title: Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt.
abstract: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour.
  
  
  
  Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence.
  
  
  
  We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids.
  
  
  
  The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Nobukatsu
authors.initials: N
authors.lastname: Horita
authors.affiliation: Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University, Japan. Electronic address: kii.gast@tmd.ac.jp.
authors.forename: Kiichiro
authors.initials: K
authors.lastname: Tsuchiya
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan; Department of Gastroenterology and Metabolism, Hiroshima University, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Hayashi
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Fukushima
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Hibiya
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Fukuda
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Kano
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Mizutani
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Nemoto
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Yui
authors.affiliation: Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Okamoto
authors.affiliation: Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Watanabe
mesh_terms: D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology*; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D007422:Intestines / Q000166:cytology*; D009940:Organoids / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*
keywords: Imaging of a single stem cell; Intestinal stem cells; Long-lived stem cells