Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting

Authors

  • Tetsutaro Hayashi,

    1. RIKEN Center for Developmental Biology, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan,
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  • Maki Asami,

    1. RIKEN Center for Developmental Biology, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan,
    2. Department of Biofunctional Chemistry, Faculty of Biomolecular Science, Okayama University Graduate School of Natural Science and Technology, 3-1-1 Tsushimanaka, Okayama 700-8535, Japan,
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  • Sayaka Higuchi,

    1. RIKEN Center for Developmental Biology, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan,
    2. Department of Biology, Faculty of Science, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan,
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  • Norito Shibata,

    1. RIKEN Center for Developmental Biology, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan,
    2. Division for Morphogenesis, National Institute for Basic Biology, Okazaki 444-8585 Japan and
    3. Department of Biophysiscs, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
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  • Kiyokazu Agata

    Corresponding author
    1. RIKEN Center for Developmental Biology, 2-2-3, Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan,
    2. Department of Biophysiscs, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
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*To whom all correspondence should be addressed.
Email: agata@mdb.biophys.kyoto-u.ac.jp

Abstract

The remarkable capability of planarian regeneration is mediated by a group of adult stem cells referred to as neoblasts. Although these cells possess many unique cytological characteristics (e.g. they are X-ray sensitive and contain chromatoid bodies), it has been difficult to isolate them after cell dissociation. This is one of the major reasons why planarian regenerative mechanisms have remained elusive for a long time. Here, we describe a new method to isolate the planarian adult stem cells as X-ray-sensitive cell populations by fluorescence-activated cell sorting (FACS). Dissociated cells from whole planarians were labeled with fluorescent dyes prior to fractionation by FACS. We compared the FACS profiles from X-ray-irradiated and non-irradiated planarians, and thereby found two cell fractions which contained X-ray-sensitive cells. These fractions, designated X1 and X2, were subjected to electron microscopic morphological analysis. We concluded that X-ray-sensitive cells in both fractions possessed typical stem cell morphology: an ovoid shape with a large nucleus and scant cytoplasm, and chromatoid bodies in the cytoplasm. This method of isolating X-ray-sensitive cells using FACS may provide a key tool for advancing our understanding of the stem cell system in planarians.

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