the tetracycline-repressor responsive element sequence, and a reporter/therapeutic gene expression plasmid was constructed by placing a reporter/therapeutic gene under the control of this hybrid promoter. Results: When the transactivator expression plasmid harbouring an expression cassette of the tetracycline-responsive transactivator gene was co-transfected with a reporter gene expression plasmid, the reporter gene expression was controlled by heat treatment. With this system, high levels of heat-induced transgene expression were observed compared to that from the HSP promoter alone without the transactivator. Evaluation of in vitro therapeutic effects using cancer cell lines revealed that therapeutic gene expression effectively caused cell death in a greater percentage of the cells. Conclusion: These findings indicate that this strategy improves the efficacy of cancer gene therapy..
110. Akira Ito, Masaki Yamaguchi, Noriaki Okamoto, Yuji Sanematsu, Kawabe Yoshinori, Kazumasa Wakamatsu, Shosuke Ito, Hiroyuki Honda, Takeshi Kobayashi, Eiichi Nakayama, Yasuaki Tamura, Masae Okura, Toshiharu Yamashita, Kowichi Jimbow, Masamichi Kamihira, T-cell receptor repertoires of tumor-infiltrating lymphocytes after hyperthermia using functionalized magnetite nanoparticles, Nanomedicine, 10.2217/nnm.12.142, 8, 6, 891-902, 2013, [URL], Aim: Accumulating evidence has indicated that hyperthermia using magnetite nanoparticles induces antitumor immunity. This study investigated the diversity of T-cell receptors (TCRs) in tumor-infiltrating lymphocytes after hyperthermia using magnetite nanoparticles.Materials & methods: Functionalized magnetite nanoparticles, N-propionyl-4-S-cysteaminylphenol (NPrCAP)/magnetite, were synthesized by conjugating the melanogenesis substrate NPrCAP with magnetite nanoparticles. NPrCAP/magnetite nanoparticles were injected into B16 melanomas in C57BL/6 mice, which were subjected to an alternating magnetic field for hyperthermia treatment. Results: Enlargement of the tumor-draining lymph nodes was observed after hyperthermia. The TCR repertoire was restricted in tumor-infiltrating lymphocytes, and expansion of Vβ11+ T cells was preferentially found. DNA sequences of the third complementaritydetermining regions revealed the presence of clonally expanded T cells. Conclusion: These results indicate that the T-cell response in B16 melanomas after hyperthermia is dominated by T cells directed toward a limited number of epitopes and that epitope-specific T cells frequently use a restricted TCR repertoire..
111. Sato Masanori, Akira Ito, Akiyama H, Kawabe Yoshinori, Masamichi Kamihira, Effects of Bcl-2 Gene Transfer to Myoblast Cells on Skeletal Muscle Tissue Formation Using Magnetic Force-based Tissue Engineering, Tissue Eng Part A, doi:10.1089/ten.TEA.2011.0728, 19(1-2):307-15, 2013.01.
112. Masanori Sato, Akira Ito, Hirokazu Akiyama, Kawabe Yoshinori, Masamichi Kamihira, Effects of B-cell lymphoma 2 gene transfer to myoblast cells on skeletal muscle tissue formation using magnetic force-based tissue engineering, Tissue Engineering - Part A., 10.1089/ten.tea.2011.0728, 19, 1-2, 307-315, 2013.01, [URL], Tissue-engineered skeletal muscle should possess a high cell-dense structure with unidirectional cell alignment. However, limited nutrient and/or oxygen supply within the artificial tissue constructs might restrict cell viability and muscular functions. In this study, we genetically modified myoblast cells with the anti-apoptotic B-cell lymphoma 2 (Bcl-2) gene and evaluated their function in artificial skeletal muscle tissue constructs. Magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of skeletal muscle bundles by applying a magnetic force. Bcl-2-overexpressing muscle bundles formed highly cell-dense and viable tissue constructs, while muscle bundles without Bcl-2 overexpression exhibited substantial necrosis/apoptosis at the central region of the bundle. Bcl-2-overexpressing muscle bundles contracted in response to electrical pulses and generated a significantly higher physical force. These findings indicate that the incorporation of anti-apoptotic gene-transduced myoblast cells into tissue constructs significantly enhances skeletal muscle formation and function..
113. Masanori Sato, Akira Ito, Hirokazu Akiyama, Yoshinori Kawabe, Masamichi Kamihira, Effects of B-cell lymphoma 2 gene transfer to myoblast cells on skeletal muscle tissue formation using magnetic force-based tissue engineering, Tissue Engineering - Part A, 10.1089/ten.tea.2011.0728, 19, 1-2, 307-315, 2013.01, Tissue-engineered skeletal muscle should possess a high cell-dense structure with unidirectional cell alignment. However, limited nutrient and/or oxygen supply within the artificial tissue constructs might restrict cell viability and muscular functions. In this study, we genetically modified myoblast cells with the anti-apoptotic B-cell lymphoma 2 (Bcl-2) gene and evaluated their function in artificial skeletal muscle tissue constructs. Magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of skeletal muscle bundles by applying a magnetic force. Bcl-2-overexpressing muscle bundles formed highly cell-dense and viable tissue constructs, while muscle bundles without Bcl-2 overexpression exhibited substantial necrosis/apoptosis at the central region of the bundle. Bcl-2-overexpressing muscle bundles contracted in response to electrical pulses and generated a significantly higher physical force. These findings indicate that the incorporation of anti-apoptotic gene-transduced myoblast cells into tissue constructs significantly enhances skeletal muscle formation and function. © Mary Ann Liebert, Inc..
114. Masanori Sato, Akira Ito, Hirokazu Akiyama, Yoshinori Kawabe, Masamichi Kamihira, Effects of B-cell lymphoma 2 gene transfer to myoblast cells on skeletal muscle tissue formation using magnetic force-based tissue engineering., Tissue engineering. Part A, 10.1089/ten.TEA.2011.0728, 19, 1-2, 307-15, 2013.01, Tissue-engineered skeletal muscle should possess a high cell-dense structure with unidirectional cell alignment. However, limited nutrient and/or oxygen supply within the artificial tissue constructs might restrict cell viability and muscular functions. In this study, we genetically modified myoblast cells with the anti-apoptotic B-cell lymphoma 2 (Bcl-2) gene and evaluated their function in artificial skeletal muscle tissue constructs. Magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of skeletal muscle bundles by applying a magnetic force. Bcl-2-overexpressing muscle bundles formed highly cell-dense and viable tissue constructs, while muscle bundles without Bcl-2 overexpression exhibited substantial necrosis/apoptosis at the central region of the bundle. Bcl-2-overexpressing muscle bundles contracted in response to electrical pulses and generated a significantly higher physical force. These findings indicate that the incorporation of anti-apoptotic gene-transduced myoblast cells into tissue constructs significantly enhances skeletal muscle formation and function..
115. Akira Ito, Masaki Yamaguchi, Noriaki Okamoto, Yuji Sanematsu, Yoshinori Kawabe, Kazumasa Wakamatsu, Shosuke Ito, Hiroyuki Honda, Takeshi Kobayashi, Eiichi Nakayama, Yasuaki Tamura, Masae Okura, Toshiharu Yamashita, Kowichi Jimbow, Masamichi Kamihira, T-cell receptor repertoires of tumor-infiltrating lymphocytes after hyperthermia using functionalized magnetite nanoparticles, NANOMEDICINE, 10.2217/NNM.12.142, 8, 6, 891-902, 2013.06, Aim: Accumulating evidence has indicated that hyperthermia using magnetite nanoparticles induces anti-tumor immunity. This study investigated the diversity of T-cell receptors (TCRs) in tumor-infiltrating lymphocytes after hyperthermia using magnetite nanoparticles. Materials & methods: Functionalized magnetite nanoparticles, N-propionyl-4-S-cysteaminylphenol (NPrCAP)/magnetite, were synthesized by conjugating the melanogenesis substrate NPrCAP with magnetite nanoparticles. NPrCAP/magnetite nanoparticles were injected into B16 melanomas in C57BL/6 mice, which were subjected to an alternating magnetic field for hyperthermia treatment. Results: Enlargement of the tumor-draining lymph nodes was observed after hyperthermia. The TCR repertoire was restricted in tumor-infiltrating lymphocytes, and expansion of V beta 11(+) T cells was preferentially found. DNA sequences of the third complementarity-determining regions revealed the presence of clonally expanded T cells. Conclusion: These results indicate that the T-cell response in B16 melanomas after hyperthermia is dominated by T cells directed toward a limited number of epitopes and that epitope-specific T cells frequently use a restricted TCR repertoire..
116. Masaki Yamaguchi, Akira Ito, Akihiko Ono, Kawabe Yoshinori, Masamichi Kamihira, Heat-Inducible Gene Expression System by Applying Alternating Magnetic Field to Magnetic Nanoparticles., ACS Synth Biol, DOI: 10.1021/sb4000838, in press, 2013.10.
117. Kazuhiro Yoshida, Yuya Okuzaki, Ken Ichi Nishijima, Kenji Kyogoku, Takashi Yamashita, Kawabe Yoshinori, Makoto Motono, Masamichi Kamihira, Shinji Iijima, Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan, Cytotechnology, 10.1007/s10616-013-9613-z, 65, 6, 985-992, 2013.12, [URL], The transgenic chicken is a candidate for the production of biopharmaceutical proteins with several economic superiorities. In general, the addition of sialic acid at the terminal of N-glycan is important for the bioactivity of biopharmaceuticals including plasma half-life; however, sialic acid has not been detected in the N-glycan of proteins produced in the egg white of genetically manipulated chickens. In this study, the extracellular domain of the TNF receptor and single chain Fv fused to Fc (referred to as TNFR/Fc and scFv/Fc, respectively) were purified from the egg yolk of genetically manipulated chickens and their sialylation in N-glycan was examined. In contrast to the glycan in egg white, yolk-derived proteins were partly sialylated. Lectin blot showed the existence of α2,6-sialic acid on TNFR/Fc, which disappeared with the removal of N-glycan by PNGase. In scFv/Fc, up to 7 % of N-glycan contained sialic acid. Disialyl glycans, which were detected in serum-derived scFv/Fc in a previous study, were not found in the yolk sample. Ovarian follicular tissue, which surrounds growing yolk, expressed several neuraminidases, suggesting the partial truncation of glycan during the yolk transfer process from the blood..
118. Kazuhiro Yoshida, Yuya Okuzaki, Ken-Ichi Nishijima, Kenji Kyogoku, Takashi Yamashita, Yoshinori Kawabe, Makoto Motono, Masamichi Kamihira, Shinji Iijima, Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan., Cytotechnology, 10.1007/s10616-013-9613-z, 65, 6, 985-92, 2013.12, The transgenic chicken is a candidate for the production of biopharmaceutical proteins with several economic superiorities. In general, the addition of sialic acid at the terminal of N-glycan is important for the bioactivity of biopharmaceuticals including plasma half-life; however, sialic acid has not been detected in the N-glycan of proteins produced in the egg white of genetically manipulated chickens. In this study, the extracellular domain of the TNF receptor and single chain Fv fused to Fc (referred to as TNFR/Fc and scFv/Fc, respectively) were purified from the egg yolk of genetically manipulated chickens and their sialylation in N-glycan was examined. In contrast to the glycan in egg white, yolk-derived proteins were partly sialylated. Lectin blot showed the existence of α2,6-sialic acid on TNFR/Fc, which disappeared with the removal of N-glycan by PNGase. In scFv/Fc, up to 7 % of N-glycan contained sialic acid. Disialyl glycans, which were detected in serum-derived scFv/Fc in a previous study, were not found in the yolk sample. Ovarian follicular tissue, which surrounds growing yolk, expressed several neuraminidases, suggesting the partial truncation of glycan during the yolk transfer process from the blood..
119. Masanori Sato, Kazushi Ikeda, Shota Kanno, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Enhancement of contractile force generation of artificial skeletal muscle tissues by mild and transient heat treatment, Current Pharmaceutical Biotechnology, 10.2174/1389201015666140408125231, 14, 13, 1083-1087, 2014, [URL], Artificial skeletal muscle tissues composed of cells are expected to be used for applications of regenerative medicine and drug screening. Generally, however, the physical forces generated by tissue-engineered skeletal muscle are lower than those of skeletal muscle tissues found in the body. Local hyperthermia is used for many diseases including muscle injuries. It was recently reported that mild heat treatment improved skeletal muscle functions. In this study, we investigated the effects of mild heat treatment on the tissue-engineered skeletal muscle tissues in vitro. We used magnetite cationic liposomes to label C2C12 myoblast cells magnetically, and constructed densely packed artificial skeletal muscle tissues by using magnetic force. Cell culture at 39°C promoted the differentiation of myoblast cells into myotubes. Moreover, the mild and transient heat treatment improved the contractile properties of artificial skeletal muscle tissue constructs. These findings indicate that the culture method using heat treatment is a useful approach to enhance functions of artificial skeletal muscle tissue..
120. Akira Ito, Yasunori Yamamoto, Masanori Sato, Kazushi Ikeda, Masahiro Yamamoto, Hideaki Fujita, Eiji Nagamori, Yoshinori Kawabe, Masamichi Kamihira, Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation, Sci Rep, doi: 10.1038/srep04781., 24, 4, 4781, 2014.04.
121. Akira Ito, Yasunori Yamamoto, Masanori Sato, Kazushi Ikeda, Masahiro Yamamoto, Hideaki Fujita, Eiji Nagamori, Kawabe Yoshinori, Masamichi Kamihira, Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation, Scientific Reports, 10.1038/srep04781, 4, 2014.04, [URL], Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3â €...V/mm amplitude, 4â €...ms width, and 1â €...Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50-60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs..
122. Akira Ito, Yasunori Yamamoto, Masanori Sato, Kazushi Ikeda, Masahiro Yamamoto, Hideaki Fujita, Eiji Nagamori, Yoshinori Kawabe, Masamichi Kamihira, Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation., Scientific reports, 10.1038/srep04781, 4, 4781-4781, 2014.04, Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3 V/mm amplitude, 4 ms width, and 1 Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50-60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs..
123. Masaki Yamaguchi, Akira Ito, Akihiko Ono, Kawabe Yoshinori, Masamichi Kamihira, Heat-inducible gene expression system by applying alternating magnetic field to magnetic nanoparticles, ACS Synthetic Biology, 10.1021/sb4000838, 3, 5, 273-279, 2014.05, [URL], By combining synthetic biology with nanotechnology, we demonstrate remote controlled gene expression using a magnetic field. Magnetite nanoparticles, which generate heat under an alternating magnetic field, have been developed to label cells. Magnetite nanoparticles and heat-induced therapeutic genes were introduced into tumor xenografts. The magnetically triggered gene expression resulted in tumor growth inhibition. This system shows great potential for controlling target gene expression in a space and time selective manner and may be used for remote control of cell functions via gene expression..
124. Masaki Yamaguchi, Akira Ito, Akihiko Ono, Yoshinori Kawabe, Masamichi Kamihira, Heat-inducible gene expression system by applying alternating magnetic field to magnetic nanoparticles., ACS synthetic biology, 10.1021/sb4000838, 3, 5, 273-9, 2014.05, By combining synthetic biology with nanotechnology, we demonstrate remote controlled gene expression using a magnetic field. Magnetite nanoparticles, which generate heat under an alternating magnetic field, have been developed to label cells. Magnetite nanoparticles and heat-induced therapeutic genes were introduced into tumor xenografts. The magnetically triggered gene expression resulted in tumor growth inhibition. This system shows great potential for controlling target gene expression in a space and time selective manner and may be used for remote control of cell functions via gene expression..
125. Akira Ito, Masahiro Yamamoto, Kazushi Ikeda, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, Effects of type IV collagen on myogenic characteristics of IGF-I gene-engineered myoblasts., J Biosci Bioeng, doi: 10.1016/j.jbiosc.2014.10.008. , 119, 5, 596-603, 2014.11.
126. Akira Ito, Masahiro Yamamoto, Kazushi Ikeda, Masanori Sato, Kawabe Yoshinori, Masamichi Kamihira, Effects of type IV collagen on myogenic characteristics of IGF-I gene-engineered myoblasts, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2014.10.008, 119, 5, 596-603, 2015.01, [URL], Skeletal muscle regeneration requires migration, proliferation and fusion of myoblasts to form multinucleated myotubes. In our previous study, we showed that insulin-like growth factor (IGF)-I gene delivery stimulates the proliferation and differentiation of mouse myoblast C2C12 cells and promotes the contractile force generated by tissue-engineered skeletal muscles. The aim of this study was to investigate the effects of the extracellular matrix on IGF-I gene-engineered C2C12 cells in vitro. Retroviral vectors for doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into C2C12 cells. When cultured on a type IV collagen-coated surface, we observed significant increases in the migration speed and number of IGF-I gene-engineered C2C12 cells with Dox addition, designated as C2C12/IGF (+) cells. Co-culture of C2C12/IGF (+) cells and parental C2C12 cells, which had been cultured in differentiation medium for 3 days, greatly enhanced myotube formation. Moreover, type IV collagen supplementation promoted the fusion of C2C12/IGF (+) cells with differentiated C2C12 cells and increased the number of myotubes with striations. Myotubes formed by C2C12/IGF (+) cells cultured on type IV collagen showed a dynamic contractile activity in response to electrical pulse stimulation. These findings indicate that type IV collagen promotes skeletal muscle regeneration mediated by IGF-I-expressing myoblasts, which may have important clinical implications in the design of myoblast-based therapies..
127. Masanobu Horie, Akira Ito, Takeshi Maki, Kawabe Yoshinori, Masamichi Kamihira, Magnetically labeled feeder system for mouse pluripotent stem cell culture, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2014.10.020, 119, 5, 614-616, 2015.01, [URL], We report here a magnetically labeled feeder system for mouse embryonic stem/induced pluripotent stem (ES/iPS) cells. Magnetic attraction of feeder cells labeled with magnetite nanoparticles significantly increased ES/iPS colony-forming efficiency. Magnetic labeling of feeder cells also facilitated separation of ES/iPS cells from feeder cells..
128. Masanobu Horie, Akira Ito, Takeshi Maki, Yoshinori Kawabe, Masamichi Kamihira, Magnetically labeled feeder system for mouse pluripotent stem cell culture, J Biosci Bioeng, doi: 10.1016/j.jbiosc.2014.10.020. , 119, 5, 614-616, 2015.05.
129. Akira Ito, Masahiro Yamamoto, Kazushi Ikeda, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, Effects of type IV collagen on myogenic characteristics of IGF-I gene-engineered myoblasts., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2014.10.008, 119, 5, 596-603, 2015.05, Skeletal muscle regeneration requires migration, proliferation and fusion of myoblasts to form multinucleated myotubes. In our previous study, we showed that insulin-like growth factor (IGF)-I gene delivery stimulates the proliferation and differentiation of mouse myoblast C2C12 cells and promotes the contractile force generated by tissue-engineered skeletal muscles. The aim of this study was to investigate the effects of the extracellular matrix on IGF-I gene-engineered C2C12 cells in vitro. Retroviral vectors for doxycycline (Dox)-inducible expression of the IGF-I gene were transduced into C2C12 cells. When cultured on a type IV collagen-coated surface, we observed significant increases in the migration speed and number of IGF-I gene-engineered C2C12 cells with Dox addition, designated as C2C12/IGF (+) cells. Co-culture of C2C12/IGF (+) cells and parental C2C12 cells, which had been cultured in differentiation medium for 3 days, greatly enhanced myotube formation. Moreover, type IV collagen supplementation promoted the fusion of C2C12/IGF (+) cells with differentiated C2C12 cells and increased the number of myotubes with striations. Myotubes formed by C2C12/IGF (+) cells cultured on type IV collagen showed a dynamic contractile activity in response to electrical pulse stimulation. These findings indicate that type IV collagen promotes skeletal muscle regeneration mediated by IGF-I-expressing myoblasts, which may have important clinical implications in the design of myoblast-based therapies..
130. Masanobu Horie, Akira Ito, Takeshi Maki, Yoshinori Kawabe, Masamichi Kamihira, Magnetically labeled feeder system for mouse pluripotent stem cell culture., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2014.10.020, 119, 5, 614-6, 2015.05, We report here a magnetically labeled feeder system for mouse embryonic stem/induced pluripotent stem (ES/iPS) cells. Magnetic attraction of feeder cells labeled with magnetite nanoparticles significantly increased ES/iPS colony-forming efficiency. Magnetic labeling of feeder cells also facilitated separation of ES/iPS cells from feeder cells..
131. Kazushi Ikeda, Akira Ito, Masanori Sato, Shota Kanno, Yoshinori Kawabe, Masamichi Kamihira, Effects of heat stimulation and l-ascorbic acid 2-phosphate supplementation on myogenic differentiation of artificial skeletal muscle tissue constructs., J Tissue Eng Regen Med., 10.1002/term.2030, 11, 1322-1331, 2015.06.
132. Takanori Inao, Yoshinori Kawabe, Takuro Yamashiro, Yujiro Kameyama, Xue Wang, Akira Ito, Masamichi Kamihira, Improved transgene integration into the Chinese hamster ovary cell genome using the Cre-loxP system, J Biosci Bioeng, 10.1016/j.jbiosc.2014.11.019., 120, 1, 99-106, 2015.07.
133. Takanori Inao, Kawabe Yoshinori, Takuro Yamashiro, Yujiro Kameyama, Xue Wang, Akira Ito, Masamichi Kamihira, Improved transgene integration into the Chinese hamster ovary cell genome using the Cre-loxP system, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2014.11.019, 120, 1, 99-106, 2015.07, [URL], Genetic engineering of cellular genomes has provided useful tools for biomedical and pharmaceutical studies such as the generation of transgenic animals and producer cells of biopharmaceutical proteins. Gene integration using site-specific recombinases enables precise transgene insertion into predetermined genomic sites if the target site sequence is introduced into a specific chromosomal locus. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre and mutated loxPs. The system enabled the repeated integration of multiple transgenes into a predetermined locus of a genome. In this study, we explored applicable mutated loxP pairs for AGIS to improve the integration efficiency. The integration efficiencies of 52 mutated loxP sequences, including novel sequences, were measured using an invitro evaluation system. Among mutated loxP pairs that exhibited a high integration efficiency, the applicability of the selected pairs to AGIS was confirmed for transgene integration into the Chinese hamster ovary cell genome. The newly found mutated loxP pairs should be useful for Cre-mediated integration of transgenes and AGIS..
134. Takanori Inao, Yoshinori Kawabe, Takuro Yamashiro, Yujiro Kameyama, Xue Wang, Akira Ito, Masamichi Kamihira, Improved transgene integration into the Chinese hamster ovary cell genome using the Cre-loxP system., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2014.11.019, 120, 1, 99-106, 2015.07, Genetic engineering of cellular genomes has provided useful tools for biomedical and pharmaceutical studies such as the generation of transgenic animals and producer cells of biopharmaceutical proteins. Gene integration using site-specific recombinases enables precise transgene insertion into predetermined genomic sites if the target site sequence is introduced into a specific chromosomal locus. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre and mutated loxPs. The system enabled the repeated integration of multiple transgenes into a predetermined locus of a genome. In this study, we explored applicable mutated loxP pairs for AGIS to improve the integration efficiency. The integration efficiencies of 52 mutated loxP sequences, including novel sequences, were measured using an in vitro evaluation system. Among mutated loxP pairs that exhibited a high integration efficiency, the applicability of the selected pairs to AGIS was confirmed for transgene integration into the Chinese hamster ovary cell genome. The newly found mutated loxP pairs should be useful for Cre-mediated integration of transgenes and AGIS..
135. Akihiko Ono, Akira Ito, Taiga Suzuki, Masaki Yamaguchi, Yoshinori Kawabe, Masamichi Kamihira, DNA damage-responsive transgene expression mediated by the p53 promoter with transcriptional amplification, J Biosci Bioeng. , doi: 10.1016/j.jbiosc.2015.02.009., 120(4):463-6, 2015.10.
136. Tetsushi Sakuma, Mitsumasa Takenaga, Yoshinori Kawabe, Takahiro Nakamura, Masamichi Kamihira, Takeshi Yamamoto, Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids., Int J Mol Sci., 10.3390/ijms161023849., 16(10):23849-66., 2015.10.
137. Akihiko Ono, Akira Ito, Taiga Suzuki, Masaki Yamaguchi, Kawabe Yoshinori, Masamichi Kamihira, DNA damage-responsive transgene expression mediated by the p53 promoter with transcriptional amplification, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2015.02.009, 120, 4, 463-466, 2015.10, [URL], We constructed a DNA damage-responsive transgene expression system mediated by the p53 promoter. We incorporated a transactivation system to generate transcriptional amplification via a positive feedback loop. Higher levels of DNA damage-responsive transgene expression were observed when transactivation was active..
138. Tetsushi Sakuma, Mitsumasa Takenaga, Kawabe Yoshinori, Takahiro Nakamura, Masamichi Kamihira, Takashi Yamamoto, Homologous recombination-independent large gene cassette knock-in in CHO cells using TALEN and MMEJ-directed donor plasmids, International Journal of Molecular Sciences, 10.3390/ijms161023849, 16, 10, 23849-23866, 2015.10, [URL], Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins..
139. Akihiko Ono, Akira Ito, Taiga Suzuki, Masaki Yamaguchi, Yoshinori Kawabe, Masamichi Kamihira, DNA damage-responsive transgene expression mediated by the p53 promoter with transcriptional amplification., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2015.02.009, 120, 4, 463-6, 2015.10, We constructed a DNA damage-responsive transgene expression system mediated by the p53 promoter. We incorporated a transactivation system to generate transcriptional amplification via a positive feedback loop. Higher levels of DNA damage-responsive transgene expression were observed when transactivation was active..
140. Tetsushi Sakuma, Mitsumasa Takenaga, Yoshinori Kawabe, Takahiro Nakamura, Masamichi Kamihira, Takashi Yamamoto, Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids., International journal of molecular sciences, 10.3390/ijms161023849, 16, 10, 23849-66, 2015.10, Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins..
141. Momoko Kitaoka, Yoko Shin, Noriho Kamiya, Yoshinori Kawabe, Masamichi Kamihira, Masahiro Goto, Transcutaneous Peptide Immunotherapy of Japanese Cedar Pollinosis Using Solid-in-Oil Nanodispersion Technology., AAPS PharmSciTech, 10.1208/s12249-015-0333-x, 16(6):1418-24, 2015.12.
142. Yoshinori Kawabe, Takanori Inao, Shodai Komatsu, Akira Ito, Masamichi Kamihira, Cre-mediated cellular modification for establishing producer CHO cells of recombinant scFv-Fc, BMC Proceedings, 10.1186/1753-6561-9-S9-P5, 9(Suppl 9):P5 , 2015.12.
143. Momoko Kitaoka, Yoko Shin, Noriho Kamiya, Yoshinori Kawabe, Masamichi Kamihira, Masahiro Goto, Transcutaneous Peptide Immunotherapy of Japanese Cedar Pollinosis Using Solid-in-Oil Nanodispersion Technology, AAPS PHARMSCITECH, 10.1208/s12249-015-0333-x, 16, 6, 1418-1424, 2015.12, Peptide immunotherapy is an attractive approach to relieve allergic symptoms such as rhinitis and asthma. Treatment of Japanse cedar pollinosis (Cryptomeria japonica; Cj), from which over one quarter of Japanese population suffer, is becoming a great concern. Recently, oral feeding of a peptide (7crp) consisting of seven immunodominant human T cell epitopes derived from two enzymes present in Cj pollen was demonstrated to have a benefit in treating Cj pollinosis. In this work, we aimed to apply a novel transcutaneous administration system as a simple and easy peptide delivery for an immunotherapy using a T cell epitope peptide. A modified 7crp peptide (7crpR) which contained triarginine linkers between each epitopes was designed to increase water solubility and was encapsulated in a unique solid-in-oil (S/O) nanodispersion. The S/O nanodispersion consists of a nano-sized peptide-surfactant complex dispersed in an oil vehicle. The S/O nanopartilces having an average diameter of 230 nm facilitated the permeation of the peptide 7crpR into the skin and suppressed serum total IgE and antigen-specific IgE levels in a Cj pollinosis mouse model. Transcutaneous administration of the T cell epitope peptide using the S/O nanodispersion system has potential for future simple and easy immunotherapy of Cj pollinosis..
144. Yoshinori Kawabe, Takuya Shimomura, Shuohao Huang, Suguru Imanishi, Akira Ito, Masamichi Kamihira, Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors., Biotechnol Bioeng., 10.1002/bit.25923, 113, 7, 1600-1610, 2016.01.
145. Kazushi Ikeda, Akira Ito, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, Improved contractile force generation of tissue-engineered skeletal muscle constructs by IGF-I and Bcl-2 gene transfer with electrical pulse stimulation, Regenerative Therapy, 10.1016/j.reth.2015.12.004, 3, 38-44, 2016.03, [URL], Introduction: Tissue-engineered skeletal muscle constructs should be designed to generate contractile force with directional movement. Because electrical impulses from a somatic nervous system are crucial for in vivo skeletal muscle development, electrical pulse stimulation (EPS) culture as an artificial exercise is essential to fabricate functional skeletal muscle tissues in vitro. To further improve muscle functions, the activation of cell-signaling pathways from myogenic growth factors, such as insulin-like growth factor (IGF)-I, is also important. Because tissue-engineered skeletal muscle constructs should maintain a high cell-dense structure, the expression of an anti-apoptotic factor, such as B-cell lymphoma 2 (Bcl-2), could be effective in preventing cell death. Methods: In the present study, myoblasts were genetically modified with inducible expression units of IGF-I and Bcl-2 genes, and the tissue-engineered skeletal muscle constructs fabricated from the myoblasts were cultured under continuous EPS. Results: Overexpression of IGF-I gene induced muscular hypertrophy in the muscle tissue constructs, and Bcl-2-overexpressing myoblasts formed significantly cell-dense and viable muscle tissue constructs. Furthermore, the combination of IGF-I and Bcl-2 gene transfer with EPS culture highly improved the force generation of the tissue-engineered skeletal muscle constructs. Conclusions: This approach has the potential to yield functional skeletal muscle substitutes with high force generation ability..
146. Kazushi Ikeda, Akira Ito, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, Improved contractile force generation of tissue-engineered skeletal muscle constructs by IGF-I and Bcl-2 gene transfer with electrical pulse stimulation, REGENERATIVE THERAPY, 10.1016/j.reth.2015.12.004, 3, 38-44, 2016.03, Introduction: Tissue-engineered skeletal muscle constructs should be designed to generate contractile force with directional movement. Because electrical impulses from a somatic nervous system are crucial for in vivo skeletal muscle development, electrical pulse stimulation (EPS) culture as an artificial exercise is essential to fabricate functional skeletal muscle tissues in vitro. To further improve muscle functions, the activation of cell-signaling pathways from myogenic growth factors, such as insulin-like growth factor (IGF)-I, is also important. Because tissue-engineered skeletal muscle constructs should maintain a high cell-dense structure, the expression of an anti-apoptotic factor, such as B-cell lymphoma 2 (Bcl-2), could be effective in preventing cell death.Methods: In the present study, myoblasts were genetically modified with inducible expression units of IGF-I and Bcl-2 genes, and the tissue-engineered skeletal muscle constructs fabricated from the myoblasts were cultured under continuous EPS.Results: Overexpression of IGF-I gene induced muscular hypertrophy in the muscle tissue constructs, and Bcl-2-overexpressing myoblasts formed significantly cell-dense and viable muscle tissue constructs. Furthermore, the combination of IGF-I and Bcl-2 gene transfer with EPS culture highly improved the force generation of the tissue-engineered skeletal muscle constructs.Conclusions: This approach has the potential to yield functional skeletal muscle substitutes with high force generation ability. (c) 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V..
147. Kawabe Yoshinori, Takuya Shimomura, Shuohao Huang, Suguru Imanishi, Akira Ito, Masamichi Kamihira, Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors, Biotechnology and Bioengineering, 10.1002/bit.25923, 113, 7, 1600-1610, 2016.07, [URL], Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre-based cell engineering..
148. Yoshinori Kawabe, Takuya Shimomura, Shuohao Huang, Suguru Imanishi, Akira Ito, Masamichi Kamihira, Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors., Biotechnology and bioengineering, 10.1002/bit.25923, 113, 7, 1600-10, 2016.07, Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre-based cell engineering. Biotechnol. Bioeng. 2016;113: 1600-1610. © 2016 Wiley Periodicals, Inc..
149. Yoshinori Kawabe, Takanori Inao, Shodai Komatsu, Guan Huang, Akira Ito, Takeshi Omasa, Masamichi Kamihira, Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site., J Biosci Bioeng., 10.1016/j.jbiosc.2016.10.011, 123, 3, 390-397, 2016.11.
150. Hideaki Yamamoto, Jane Marie Tonello, Takanori Sambuichi, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Characterization of genetically engineered mouse hepatoma cells with inducible liver functions by overexpression of liver-enriched transcription factors, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.07.011, 2017.01, [URL], New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1β, HNF3β [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research..
151. Masanobu Horie, Anuj Tripathi, Akira Ito, Kawabe Yoshinori, Masamichi Kamihira, Magnetic nanoparticles
Functionalization and manufacturing of pluripotent stem cells, Advanced Structured Materials, 10.1007/978-981-10-3328-5_9, 66, 363-383, 2017.01, [URL], Regenerative medicine uses cell alone or in combination with carrier to deliver at the required site for restoring the normal functions of diseased or degenerated tissue. Various strategies to restore tissue functions involve specific cell types, scaffolds and delivery processes that are still in developmental stage. Obtaining sufficient quantity of cells by non-invasive approach for the application in regenerative medicine is still a challenge. Pluripotent stem cells (PSCs), including embryonic stem cells and induced pluripotent stem cells (iPSCs), possess the inherent ability of self-renewal and differentiation into many cell types. In particular, iPSCs are of a special interest because patient-derived iPSCs have the ability to reproduce patient-specific clinical conditions. The development of manufacturing systems for PSCs, including cell culture engineering, is a challenging research field for the clinical application of PSCs such as in regenerative medicine. One of these manufacturing systems uses magnetic nanoparticles which are well known for their application in magnetic resonance imaging and magnetic hyperthermia. Besides, this chapter is focused on the basics of magnetic nanoparticles, its functionalization and further applications of a magnetic force-based cell manufacturing system for pluripotent stem cells. Indeed, we have developed a procedure in which cells are labeled with magnetite cationic liposomes via electrostatic interaction between the positively charged liposomes and the target cells. The culture system may provide a useful tool for studying the behavior of PSCs and an efficient way of PSCs manufacturing for clinical applications..
152. Kawabe Yoshinori, Shinya Komatsu, Shodai Komatsu, Mai Murakami, Akira Ito, Tetsushi Sakuma, Takahiro Nakamura, Takashi Yamamoto, Masamichi Kamihira, Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.12.003, 2017.01, [URL], Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering..
153. Kawabe Yoshinori, Takanori Inao, Shodai Komatsu, Guan Huang, Akira Ito, Takeshi Omasa, Masamichi Kamihira, Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2016.10.011, 123, 3, 390-397, 2017.03, [URL], Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production..
154. Yoshinori Kawabe, Takanori Inao, Shodai Komatsu, Guan Huang, Akira Ito, Takeshi Omasa, Masamichi Kamihira, Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2016.10.011, 123, 3, 390-397, 2017.03, Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production..
155. Kazushi Ikeda, Akira Ito, Ryusuke Imada, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, In vitro drug testing based on contractile activity of C2C12 cells in an epigenetic drug model., Scientific reports, 10.1038/srep44570, 7, 44570-44570, 2017.03, Skeletal muscle tissue engineering holds great promise for pharmacological studies. Herein, we demonstrated an in vitro drug testing system using tissue-engineered skeletal muscle constructs. In response to epigenetic drugs, myotube differentiation of C2C12 myoblast cells was promoted in two-dimensional cell cultures, but the levels of contractile force generation of tissue-engineered skeletal muscle constructs prepared by three-dimensional cell cultures were not correlated with the levels of myotube differentiation in two-dimensional cell cultures. In contrast, sarcomere formation and contractile activity in two-dimensional cell cultures were highly correlated with contractile force generation of tissue-engineered skeletal muscle constructs. Among the epigenetic drugs tested, trichostatin A significantly improved contractile force generation of tissue-engineered skeletal muscle constructs. Follistatin expression was also enhanced by trichostatin A treatment, suggesting the importance of follistatin in sarcomere formation of muscular tissues. These observations indicate that contractility data are indispensable for in vitro drug screening..
156. Momoko Kitaoka, Ayaka Naritomi, Yoshinori Kawabe, Masamichi Kamihira, Noriho Kamiya, Masahiro Goto, Transcutaneous pollinosis immunotherapy using a solid-in-oil nanodispersion system carrying T cell epitope peptide and R848, BIOENGINEERING & TRANSLATIONAL MEDICINE, 10.1002/btm2.10048, 2, 1, 102-108, 2017.03, Antigen-specific immunotherapy is the only curative approach for the treatment of allergic diseases such as Japanese cedar pollinosis. Immunotherapy using a T cell epitope vaccine in combination with the adjuvant R848 is of particular interest as a safe and effective approach to treat allergic diseases. Herein, we propose a simple and easy to handle vaccine administration method using the original solid-in-oil (S/O) nanodispersion system that permeates through the skin. The S/O nanodispersion system is composed of nanoparticles of hydrophilic molecules surrounded with hydrophobic surfactants that are dispersed in an oil vehicle. The system has potential to carry and deliver both hydrophilic and hydrophobic bioactives. Hydrophilic T cell epitope peptide was efficiently delivered through mouse skin using the S/O nanodispersion system and lowered antigen-specific IgE levels in pollinosis model mice. Addition of the hydrophobic adju1vant R848 significantly lowered the antibody secretion and shifted the Th1/Th2-balance toward Th1-type immunity in the model mice, showing the potential to alleviate Japanese cedar pollinosis..
157. Kazushi Ikeda, Akira Ito, Ryusuke Imada, Masanori Sato, Yoshinori Kawabe, Masamichi Kamihira, In vitro drug testing based on contractile activity of C2C12 cells in an epigenetic drug model, Sci Rep, 10.1038/srep44570, 7, 44570, 2017.05.
158. Kazushi Ikeda, Akira Ito, Masanori Sato, Shota Kanno, Yoshinori Kawabe, Masamichi Kamihira, Effects of heat stimulation and l-ascorbic acid 2-phosphate supplementation on myogenic differentiation of artificial skeletal muscle tissue constructs., Journal of tissue engineering and regenerative medicine, 10.1002/term.2030, 11, 5, 1322-1331, 2017.05, Although skeletal muscle tissue engineering has been extensively studied, the physical forces produced by tissue-engineered skeletal muscles remain to be improved for potential clinical utility. In this study, we examined the effects of mild heat stimulation and supplementation of a l-ascorbic acid derivative, l-ascorbic acid 2-phosphate (AscP), on myoblast differentiation and physical force generation of tissue-engineered skeletal muscles. Compared with control cultures at 37°C, mouse C2C12 myoblast cells cultured at 39°C enhanced myotube diameter (skeletal muscle hypertrophy), whereas mild heat stimulation did not promote myotube formation (differentiation rate). Conversely, AscP supplementation resulted in an increased differentiation rate but did not induce skeletal muscle hypertrophy. Following combined treatment with mild heat stimulation and AscP supplementation, both skeletal muscle hypertrophy and differentiation rate were enhanced. Moreover, the active tension produced by the tissue-engineered skeletal muscles was improved following combined treatment. These findings indicate that tissue culture using mild heat stimulation and AscP supplementation is a promising approach to enhance the function of tissue-engineered skeletal muscles. Copyright © 2015 John Wiley & Sons, Ltd..
159. Akihiko Ono, Akira Ito, Tomonaga Sato, Masaki Yamaguchi, Taiga Suzuki, Yoshinori Kawabe, Masamichi Kamihira, Hypoxia-responsive transgene expression system using RTP801 promoter and synthetic transactivator fused with oxygen-dependent degradation domain., J Biosci Bioeng, 10.1016/j.jbiosc.2017.02.012, 124, 1, 115-124, 2017.07.
160. Akihiko Ono, Akira Ito, Tomonaga Sato, Masaki Yamaguchi, Taiga Suzuki, Yoshinori Kawabe, Masamichi Kamihira, Hypoxia-responsive transgene expression system using RTP801 promoter and synthetic transactivator fused with oxygen-dependent degradation domain., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2017.02.012, 124, 1, 115-124, 2017.07, Precise control of gene expression using an artificial gene circuit is a major challenge in the application of synthetic biology. Here, we designed a hypoxia-responsive transgene expression system by combining a hypoxia-inducible RTP801 promoter and a tetracycline-responsive transactivator fused with an oxygen-dependent degradation domain (TA-ODD). The reporter gene expression was highly induced by hypoxia when a transactivator-expression plasmid, pRTP801/TA-ODD, harboring a TA-ODD gene driven by the RTP801 promoter, was cotransfected with a reporter plasmid, pTRE/EGFP, harboring an EGFP gene controlled under the transactivator-responsive promoter. A stable cell line into which the expression units RTP801/TA-ODD and TRE/EGFP had been introduced responded to hypoxia and expressed the reporter gene in an oxygen-concentration-dependent manner. Moreover, the cells demonstrated potential as sensors to detect hypoxic conditions in a three-dimensional tissue culture in vitro. These results indicate that the hypoxia-responsive transgene expression system is useful for constructing cell-based hypoxia detection systems..
161. Xue Wang, Kawabe Yoshinori, Risa Kato, Takeshi Hada, Akira Ito, Yoshimasa Yamana, Masako Kondo, Masamichi Kamihira, Accumulative scFv-Fc antibody gene integration into the hprt chromosomal locus of Chinese hamster ovary cells, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.05.017, 124, 5, 583-590, 2017.11, [URL], We have previously developed an accumulative site-specific gene integration system (AGIS) using Cre-recombinase and mutated loxP sites. AGIS enables repeated transgene integration into a predetermined chromosomal site in mammalian cells. However, the process of establishing cells with multiple integrated copies of the transgene is still time-consuming. In the present study, we describe an improved version of AGIS that facilitates and accelerates the establishment of high-producer Chinese hamster ovary (CHO) cells. Two donor vectors were simultaneously introduced into the cells in a single transfection. Cells with successfully targeted transgene integration were screened based on a change in the color of the reporter fluorescent protein that they express. Repeated rounds of integration allowed the transgene copy number to be increased. As a model, an scFv-Fc antibody gene was integrated into the hprt locus of the CHO cell genome. After three rounds of integration, a high-producer CHO cell clone with six copies of the scFv-Fc gene was successfully established. scFv-Fc productivity was approximately four-fold greater than a control cell line harboring a single copy of the transgene. This newly designed AGIS procedure should facilitate the development of producer cells suitable for biopharmaceutical protein production..
162. Xue Wang, Yoshinori Kawabe, Risa Kato, Takeshi Hada, Akira Ito, Yoshimasa Yamana, Masako Kondo, Masamichi Kamihira, Accumulative scFv-Fc antibody gene integration into the hprt chromosomal locus of Chinese hamster ovary cells., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2017.05.017, 124, 5, 583-590, 2017.11, We have previously developed an accumulative site-specific gene integration system (AGIS) using Cre-recombinase and mutated loxP sites. AGIS enables repeated transgene integration into a predetermined chromosomal site in mammalian cells. However, the process of establishing cells with multiple integrated copies of the transgene is still time-consuming. In the present study, we describe an improved version of AGIS that facilitates and accelerates the establishment of high-producer Chinese hamster ovary (CHO) cells. Two donor vectors were simultaneously introduced into the cells in a single transfection. Cells with successfully targeted transgene integration were screened based on a change in the color of the reporter fluorescent protein that they express. Repeated rounds of integration allowed the transgene copy number to be increased. As a model, an scFv-Fc antibody gene was integrated into the hprt locus of the CHO cell genome. After three rounds of integration, a high-producer CHO cell clone with six copies of the scFv-Fc gene was successfully established. scFv-Fc productivity was approximately four-fold greater than a control cell line harboring a single copy of the transgene. This newly designed AGIS procedure should facilitate the development of producer cells suitable for biopharmaceutical protein production..
163. Jane Marie Tonello, Saori Kawashima, Kazuki Sato, Kawabe Yoshinori, Akira Ito, Masamichi Kamihira, Three-dimensional culture of a genetically modified hepatoma cell line using macroporous gelatin beads, Cytotechnology, 10.1007/s10616-017-0117-0, 69, 6, 925-931, 2017.12, [URL], Hepatoma cells are a candidate cell source for bio-artificial livers. However, they exhibit reduced liver functions compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells were created by transduction with vectors mediating inducible overexpression of eight liver-enriched transcription factors. Upon the induction of the liver-enriched transcription factors transduced, the cells expressed both phenotypic and genotypic liver functions at high levels. In the present study, we performed three-dimensional culture of these cells using macroporous gelatin beads. When immobilized on the macroporous gelatin beads, these cells exhibited further enhancement in liver functionality, including increased albumin secretion, ammonia removal and cytochrome P450 activity. The levels of these functions were significantly enhanced compared to monolayer culture. The method is simple and scalable, and provides highly functional cells that can be used in basic and applied fields of hepatic research..
164. Jane Marie Tonello, Saori Kawashima, Kazuki Sato, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Three-dimensional culture of a genetically modified hepatoma cell line using macroporous gelatin beads., Cytotechnology, 10.1007/s10616-017-0117-0, 69, 6, 925-931, 2017.12, Hepatoma cells are a candidate cell source for bio-artificial livers. However, they exhibit reduced liver functions compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells were created by transduction with vectors mediating inducible overexpression of eight liver-enriched transcription factors. Upon the induction of the liver-enriched transcription factors transduced, the cells expressed both phenotypic and genotypic liver functions at high levels. In the present study, we performed three-dimensional culture of these cells using macroporous gelatin beads. When immobilized on the macroporous gelatin beads, these cells exhibited further enhancement in liver functionality, including increased albumin secretion, ammonia removal and cytochrome P450 activity. The levels of these functions were significantly enhanced compared to monolayer culture. The method is simple and scalable, and provides highly functional cells that can be used in basic and applied fields of hepatic research..
165. Hideaki Yamamoto, Jane Marie Tonello, Takanori Sambuichi, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Characterization of genetically engineered mouse hepatoma cells with inducible liver functions by overexpression of liver-enriched transcription factors., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2017.07.011, 125, 1, 131-139, 2018.01, New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1β, HNF3β [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research..
166. Yoshinori Kawabe, Takuya Shimomura, Shuohao Huang, Suguru Imanishi, Akira Ito, Masamichi Kamihira, Development of retroviral vectors capable of site-specific gene insertion together with protein delivery, BMC Proceedings, https://doi.org/10.1186/s12919-018-0097-x, 2018, 12(Suppl 1):P-327, 2018.03.
167. Yoshinori Kawabe, Shinya Komatsu, Shodai Komatsu, Mai Murakami, Akira Ito, Tetsushi Sakuma, Takahiro Nakamura, Takashi Yamamoto, Masamichi Kamihira, Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems, J Biosci Bioeng, 10.1016/j.jbiosc.2017.12.003, 125, 5, 599-605, 2018.05.
168. Xue Wang, Yoshinori Kawabe, Takeshi Hada, Akira Ito, Masamichi Kamihira, Cre-mediated transgene integration in Chinese hamster ovary cells using minicircle DNA vectors, Biotechnology Journal, 10.1002/biot.201800063, 13, 7, e1800063, 2018.05.
169. Yoshinori Kawabe, Shinya Komatsu, Shodai Komatsu, Mai Murakami, Akira Ito, Tetsushi Sakuma, Takahiro Nakamura, Takashi Yamamoto, Masamichi Kamihira, Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2017.12.003, 125, 5, 599-605, 2018.05, Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering..
170. Xue Wang, Yoshinori Kawabe, Takeshi Hada, Akira Ito, Masamichi Kamihira, Cre-Mediated Transgene Integration in Chinese Hamster Ovary Cells Using Minicircle DNA Vectors., Biotechnology journal, 10.1002/biot.201800063, 13, 7, e1800063, 2018.07, Bacterial backbone sequences of conventional plasmid vectors have been reported to exhibit negative effects on transgene expression in mammalian cells, such as cytotoxicity and gene silencing. Minicircle DNA vectors can be employed to overcome these issues and to improve the transfection efficiency because of their smaller size. In this study, transgenes are integrated into the hypoxanthine phosphoribosyltransferase (hprt) locus of Chinese hamster ovary (CHO) cells by the Cre-loxP system using minicircle DNA vectors as transgene donors. The targeted transgene integration efficiency is improved 2-3-fold (≈1.4%) using minicircle DNA vectors compared with conventional plasmid vectors. Moreover, clones with expected structures after transgene integration are obtained with a high frequency. When a transgene together with bacterial sequences derived from a plasmid vector is integrated into the hprt locus, the cell growth rate and antibody titer decrease. These results indicate that minicircle DNA vectors are more suitable than conventional plasmid vectors for transgene delivery in recombinant protein production using CHO cells..
171. Arifuzzaman M, Ito A, Ikeda K, Kawabe Y, Kamihira M., Fabricating Muscle-Neuron Constructs with Improved Contractile Force Generation, Tissue Eng Part A, http://doi.org/10.1089/ten.tea.2018.0165, 25, 7-8, 563-574, 2018.10.
172. Paerhati P, Ito A, Yoshioka K, Iwamoto K, Fujiwara S, Horie M, Kawabe Y, Kamihira M., Neural differentiation of mouse induced pluripotent stem cells using cadherin gene-engineered PA6 feeder cells., J Biosci Bioeng, https://doi.org/10.1016/j.jbiosc.2018.10.009, 127, 5, 633-640, 2018.11.
173. Md Arifuzzaman, Akira Ito, Kazushi Ikeda, Yoshinori Kawabe, Masamichi Kamihira, Fabricating Muscle-Neuron Constructs with Improved Contractile Force Generation., Tissue engineering. Part A, 10.1089/ten.TEA.2018.0165, 25, 7-8, 563-574, 2019.04, IMPACT STATEMENT: In this study, we fabricated innervated skeletal muscle tissue constructs comprising C2C12 myoblasts and PC12 neural cells using a magnetic force-based tissue engineering technique. We found that the C2C12/PC12 co-culture enhanced neural differentiation of PC12 cells and sarcomere formation of C2C12 myotubes, accompanying with neuromuscular junction formation. The innervated skeletal muscle tissue constructs generated significantly higher contractile forces compared with aneural (C2C12 monoculture) skeletal muscle tissue constructs. These innervated skeletal muscle tissue constructs can be a useful tool for drug testing and biological research for neuromuscular diseases..
174. Paerwen Paerhati, Akira Ito, Kantaro Yoshioka, Kaori Iwamoto, Sho Fujiwara, Masanobu Horie, Yoshinori Kawabe, Masamichi Kamihira, Neural differentiation of mouse induced pluripotent stem cells using cadherin gene-engineered PA6 feeder cells., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2018.10.009, 127, 5, 633-640, 2019.05, Investigating neural differentiation of pluripotent stem cells, including induced pluripotent stem (iPS) cells, is of importance for studying early neural development and providing a potential source of cells for nerve regeneration. Stromal cell-derived inducing activity (SDIA) using PA6 stromal cells promotes neural differentiation of iPS cells. Thus, we hypothesized that cadherin gene-engineered PA6 feeder cells will enhance the performance of SDIA by facilitating cell-cell interactions. Consequently, we created cadherin gene-engineered PA6 cells. Efficiency of neural differentiation from mouse iPS cells on PA6 feeder cells overexpressing E-cadherin gene (46%) or N-cadherin gene (27%) was significantly higher compared with parental PA6 feeder cells (19%). In addition, efficiency of motor neuron differentiation from mouse iPS cells on cadherin-gene engineered feeder cells (E-cadherin, 7.4%; N-cadherin, 11%) was significantly higher compared with parental PA6 feeder cells (4.1%). Altogether, these results indicate that cadherin gene-engineered feeder cells are a potent tool for promoting neural differentiation of pluripotent stem cells..
175. Akira Ito, Ryoji Teranishi, Kazuki Kamei, Masaki Yamaguchi, Akihiko Ono, Shinya Masumoto, Yuto Sonoda, Masanobu Horie, Kawabe Yoshinori, Masamichi Kamihira, Magnetically triggered transgene expression in mammalian cells by localized cellular heating of magnetic nanoparticles, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2019.03.008, 128, 3, 355-364, 2019.09, [URL], To develop a remote control system of transgene expression through localized cellular heating of magnetic nanoparticles, a heat-inducible transgene expression system was introduced into mammalian cells. Cells were labeled with magnetic nanoparticles and exposed to an alternating magnetic field. The magnetically labeled cells expressed the transgene in a monolayer and multilayered cell sheets in which cells were heated around the magnetic nanoparticles without an apparent temperature increase in the culture medium. Magnetic cells were also generated by genetically engineering with a ferritin gene, and transgene expression could be induced by exposure to an alternating magnetic field. This approach may be applicable to the development of novel gene therapies in cell-based medicine..
176. Akira Ito, Ryoji Teranishi, Kazuki Kamei, Masaki Yamaguchi, Akihiko Ono, Shinya Masumoto, Yuto Sonoda, Masanobu Horie, Yoshinori Kawabe, Masamichi Kamihira, Magnetically triggered transgene expression in mammalian cells by localized cellular heating of magnetic nanoparticles., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2019.03.008, 128, 3, 355-364, 2019.09, To develop a remote control system of transgene expression through localized cellular heating of magnetic nanoparticles, a heat-inducible transgene expression system was introduced into mammalian cells. Cells were labeled with magnetic nanoparticles and exposed to an alternating magnetic field. The magnetically labeled cells expressed the transgene in a monolayer and multilayered cell sheets in which cells were heated around the magnetic nanoparticles without an apparent temperature increase in the culture medium. Magnetic cells were also generated by genetically engineering with a ferritin gene, and transgene expression could be induced by exposure to an alternating magnetic field. This approach may be applicable to the development of novel gene therapies in cell-based medicine..
177. Ming Shi, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2019.09.011, 129, 3, 363-370, 2020.03, [URL], It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters..
178. Ming Shi, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2019.09.011, 129, 3, 363-370, 2020.03, It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters..
179. Kantaro Yoshioka, Akira Ito, Yoshinori Kawabe, Masamichi Kamihira., Novel neuromuscular junction model in 2D and 3D myotubes co-cultured with induced pluripotent stem cell-derived motor neurons, J Biosci Bioeng, 10.1016/j.jbiosc.2019.10.004, 129, 4, 486-493, 2020.04.
180. Kantaro Yoshioka, Akira Ito, Yoshinori Kawabe, Masamichi Kamihira, Novel neuromuscular junction model in 2D and 3D myotubes co-cultured with induced pluripotent stem cell-derived motor neurons., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2019.10.004, 129, 4, 486-493, 2020.04, Motor neurons differentiated from induced pluripotent stem (iPS) cells have attracted attention for use in the construction of drug screening systems for neuronal diseases, such as amyotrophic lateral sclerosis. However, conventional drug screening systems using 2-dimensional (2D) cultures of iPS cell-derived motor neurons often evaluate the cell survival rate, morphological changes in the cells and/or gene expression analysis, and these parameters do not always reflect the actual functions of motor neurons, i.e., the induction of muscle contractions. In the present study, we developed a neuromuscular junction model comprising motor neurons and myotubes, which were differentiated from iPS cells and C2C12 myoblasts, respectively. Using this model, the contractile activity and force generation of the myotubes via the neuromuscular junction were successfully measured in both two- and three-dimensional (3D) cell culture systems. The results suggested that this neuromuscular junction model can be used to construct a drug candidate screening system for neuronal diseases..
181. Ryusei Iwao, Yoshinori Kawabe, Mai Murakami, Akira Ito, Masamichi Kamihira, Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307001, 333, 07001, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01.
182. Feiyang Zheng, Yoshinori Kawabe, Mai Murakami, Mamika Takahashi, Shoichiro Yoshida, Akira Ito, Masamichi Kamihira, Retrotransposon-mediated Gene Transfer for Animal Cells, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307002, 333, 07002, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01.
183. Kazuki Shirakawa, Yoshinori Kawabe, Guan Huang, Akira Ito, Masamichi Kamihira, Targeted Gene Integration into Nuclear Genome of Microalgae Using Cre/loxP Recombination System, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307003, 333, 07003, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01.
184. Shinya Masumoto, Akira Ito, Akihiko Ono, Tomonaga Sato, Masaki Yamaguchi, Yoshinori Kawabe, Masamichi Kamihira, Construction of Hypoxia-Responsive VEGF Gene-Expression System Using Synthetic Biological Approach, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307005, 333, 07005, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01.
185. Hiroyuki Kitano, Manuel Souvervielle Soto, Yuto Sonoda, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Generation of Gene-Engineered Human Hepatoma Cells with Heat-Inducible Liver Functions, MATEC Web of Conferences, https://doi.org/10.1051/matecconf/202133307007, 333, 07007, The 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2021.01.
186. Kantaro Yoshioka, Akira Ito, Md Arifuzzaman, Taichi Yoshigai, Fangming Fan, Kei-Ichiro Sato, Kazunori Shimizu, Yoshinori Kawabe, Masamichi Kamihira, Miniaturized skeletal muscle tissue fabrication for measuring contractile activity, J Biosci Bioeng, 10.1016/j.jbiosc.2020.11.014, 131, 4, 434-441, 2021.04.
187. Kantaro Yoshioka, Akira Ito, Md Arifuzzaman, Taichi Yoshigai, Fangming Fan, Kei-Ichiro Sato, Kazunori Shimizu, Yoshinori Kawabe, Masamichi Kamihira, Miniaturized skeletal muscle tissue fabrication for measuring contractile activity., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2020.11.014, 131, 4, 434-441, 2021.04, The contractile function of skeletal muscle is essential for maintaining the vital activity of life. Muscular diseases such as muscular dystrophy severely compromise the quality of life of patients and ultimately lead to death. There is therefore an urgent need to develop therapeutic agents for these diseases. In a previous study, we showed that three-dimensional skeletal muscle tissues fabricated using the magnetic force-based tissue engineering technique exhibited contractile activity, and that drug effects could be evaluated based on the contractile activity of the skeletal muscle tissues. However, the reported method requires a large number of cells and the tissue preparation procedure is complex. It is therefore necessary to improve the tissue preparation method. In this study, a miniature device made of polydimethylsiloxane was used to simplify the production of contracting skeletal muscle tissues applicable to high-throughput screening. The effects of model drugs on the contractile force generation of skeletal muscle tissues prepared from mouse C2C12 myoblast and human induced pluripotent stem cells were evaluated using the miniature muscle device. The results indicated that the muscle device system could provide a useful tool for drug screening..
188. Feiyang Zheng, Yoshinori Kawabe, Mai Murakami, Mamika Takahashi, Kyoka Nishihata, Souichiro Yoshida, Akira Ito, Masamichi Kamihira, LINE-1 vectors mediate recombinant antibody gene transfer by retrotransposition in Chinese hamster ovary cells., Biotechnology journal, 10.1002/biot.202000620, e2000620, 2021.05, Retrotransposons, such as long interspersed element-1 (LINE-1), can copy themselves to other genomic loci via a transposition event (termed retrotransposition). Retrotransposons, therefore, have potential use as an efficient gene delivery tool to integrate multiple copies of a target gene into a host genome. Here, we developed a retrotransposon vector based on LINE-1 that achieves target gene integration of multiple transgene copies. The retrotransposon vector contains a neomycin resistance gene split by an intron as a marker gene, and a gene encoding an antibody single-chain variable fragment (Fv) fused with the constant antibody region (Fc) (scFv-Fc) as a model target gene. G418-resistant Chinese hamster ovary cells were generated using this retrotransposon vector, and scFv-Fc was produced in the culture medium. To regulate retrotransposition, we developed a retrotransposon vector system that separately expressed the two open reading frames (ORF1 and ORF2) of LINE-1. Genomic PCR analysis detected the transgene sequence in almost all tested clones. Compared with clones established using the intact LINE-1 vector, clones generated with the split ORF1 and ORF2 system showed similar specific scFv-Fc productivity and retrotransposition efficiency. This approach of using a retrotransposon-based vector system has the potential to provide a new gene delivery tool for mammalian cells..
189. Hiroyuki Kitano, Yuki Nagae, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Development of a genetically modified hepatoma cell line with heat-inducible high liver function, Cytotechnology, https://doi.org/10.1007/s10616-021-00457-4, 73, 3, 353-362, 2021.06.
190. Hiroyuki Kitano, Yuki Nagae, Yoshinori Kawabe, Akira Ito, Masamichi Kamihira, Development of a genetically modified hepatoma cell line with heat-inducible high liver function., Cytotechnology, 10.1007/s10616-021-00457-4, 73, 3, 353-362, 2021.06, Hepatoma cells are a promising cell source for the construction of bioartificial liver (BAL) systems owing to their high proliferative capability. However, their low liver function compared with primary hepatocytes is a major problem. In a previous study, we established a genetically modified hepatoma cell line, Hepa/8F5, in which eight liver-enriched transcription factor (LETF) genes were transduced into mouse hepatoma Hepa1-6 cells using a drug-inducible transactivator system. These cells proliferate actively under normal culture conditions, meaning that large quantities can be prepared easily. When the overexpression of the LETFs is induced by the addition of an inducer drug, cell growth stops and cell morphology changes with concomitant high expression of liver functions. However, the liver functions largely depend on the presence of the inducer drug, which must be continuously added to maintain these enhanced functions. In the present study, we attempted to modify the method of induction of LETF overexpression in Hepa/8F5 cells to remove the requirement for continual drug addition. To this end, we constructed a system in which the artificial transactivator was transcribed and amplified under the control of a heat-shock protein promoter, and introduced the system into the genome of Hepa/8F5 cells. In our modified cell line, heat-triggered LETF expression was confirmed to induce high liver function. After drug-screening of transfected cells, we established a hepatoma cell line (Hepa/HS), which exhibited high, heat-inducible liver functions. The Hepa/HS cells may represent a new cell source for hepatic studies such as the construction of BAL systems. Supplementary Information: The online version of this article (10.1007/s10616-021-00457-4) contains supplementary material, which is available to authorized users..
191. Feiyang Zheng, Yoshinori Kawabe, Mai Murakami, Mamika Takahashi, Kyoka Nishihata, Souichiro Yoshida, Akira Ito, Masamichi Kamihira, LINE-1 vectors mediate recombinant antibody gene transfer by retrotransposition in Chinese hamster ovary cells, Biotechnol J, https://doi.org/10.1002/biot.202000620, 16, 7, e2000620, 2021.07.
192. Guan Huang$, Yoshinori Kawabe$, Kazuki Shirakawa, Tatsuki Akiyama, Masamichi Kamihira ($Co-1st author), Novel transgenic Chlamydomonas reinhardtii strain with retargetable genomic transgene integration using Cre-loxP system  , J Biosci Bioeng, https://doi.org/10.1016/j.jbiosc.2021.07.006, in press, 2021.07.
193. Kantaro Yoshioka, Akira Ito, Masanobu Horie, Kazushi Ikeda, Sho Kataoka, Keiichiro Sato, Taichi Yoshigai, Hidetoshi Sakurai, Akitsu Hotta, Yoshinori Kawabe, Masamichi Kamihira, Contractile Activity of Myotubes Derived from Human Induced Pluripotent Stem Cells: A Model of Duchenne Muscular Dystrophy., Cells, 10.3390/cells10102556, 10, 10, 2021.09, Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to repair the dystrophin gene has been proposed as a new treatment method for DMD. However, it is not known whether the contractile function of myotubes derived from gene-repaired iPS cells can be restored. We therefore investigated the maturation of myotubes in electrical pulse stimulation culture and examined the effect of gene repair by observing the contractile behaviour of myotubes. The contraction activity of myotubes derived from dystrophin-gene repaired iPS cells was improved by electrical pulse stimulation culture. The iPS cell method used in this study for evaluating muscle contractile activity is a useful technique for analysing the mechanism of hereditary muscular disease pathogenesis and for evaluating the efficacy of new drugs and gene therapy..
194. Shinya Masumoto, Akihiko Ono, Akira Ito, Yoshinori Kawabe, Masamichi Kamihira, Hypoxia-responsive expression of vascular endothelial growth factor for induction of angiogenesis in artificial three-dimensional tissues, J Biosci Bioeng, 10.1016/j.jbiosc.2021.06.010, 132, 4, 399-407, 2021.10.
195. Kantaro Yoshioka, Akira Ito, Masanobu Horie, Kazushi Ikeda, Sho Kataoka, Keiichiro Sato, Taichi Yoshigai, Hidetoshi Sakurai, Akitsu Hotta, Yoshinori Kawabe, Masamichi Kamihira, Contractile activity of myotubes derived from human induced pluripotent stem cells: A model of duchenne muscular dystrophy, Cells, https://doi.org/10.3390/cells10102556, 10, 10, 2556, 2021.10.
196. Guan Huang, Yoshinori Kawabe, Kazuki Shirakawa, Tatsuki Akiyama, Masamichi Kamihira, Novel transgenic Chlamydomonas reinhardtii strain with retargetable genomic transgene integration using Cre-loxP system., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2021.07.006, 132, 5, 469-478, 2021.11, The use of Chlamydomonas for biofuel and biopharmaceutical production has been anticipated. However, the genetic engineering technology for Chlamydomonas is not as advanced as that for other organisms. Here, we established transgenic Chlamydomonas strains capable of high and stable transgene expression. The established cells exhibited stable reporter gene expression at a high level throughout long-term culture (∼60 days), even in the absence of drug pressure. The transgene insertion sites in the cell genome that may be suitable for exogenous gene expression were identified. Because the transgene contains a loxP site, the cells can be used as founders for retargeting other transgenes using the Cre-loxP system to generate transgenic Chlamydomonas producing useful substances. As a model biopharmaceutical gene, an interferon expression cassette was integrated into the genomic locus of the cells using Cre recombinase. The transgenic cells stably produced interferon protein in medium for 12 passages under non-selective conditions. These results indicate that the Chlamydomonas cells established in this study can serve as valuable and powerful tools not only for basic research on microalgae but also for the rapid establishment of cell lines expressing exogenous genes..

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pure2017年10月2日から、「九州大学研究者情報」を補完するデータベースとして、Elsevier社の「Pure」による研究業績の公開を開始しました。
 
 
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