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Title: Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

Abstract

Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: 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. Methods: 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. Results: 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 >1 year, which suggested the presence of long-lived mCherry-positivemore » 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. Conclusions: 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.« less

Authors:
 [1];  [2];  [1];  [3]; ; ; ; ; ; ;  [1]; ;  [2];  [1]
  1. Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)
  2. Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan)
  3. (Japan)
Publication Date:
OSTI Identifier:
22416840
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 454; Journal Issue: 4; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; FLUORESCENCE; GENES; GOLGI COMPLEXES; GTP-ASES; LABELLING; LEUCINE; MAINTENANCE; MICE; MICROSCOPY; RECEPTORS; SMALL INTESTINE; STEM CELLS

Citation Formats

Horita, Nobukatsu, Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp, Hayashi, Ryohei, Department of Gastroenterology and Metabolism, Hiroshima University, Fukushima, Keita, Hibiya, Shuji, Fukuda, Masayoshi, Kano, Yoshihito, Mizutani, Tomohiro, Nemoto, Yasuhiro, Yui, Shiro, Okamoto, Ryuichi, Nakamura, Tetsuya, and Watanabe, Mamoru. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt. United States: N. p., 2014. Web. doi:10.1016/J.BBRC.2014.10.091.
Horita, Nobukatsu, Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp, Hayashi, Ryohei, Department of Gastroenterology and Metabolism, Hiroshima University, Fukushima, Keita, Hibiya, Shuji, Fukuda, Masayoshi, Kano, Yoshihito, Mizutani, Tomohiro, Nemoto, Yasuhiro, Yui, Shiro, Okamoto, Ryuichi, Nakamura, Tetsuya, & Watanabe, Mamoru. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt. United States. doi:10.1016/J.BBRC.2014.10.091.
Horita, Nobukatsu, Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp, Hayashi, Ryohei, Department of Gastroenterology and Metabolism, Hiroshima University, Fukushima, Keita, Hibiya, Shuji, Fukuda, Masayoshi, Kano, Yoshihito, Mizutani, Tomohiro, Nemoto, Yasuhiro, Yui, Shiro, Okamoto, Ryuichi, Nakamura, Tetsuya, and Watanabe, Mamoru. 2014. "Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt". United States. doi:10.1016/J.BBRC.2014.10.091.
@article{osti_22416840,
title = {Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt},
author = {Horita, Nobukatsu and Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp and Hayashi, Ryohei and Department of Gastroenterology and Metabolism, Hiroshima University and Fukushima, Keita and Hibiya, Shuji and Fukuda, Masayoshi and Kano, Yoshihito and Mizutani, Tomohiro and Nemoto, Yasuhiro and Yui, Shiro and Okamoto, Ryuichi and Nakamura, Tetsuya and Watanabe, Mamoru},
abstractNote = {Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: 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. Methods: 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. Results: 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 >1 year, 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. Conclusions: 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.},
doi = {10.1016/J.BBRC.2014.10.091},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 454,
place = {United States},
year = 2014,
month =
}
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