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.

Defining hierarchies of stemness in the intestine: evidence from biomarkers and regulatory pathways

“…As a first step toward bringing ISC-focused therapies to clinic, several recent studies have utilized the groundwork laid by ISC biomarker discovery in mouse models to identify, isolate, and culture intestinal crypts and ISCs from human tissue (30,74,101). Human ISCs have been isolated from adult tissues using a combinatorial surface marker approach, based on previous observations in mice demonstrating enrichment for functional Lgr5ϩ ISCs in cell populations FACS-sorted for CD24 (31,99). Interestingly, in human intestinal epithelium, LGR5 is most significantly enriched in CD44ϩ/CD24Ϫ or CD44ϩ/CD24Ϫ/CD166ϩ populations, which also demonstrate functional stemness in basal culture conditions (30,101).…”

Paneth cells expand from newly created and preexisting cells during repair after doxorubicin-induced damage

“…Flow cytometry of Paneth cells was performed using the method described by von Furstenberg et al (41). Briefly, jejunal epithelial cells were isolated using the EDTA-dispase method described by Formeister et al (12).…”

Distinct levels of Sox9 expression mark colon epithelial stem cells that form colonoids in culture

“…In vivo genetic lineage tracing studies in mice have demonstrated that the G protein-coupled receptor Lgr5, cell surface marker CD133, polycomb ring finger oncogene Bmi1, and telomerase (Tert) mark a population of IESCs in the crypt base of the small intestine. Additionally, the cell surface marker CD24 has been shown to be useful for fluorescent-activated cell sorting (FACS) enrichment of a small intestine IESC population that possesses stem cell characteristics, which are defined by multipotency and self-renewal capacity in vitro (15,44). Other studies in the small intestine have correlated gene expression patterns of Ascl2, Olfm4, Musashi1, and Dcamkl1 to IESC zone (17,22,30,42,43); however, it is still unclear whether these genes mark cells that possess functional stem cell properties based on in vivo lineage tracing or in vitro culture of FACS isolated cells.…”

“…While considerable efforts have been made to define the genetic signature of small intestine epithelial cells (2,9,10,15,17,18,25,26,30,37,(41)(42)(43)(44), fewer studies have focused on identifying genetic biomarkers of colonic epithelial stem cells (CESCs) (11,13,36,39). The concept of two populations of CESCs has been postulated: one is a slowly dividing labelretaining population, and the other is an actively dividing high-turnover population (19).…”