Journal of Cellular Biochemistry

Activin A is essential for Feeder-free culture of human induced pluripotent stem cells

Authors

  • Minoru Tomizawa MD, PhD,

    Corresponding author
    1. Department of Gastroenterology, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
    • Department of Gastroenterology, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan.
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  • Fuminobu Shinozaki,

    1. Department of Radiology, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
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  • Takao Sugiyama,

    1. Department of Rheumatology, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
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  • Shigenori Yamamoto,

    1. Department of Pediatrics, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
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  • Makoto Sueishi,

    1. Department of Rheumatology, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
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  • Takanobu Yoshida

    1. Department of Internal Medicine, National Hospital Organization Shimoshizu Hospital, 934-5 Shikawatashi, Yotsukaido City, Chiba 284-0003, Japan
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Abstract

Feeder-free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder-free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA-1-60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA-1-60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder-free culture of hiPS cells requires A or AC to maintain pluripotency. J. Cell. Biochem. 114: 584–588, 2013. © 2012 Wiley Periodicals, Inc.

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