Original ResearchFull Report: Basic and Translational—Alimentary TractIsolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay
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Mice and Human Tissues
The Lgr5–enhanced GFP mouse model was provided by Hans Clevers. Adult mice (6–12 weeks old) were used in all experiments. All mice were housed in the animal facility at Stowers Institute for Medical Research and handled according to Stowers Institute for Medical Research and National Institutes of Health guidelines. All procedures were approved by the Institutional Animal Care and Use Committee of Stowers Institute for Medical Research. Use of human tissues was approved by the institutional
A Combination of Antibodies to CD44, CD24, and CD166 Purified Putative ISCs by Excluding Differentiated Cells From the Villus and Crypt
We used a general strategy to enrich ISCs and to exclude differentiated cells with a combination of positive and negative markers, respectively (Figure 1A). Besides confirming a broad crypt-restricted CD44 expression (Figure 1B), we further found that CD44 expression was higher at the interface between 2 adjacent Lgr5-GFPhi CBCs than that between 2 adjacent PCs (Figure 1C and Supplementary Figures 1 and 3). Furthermore, 2 additional ISC surface markers, CD24 and CD166 (ALCAM),8, 9, 10 were
Discussion
Identification of highly purified functional ISCs is required for understanding their properties and for translational studies using human ISCs. Using transgenic reporters to isolate ISCs has several limitations. (1) The mosaic expression pattern of the reporter gene results in an inability to distinguish a truly negative population. (2) Direct application of these studies to the human system is difficult. (3) A single reporter could exclude important ISC subpopulations. Progress in other stem
Acknowledgments
The authors thank H. Clevers for providing Lgr5-GFP mice; M. Hembree, M. McClain, R. T. Ross, T. Johnson, H. Marshall, B. Lewis, D. Dukes, C. Semerad, J. Park, S. Beckham, F. Guo, and W. McDowell for technical support; A. Venkatraman, R. Sugimura, M. Zhao, and F. Tao for scientific discussion; K. Tannen for editing; and I. Schmid and the FACS facilities at University of California, Los Angeles Jonsson Comprehensive Cancer Center and AIDS Research Center for extra sorting support.
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Sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Author names in bold designate shared co-first authorship.
Conflicts of interest The authors disclose no conflicts.
Funding This research was performed as a project of the Intestinal Stem Cell Consortium, L. Li (U01DK085507), M. H. Wong (U01DK85525), M. Martin (U01DK85535), S. T. Magness (U01DK085547: SJH/STM), and the Coordinating Center J. Niland (U01DK85532). L. Li is supported in part by Stowers Institute for Medical Research.