Sox9expression marks a subset of CD24-expressing small intestine epithelial stem cells that form organoids in vitro

Abstract

The inability to identify, isolate, and culture intestinal epithelial stem cells (IESCs) has been prohibitive to the study and therapeutic utilization of these cells. Using a Sox9(EGFP) mouse model, we demonstrate that Sox9(EGFP) fluorescence signatures can be used to differentiate between and enrich for progenitors (Sox9(EGFPsubLo)) and multipotent IESCs (Sox9(EGFPlo)). Sox9(EGFPlo) cells generate "organoids" in a recently defined culture system that mimics the native IESC niche. These organoids possess all four differentiated cell types of the small intestine epithelium, demonstrating the multipotent capacity of Sox9(EGFPlo) cells. Our results are consistent with the previously reported observation that single IESCs generate cryptlike units without a detectable mesenchymal cell component. A prospective search revealed that CD24 is expressed in the Sox9(EGFPlo) population and marks IESCs that form organoids in culture. CD24 represents the first cell surface marker that facilitates fluorescence-activated cell sorting enrichment of IESCs with widely available antibodies without requiring a specialized fluorescent reporter gene mouse model.

Insulin receptor isoform switching in intestinal stem cells, progenitors, differentiated lineages and tumors: evidence that IR-B limits proliferation

“…1A) (Formeister et al, 2009;Gracz et al, 2010;Van Landeghem et al, 2012). Evaluation of total Ir mRNA by qRT-PCR revealed approximately equal levels of total Ir expression in each cell population with a modest, but significant enrichment of Ir mRNA in Sox9-EGFP High cells versus other cell populations (Fig.…”

“…Insulin receptor isoform expression exists in a gradient from proliferative stem and progenitor cells (IR-A predominant) to post-mitotic, differentiated lineages (IR-B predominant) Single-cell suspensions were prepared from the jejunal epithelium of Sox9-EGFP reporter mice (Formeister et al, 2009;Gracz et al, 2010;Van Landeghem et al, 2012) and the four IEC populations were isolated by FACS based on distinct expression levels of Sox9-EGFP (Fig. 1A) (Formeister et al, 2009;Gracz et al, 2010;Van Landeghem et al, 2012).…”

“…Our studies used a novel Sox9-EGFP BAC transgenic reporter mouse that allows isolation and analyses of IESCs, progenitors and differentiated IECs based on distinct levels of Sox9-EGFP (Formeister et al, 2009;Gracz et al, 2010;Van Landeghem et al, 2012 (Muñoz et al, 2012;Van Landeghem et al, 2012). Sox9-EGFP Sublow cells lie in the region of highly proliferative, transit-amplifying progenitor cells and are enriched for genes associated with active proliferation.…”

“…Sox9-EGFP Sublow cells lie in the region of highly proliferative, transit-amplifying progenitor cells and are enriched for genes associated with active proliferation. Sox9-EGFP High cells are dramatically enriched for enteroendocrine cell (EEC) biomarkers, but also enriched for reported biomarkers of a quiescent IESC population (Gracz et al, 2010;Van Landeghem et al, 2012). Sox9-EGFP Negative cells on the villi are enriched for markers of post-mitotic, differentiated cells, including biomarkers of enterocytes, goblet and Paneth cells (Van Landeghem et al, 2012).…”

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells

“…Crypts were quantitated, pelleted (13 g, 7 minutes) and resuspended in 50 l Matrigel (BD Biosciences) supplemented with 50 ng/ml EGF (R&D Systems), 5 ng/ml WNT3A (R&D Systems), 1 g/ml R-spondin 1 (R&D Systems) and 100 ng/ml noggin (PeproTech). After polymerization at 37°C for 30 minutes, 0.5 ml culture medium was added to each well and the culture was maintained as described (Gracz et al, 2010).…”