Biochem Biophys Res Commun. 2014 Oct 29;454(4):493-499.

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

Horita N1, Tsuchiya K2, Hayashi R3, Fukushima K1, Hibiya S1, Fukuda M1, Kano Y1, Mizutani T1, Nemoto Y1, Yui S1, Okamoto R4, Nakamura T4, Watanabe M1.

1Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan.

2Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University, Japan. Electronic address: kii.gast@tmd.ac.jp.

3Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Japan; Department of Gastroenterology and Metabolism, Hiroshima University, Japan.

4Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University, Japan.

 

Abstract

The small intestinal epithelium is one of the most rapidly renewing tissue within an organism, which is composed of crypt and villus region. Intestinal stem cells located at the base of crypt, rapid cycling cell, which is named crypt base columnar cell, CBC stenm cell. This stem cell produce progenitor cells, which move from the stem cell compartment and differentiate into various kind of epithelial cells. On the other hand, Paneth cell derived from stem cell move down to base of crypt and reside between stem cells. Recently many advances have been reported about the small intestinal stem cell.

First, Lgr5 and other genes were identified as stem cell marker by lineage tracing, and it is also reported that there are about fourteen CBC stem cells in each crypt. Second, it has been reported that the establishment of intestinal stem cell culture as 3-dimentional organoid using Matrigel. These advances contributed to make a rapid advance of intestinal stem cell field. However, independent single stem cell have not been distinguished by any stem cell markers. Because all stem cell markers have been expressed in several stem cells but not single stem cell. That is why, in this study, we tried to identify individual stem cell by our original transduction and stem cell marking method, using organoid culture system. At first, we established lentiviral transduction and stem cell marking system by using the intestinal organoids. Second, we distinguished each stem cell by fluorescent marking. Finally, we performed dynamic analysis of single stem cell and identified independent long-lived stem cell. From these results, we hypothesize that epithelial cells in a crypt might be permanently supplied from independent stem cells but not single stem cell, which might supply cells alternately to both crypt and villus.

PMID: 25451268

 

Supplement:

The aim of this study is to distinguish individual stem cell from plural stem cells without any stem cell marker. We established the original gene transduction and stem cell marking method using intestinal organoid culture system. The important point of our gene transduction method into the intestinal organoid is to mix the lentivirus with Matrigel. Because lentivirus could not penetrate Matrigel structure. This mixing method enabled lentiviral gene transfer into the organoid and detect mCherry fluorescent. The mCherry protein was detected for over 50 weeks after gene transduction, which means long-lived mCherry positive stem cell has been producing mCherry positive living cells. Gene transduced organoids by Lentivirus have hetero expression of mCherry fluorescence from coexisting mCherry positive and negative stem cells in same crypt. Finally, we checked that each long-lived mCherry-positive and -negative single stem cell contribute to the generation of organoids and equal expression of a series of stem cell markers. Our data suggested that epithelial cells in a crypt might be permanently supplied from independent stem cells but not single stem cell. These independent stem cell might supply cells alternately to both crypt and villus and completely form intestinal epithelium with a number of functions (Figure 1).

Figure 1 W

Figure 1. Schema of our hypothesis about intestinal epithelial cell supply

 

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