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

Am J Physiol Gastrointest Liver Physiol. 2011 Mar;300(3):G409-17. doi: 10.1152/ajpgi.00453.2010. Epub 2010 Dec 23.


Intestinal stem cells (ISCs) have been studied for more than three decades; however, their isolation has remained a challenge. We hypothesized that, just as for stem cells of other tissues, one or more membrane markers would allow positive selection of ISCs by antibody-based sorting. To explore this hypothesis, microarray data of putative ISC fractions generated by side population sorting and laser capture microdissection were subjected to bioinformatic analysis to identify common membrane antigens. The microarray comparison suggested CD24 as a candidate surface marker, and immunohistochemistry showed expression of CD24 in epithelial cells of crypt bases. Flow cytometry of jejunal epithelial preparations revealed a CD24(+) CD45(-) fraction comprising ∼1% of the cells. Analysis with epithelial cell adhesion molecule and CD31 confirmed that the cell preparations were epithelial and without endothelial contamination. Cycling cells identified by prior injection with 5-ethynyl-2'-deoxyuridine were found predominantly in the CD24(lo) subfraction. Transcript analysis by real-time RT-PCR showed this subfraction to be enriched in the ISC markers leucine-rich-repeat-containing G-protein-coupled receptor 5 (40-fold) and Bmi1 (5-fold), but also enriched in lysozyme (10-fold). Flow cytometry with anti-lysozyme antibodies demonstrated that Paneth cells comprise ∼30% of the CD24(lo) subfraction. Additional flow analyses with leucine-rich-repeat-containing G-protein-coupled receptor 5-enhanced green fluorescent protein (EGFP) epithelium demonstrated colocalization of EGFP(hi) and CD24(lo). In contrast, CD24 cells were negative for the quiescent ISC marker doublecortin and CaM kinase-like-1. Culture of CD24(lo) cells in Matrigel generated organoid structures, which included all four epithelial lineages, thus giving functional evidence for the presence of ISCs. We conclude that the CD24(lo) fraction of jejunal epithelium is highly enriched with cycling ISCs. This isolation method should be useful to many investigators in the field to advance both the basic understanding of ISC biology and the therapeutic applications of ISCs.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • CD24 Antigen / genetics
  • CD24 Antigen / metabolism*
  • Cell Adhesion Molecules / metabolism
  • Cell Proliferation
  • Cell Separation / methods*
  • Cells, Cultured
  • Epithelial Cells / immunology*
  • Epithelial Cells / metabolism
  • Flow Cytometry*
  • Gene Expression Profiling / methods
  • Gene Expression Regulation
  • Immunohistochemistry
  • Jejunum / cytology
  • Jejunum / immunology*
  • Jejunum / metabolism
  • Leukocyte Common Antigens / deficiency
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Nuclear Proteins / metabolism
  • Oligonucleotide Array Sequence Analysis
  • Paneth Cells / immunology*
  • Paneth Cells / metabolism
  • Platelet Endothelial Cell Adhesion Molecule-1 / metabolism
  • Polycomb Repressive Complex 1
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / metabolism
  • RNA, Messenger / metabolism
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism
  • Repressor Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Stem Cells / immunology*
  • Stem Cells / metabolism
  • Time Factors


  • Biomarkers
  • Bmi1 protein, mouse
  • CD24 Antigen
  • Cd24a protein, mouse
  • Cell Adhesion Molecules
  • Lgr5 protein, mouse
  • Nuclear Proteins
  • Platelet Endothelial Cell Adhesion Molecule-1
  • Proto-Oncogene Proteins
  • RNA, Messenger
  • Receptors, G-Protein-Coupled
  • Repressor Proteins
  • Polycomb Repressive Complex 1
  • Dcamkl1 protein, mouse
  • Protein-Serine-Threonine Kinases
  • Leukocyte Common Antigens
  • Ptprc protein, mouse