A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation

Motohiro Yamauchi, Kensuke Otsuka, Hisayoshi Kondo, Nobuyuki Hamada, Masanori Tomita, Masayuki Takahashi, Satoshi Nakasono, Toshiyasu Iwasaki, Kazuo Yoshida

研究成果: Contribution to journalArticle査読

13 被引用数 (Scopus)

抄録

The microcolony assay developed by Withers and Elkind has been a gold standard to assess the surviving fraction of small intestinal stem cells after exposure to high (≥8 Gy) doses of ionizing radiation (IR), but is not applicable in cases of exposure to lower doses. Here, we developed a novel in vitro assay that enables assessment of the surviving fraction of small intestinal stem cells after exposure to lower IR doses. The assay includes in vitro culture of small intestinal stem cells, which allows the stem cells to develop into epithelial organoids containing all four differentiated cell types of the small intestine. We used Lgr5-EGFP-IRES-CreERT2/ROSA26-tdTomato mice to identify Lgr5+ stem cells and their progeny. Enzymatically dissociated single crypt cells from the duodenum and jejunum of mice were irradiated with 7.25, 29, 101, 304, 1000, 2000 and 4000 mGy of X-rays immediately after plating, and the number of organoids was counted on Day 12. Organoid-forming efficiency of irradiated cells relative to that of unirradiated controls was defined as the surviving fraction of stem cells. We observed a significant decrease in the surviving fraction of stem cells at ≥1000 mGy. Moreover, fluorescence-activated cell sorting analyses and passage of the organoids revealed that proliferation of stem cells surviving IR is significantly potentiated. Together, the present study demonstrates that the in vitro assay is useful for quantitatively assessing the surviving fraction of small intestinal stem cells after exposure to lower doses of IR as compared with previous examinations using the microcolony assay.

本文言語英語
ページ(範囲)381-390
ページ数10
ジャーナルJournal of radiation research
55
2
DOI
出版ステータス出版済み - 3 2014
外部発表はい

All Science Journal Classification (ASJC) codes

  • 放射線
  • 放射線学、核医学およびイメージング
  • 健康、毒物学および変異誘発

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