Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors

Abstract

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.

Fig. 1.

Hierarchical clustering of genes differentially expressed in colon top and basal crypt as identified by SAM. Cluster I is enriched in genes associated with cell proliferation, and cluster II is enriched in genes expressed in pericryptal mesenchymal cells.

Fig. 2.

Significant correlation between genes differentially expressed in colon top and basal crypt and Wnt/β-catenin signaling targets. Microarray data of inducible expression of dnTCF4 in Ls174 cells were retrieved from van de Wetering et al. (13), and overlapping clones with colon top–bottom crypt gene list as identified by SAM were selected and calculated for correlation. The x axis measures mean gene expression change (log2) 23 h after dnTCF4 induction, and the y axis measures mean fold change (log2) of bottom versus top colon crypt compartments.

Fig. 3.

Expression and localization of GREM1 and GREM2 by myofibroblast cells and smooth muscle cells at colon crypt. (A and B) In situ hybridization (ISH) for GREM1 (A) and GREM2 (B). Dark brown dots indicate positive staining. (C and D) Immunohistochemical staining of fibronectin (C) and α-smooth muscle actin (D) as markers for intestinal myofibroblasts as well as smooth muscle cells. Dark brown staining indicates positive staining. (E and F) Double labeling for GREM1 mRNA (E, red) and myofibroblast marker vimentin (F, green) at colon basal crypt region. (G) Combined image showing coexpression of GREM1 and vimentin (yellow dots indicated by white arrows) at scattered pericryptal mesenchymal cells corresponding to myofibroblasts. See SI Fig. 10 for the enlarged version of fluorescent ISH/immunostaining. (H) RT-PCR analysis of BMP antagonists expression in four intestinal myofibroblast isolates (CMF11, CMF7B, IMF11B, and 18Co) as well as three colon cancer cell lines (Caco-2, DLD-1, and HT29).

Fig. 4.

Gremlin 1 partially inhibits Caco-2 cell differentiation and activates Wnt/β-catenin signaling in normal intestinal cells. (A) Quantitative RT-PCR analysis revealed a statistically significant decrease in expression of intestinal epithelial differentiation markers ANPEP and p21 at day 7 when Caco-2 cells were cultured in growth media supplemented with gremlin 1. The analysis detected a significant up-regulation of the AXIN2 transcript in Caco-2 cells after a 4-h treatment with gremlin 1 (*, P < 0.05). (B) Quantitative RT-PCR analysis demonstrated a statistically significant increase in AXIN2 expression in normal rat intestinal cells IEC-6 and IEC-18 after 48-h treatment with gremlin 1 (*, P < 0.01). (C and D) Gremlin 1 induces nuclear/cytoplasm localization of β-catenin in IEC-18 cells.

Fig. 5.

Graphical view of human colon intestinal epithelial cell development and stem cell niche maintenance. Only genes with significant differential expression in paired t test (P < 0.05) are listed. ISEMF, intestinal subepithelial myofibroblast; SMC, smooth muscle cell.