Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its “index sorting” function for stem cell research

Authors

  • Tetsutaro Hayashi,

    1. RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Norito Shibata,

    1. RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047
    2. Department of Developmental Biology, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585
    Search for more papers by this author
    • Present address: Global COE Program, Division of Biological Science, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan

  • Ryo Okumura,

    1. Department of Biofunctional Chemistry, Faculty of Biomolecular Science, Okayama University Graduate School of Natural Science and Technology, Okayama 700-8535
    Search for more papers by this author
  • Tomomi Kudome,

    1. RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047
    Search for more papers by this author
  • Osamu Nishimura,

    1. RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047
    Search for more papers by this author
    • Present address: Global COE Program, Division of Biological Science, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan

  • Hiroshi Tarui,

    Corresponding author
    1. RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047
    Search for more papers by this author
  • Kiyokazu Agata

    Corresponding author
    1. RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047
    Search for more papers by this author
    • §

      Present address: Department of Biophysics, Faculty of Sciences, Kyoto University, Kyoto 606-8502, Japan


*Author to whom all correspondence should be addressed.
Email: agata@mdb.biophys.kyoto-u.ac.jp; tarui@cdb.riken.jp

Abstract

To achieve an integrated understanding of the stem cell system of planarians at both the cellular and molecular levels, we developed a new method by combining “fluorescent activated cell sorting (FACS) index sorting” analysis and single-cell reverse transcription–polymerase chain reaction (RT–PCR) to detect the gene expression and cell cycle state of stem cells simultaneously. Single cells were collected using FACS, and cDNAs of each cell were used for semi-quantitative RT–PCR. The results were plotted on the FACS sorting profile using the “index sorting” function, which enabled us to analyze the gene expression in combination with cell biological data (such as cell cycle phase) for each cell. Here we investigated the adult stem cells of planarians using this method and obtained findings suggesting that the stem cells might undergo commitment during S to G2/M phase. This method could be a powerful and straightforward tool for examining the stem cell biology of not only planarians but also other organisms, including vertebrates.

Ancillary