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J. Biol. Chem., Vol. 283, Issue 39, 26468-26476, September 26, 2008

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Design of the Artificial Acellular Feeder Layer for the Efficient Propagation of Mouse Embryonic Stem Cells^*

Masato Nagaoka¹, Yuko Hagiwara¹, Keiko Takemura¹, Yuta Murakami, Jixuan Li, Stephen A. Duncan, and Toshihiro Akaike²

From the Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and the Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B-57 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

Embryonic stem (ES) cells are pluripotent-undifferentiated cells that have a great interest for the investigation of developmental biology. Murine ES cells maintain their pluripotency by the supplementation of the leukemia inhibitory factor (LIF). LIF is reported to act as a matrix-anchored form, and immobilized cytokines are useful to sustain their signaling on target cells. In this study, we used the immobilizable fusion protein composed of LIF and IgG-Fc region, which was used as a model of the matrix-anchored form of LIF to establish a novel system for ES cell culture and to investigate the effect of immobilized LIF on maintenance of ES cell pluripotency. Mouse ES cells maintained their undifferentiated state on the surface coated with LIF-Fc. Furthermore, when cultured on the co-immobilized surface with LIF-Fc and E-cadherin-Fc, mouse ES cells showed characteristic scattering morphologies without colony formation, and they could maintain their undifferentiated state and pluripotency without additional LIF supplementation. The activation of LIF signaling was sustained on the co-immobilized surface. These results indicate that immobilized LIF and E-cadherin can maintain mouse ES cells efficiently and that the immobilizable LIF-Fc fusion protein is useful for the investigation of signaling pathways of an immobilized form of LIF in the maintenance of ES cell pluripotency.

Received for publication, July 2, 2008

^* This work was supported by a grant from the 21st Century Centers of Excellence Program and a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology. Tel.: 81-45-924-5790; Fax: 81-45-924-5815; E-mail: takaike{at}bio.titech.ac.jp.

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