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Updated: May 11, 2026
Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids
Published on: June 2, 2023
Characterization of anoikis-resistant cells in mouse colonic epithelium
Hideyoshi Kawasaki1, Tetsuya Yoshida, Kazuhide Horiguchi
1Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.
Abstract:
Anoikis is a form of apoptosis triggered by inadequate or inappropriate cell matrix contacts. Anoikis resistance has been utilized to isolate adult stem or progenitor cell populations from various tissues. The aim of this study was to characterize the stem or progenitor cell markers expressed in anoikis-resistant cells isolated from mouse colonic epithelium. Mouse colonic epithelial cells were isolated by Ca(2+)-depletion, and anoikis-resistant cells were obtained by culturing the cells in ultra-low attachment dishes. Flow cytometry analysis showed that anoikis-resistant cells are positive for the epithelial cell marker CD326, but negative for the hematopoietic cell marker CD45, eliminating the possibility of hematopoietic cell contamination. The majority of anoikis-resistant cells were also positive for the stem or progenitor cell markers CD133 and DCLK1 by immunofluorescent analysis. Reverse transcriptase-polymerase chain reaction analysis revealed that anoikis-resistant cells express +4 position cell marker Hopx, but did not express the other reported stem or progenitor cell markers, Lgr5, Musashi1, Bmi1, mTert and Olfm4. CD133 and DCLK1 double positive cells were observed both apical and basal crypts in mouse proximal colonic tissues. Together, anoikis-resistant cells in mouse colon epithelium were shown to be positive for CD133, DCLK1, Hopx and CD326, but negative for CD45, Lgr5, Musashi1, Bmi1, mTert and Olfm4. This study has shown a possible approach to isolating stem cells from the intestinal epithelium.
Insights
Anoikis-resistant cells from mouse colon epithelium express stem cell markers CD133, DCLK1, and Hopx. This finding offers a novel method for isolating intestinal stem cells.
Area of Science:
- Cell Biology
- Stem Cell Research
- Gastrointestinal Biology
Background:
- Anoikis, a form of programmed cell death, is triggered by disrupted cell-matrix interactions.
- Anoikis resistance is a valuable trait for isolating adult stem and progenitor cells from diverse tissues.
- Identifying specific markers on these resistant cells is crucial for understanding their stem cell potential.
Purpose of the Study:
- To identify and characterize the stem or progenitor cell markers present in anoikis-resistant cells derived from mouse colonic epithelium.
- To evaluate the expression profile of known stem cell markers in these isolated cells.
- To determine the feasibility of using anoikis resistance as a method for intestinal stem cell isolation.
Main Methods:
- Isolation of mouse colonic epithelial cells using calcium (Ca2+) depletion.
- Induction of anoikis resistance by culturing cells in ultra-low attachment dishes.
- Analysis of cell surface and intracellular markers using flow cytometry, immunofluorescence, and reverse transcriptase-polymerase chain reaction (RT-PCR).
Main Results:
- Anoikis-resistant cells were confirmed as epithelial (CD326+) and non-hematopoietic (CD45-).
- A majority of these cells expressed stem/progenitor markers CD133 and DCLK1.
- Expression analysis revealed Hopx positivity but negativity for Lgr5, Musashi1, Bmi1, mTert, and Olfm4.
- CD133 and DCLK1 double-positive cells were observed in both apical and basal regions of mouse colonic crypts.
Conclusions:
- Anoikis-resistant cells from mouse colon epithelium exhibit a distinct marker profile: CD133+, DCLK1+, Hopx+, CD326+, and CD45-.
- This specific combination of markers, excluding others like Lgr5, suggests a unique stem cell population.
- The study demonstrates a promising approach for isolating intestinal stem cells based on anoikis resistance.
