Gastroenterology

Gastroenterology

Volume 145, Issue 2, August 2013, Pages 383-395.e21
Gastroenterology

Original Research
Full Report: Basic and Translational—Alimentary Tract
Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay

https://doi.org/10.1053/j.gastro.2013.04.050Get rights and content

Background & Aims

Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues.

Methods

We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs.

Results

CD44+CD24loCD166+ cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78lo/– putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs.

Conclusions

We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44+CD24loCD166+, GRP78lo/–, and c-Kit facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44+CD24−/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.

Section snippets

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.

References (36)

  • E. Sangiorgi et al.

    Bmi1 is expressed in vivo in intestinal stem cells

    Nat Genet

    (2008)
  • R.K. Montgomery et al.

    Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells

    Proc Natl Acad Sci U S A

    (2011)
  • H. Tian et al.

    A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable

    Nature

    (2011)
  • N. Takeda et al.

    Interconversion between intestinal stem cell populations in distinct niches

    Science

    (2011)
  • A.D. Gracz et al.

    Sox9 expression marks a subset of CD24-expressing small intestine epithelial stem cells that form organoids in vitro

    Am J Physiol Gastrointest Liver Physiol

    (2010)
  • R.J. von Furstenberg et al.

    Sorting mouse jejunal epithelial cells with CD24 yields a population with characteristics of intestinal stem cells

    Am J Physiol Gastrointest Liver Physiol

    (2011)
  • T. Sato et al.

    Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche

    Nature

    (2009)
  • T. Sato et al.

    Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

    Nature

    (2011)
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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.

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