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

Abstract

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.

Fig. 1.

Cross-comparison strategy using side population (SP) and laser capture microdissection (LCM) populations to identify possible intestinal stem cell (ISC) membrane markers. Numbers of transcripts (n) are shown for each stage of the analysis.

Fig. 2.

Mouse jejunum stained with CD24 antibody at low (A) and high power (B).

Fig. 3.

Flow cytometric identification of a CD24⁺ CD45⁻ fraction from jejunal epithelium. A: cells labeled with both CD24-PE (phycoerythrin) and CD45-FITC antibodies. B: selection of the entire CD45⁺ population for back-gating. C: CD45⁺ population as it relates to forward scatter (FSC)/side scatter (SSC) properties. D: exclusion of the CD45⁺ cells in FSC/SSC. E: sorting of gated cells with anti-CD45. F: validation of reduction in CD45⁺ cells.

Fig. 4.

Flow cytometric identification of CD24⁺ CD31⁻ and CD24⁺ EpCAM⁺ (epithelial cell adhesion molecule) fractions from jejunal epithelium. A: cells labeled with both CD24-PB (Pacific Blue) and CD31-Alexa 647 antibodies. B: CD24-PB and EpCAM-FITC antibodies.

Fig. 5.

Flow cytometric analysis of isolated jejunal epithelial cells 3 h post-EdU (5-ethynyl-2′-deoxyuridine) injection. A: no stain. B: CD24-PB antibody only. C: CD24-PB antibody and Edu-Alexa 647. D: CD24-PB antibody and Edu-Alexa 647 with CD24^hi and CD24^lo gates labeled.

Fig. 6.

Quantitative RT-PCR of mRNA isolated from the CD24^lo CD45⁻ fraction of jejunal epithelium compared with intact jejunum. Transcripts shown are markers for the following: ISCs [leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) and Bmi1 polycomb ring finger oncogene (Bmi1)]; Paneth cells (lysozyme); absorptive cells [sucrase-isomaltase (SI)]; and myofibroblasts [smooth muscle actin (SMA)]. Bars show means + SE (n = 7). Dashed line indicates intact jejunum. N.D., nondetectable. *P < 0.05.

Fig. 7.

Use of Lgr5-EGFP (enhanced green fluorescent protein) mice to assess colocalization of CD24 and Lgr5. A: flow cytometric analysis of jejunal epithelial cells displaying Lgr5-EGFP^hi distribution with respect to the CD24^hi and CD24^lo fractions. B: histological section of Lgr5-EGFP mouse jejunum, showing mosaicism of EGFP-expressing crypts.

Fig. 8.

Flow cytometric analysis of jejunal epithelial cells labeled with CD24-PB and isotype-DyLight 633 antibodies (A) and CD24-PB and doublecortin and CaM kinase-like-1 (DCAMKL1)-DyLight 633 antibodies (B).

Fig. 9.

Flow cytometric analysis of jejunal epithelial cells labeled with CD24-PB and lysozyme-FITC antibodies. A: representative numbers in the positive quadrants. B: gating of the CD24^hi and CD24^lo subfractions.

Fig. 10.

Growth of CD24^lo CD45⁻ cells in 3D Matrigel culture as per Sato et al. (36). A: phase-contrast images depict progression of organoid growth over 25 days (0d to 25d) with scale as shown. B: immunofluourescent staining of a CD24^lo CD45⁻ organoids, harvested at 25 days, with markers of Paneth cells (lysozyme), enteroendocrine cells [substance P (sub P)], enterocytes (SI), and goblet cells [mucin 2 (muc2)].