Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its "index sorting" function for stem cell research
Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its "index sorting" function for stem cell research
复制标题
DOI：
10.1111/j.1440-169x.2009.01157.x
复制
发表时间：
2010-01-01
影响因子：
2.5
通讯作者：
Agata, Kiyokazu
中科院分区：
文献类型：
--
作者：
Hayashi, Tetsutaro;Shibata, Norito;Agata, Kiyokazu
为了在细胞和分子水平上全面了解涡虫的干细胞系统，我们开发了一种新方法，将“荧光激活细胞分选（FACS）指数分选”分析与单细胞逆转录 - 聚合酶链反应（RT - PCR）相结合，以同时检测干细胞的基因表达和细胞周期状态。使用FACS收集单细胞，并将每个细胞的cDNA用于半定量RT - PCR。利用“指数分选”功能将结果绘制在FACS分选图谱上，这使我们能够结合每个细胞的细胞生物学数据（如细胞周期阶段）分析基因表达。在此，我们使用这种方法研究了涡虫的成体干细胞，并获得了一些发现，表明干细胞可能在S期到G2/M期期间发生分化。这种方法不仅对于研究涡虫的干细胞生物学，而且对于研究包括脊椎动物在内的其他生物的干细胞生物学，都可能是一种强大且直接的工具。
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.