Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its "index sorting" function for stem cell research.
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.
Hayashi
Tetsutaro
T
RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047, Japan.
Shibata
Norito
N
Okumura
Ryo
R
Kudome
Tomomi
T
Nishimura
Osamu
O
Tarui
Hiroshi
H
Agata
Kiyokazu
K
D000818:Animals; D002455:Cell Division / Q000235:genetics; D002469:Cell Separation / Q000379:methods; D016000:Cluster Analysis; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D016195:G2 Phase / Q000235:genetics; D020869:Gene Expression Profiling; D017238:Genes, Helminth / Q000235:genetics; D015801:Helminth Proteins / Q000235:genetics; D017403:In Situ Hybridization; D008297:Male; D010932:Planarians / Q000166:cytology* / Q000528:radiation effects; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D012107:Research Design; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000235:genetics; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*
D015801:Helminth Proteins; D018809:Proliferating Cell Nuclear Antigen