A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation
| Autor: | Masanori Tomita, Toshiyasu Iwasaki, Satoshi Nakasono, Nobuyuki Hamada, Motohiro Yamauchi, Masayuki Takahashi, Kensuke Otsuka, Hisayoshi Kondo, Kazuo Yoshida |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
small intestinal stem cells
Technology survival assay Cell Survival Health Toxicology and Mutagenesis Cellular differentiation organoid Biology Radiation Dosage Lgr5 Mice Gentamicin protection assay in vitro culture Intestine Small Organoid medicine Animals Radiology Nuclear Medicine and imaging Cells Cultured Radiation Stem Cells LGR5 Dose-Response Relationship Radiation Cell sorting Molecular biology Small intestine In vitro medicine.anatomical_structure Immunology Biological Assay Stem cell ionizing radiation |
| Zdroj: | Journal of Radiation Research |
| ISSN: | 1349-9157 0449-3060 |
| Popis: | 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. Journal of Radiation Research, 55(2), pp.381-390; 2014 |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|