Fig. 4. In vitro villus cell clonogenicity.

(a) Non-dysplastic small intestinal crypts from wild-type and Vill-Grem1 mice readily formed branching enteroids with similar efficiency in standard conditions (ENS media). Vill-Grem1 crypts also formed lasting enteroids in the absence of Noggin in the medium (ES media), although the effect of endogenous Grem1 expression could be overcome by addition of competing recombinant BMP ligands 2, 4, and 7 (ES+rBMP2,4,7 media). Culture of Apc^Min polyp tissue resulted in non-branching spheroid generation in all media conditions, indicating dispensability of BMP antagonist in somatically mutant cells. Villi dissected from wild-type and Apc^Min/+ mice dissociated and died regardless of media supplementation. Villi dissected from Vill-Grem1 mice (in Wnt3A supplemented (ENSW) media) and Vill-Grem1/Apc^Min mice (in all media conditions) could form proliferative spheroid lesions that could be repeatedly passaged and propagated long-term. Vill-Grem1/Apc^Min mice generated villus spheroids efficiently in the absence of Noggin as a media supplement and spheroid generation could not be abrogated by addition of competing BMP ligands indicating BMP antagonist independent growth. Abbreviations of media supplements E: Epidermal growth factor; N: Noggin; S: R-Spondin; W: Wnt3A. (b) qRT-PCR analysis of effect of mouse genotype on villus Wnt target gene expression (versus wild-type) showed that a single Apc hit was sufficient to increase endogenous individual villus Wnt target gene expression including the stem-cell markers Lgr5 and Ascl2. Expression of the progenitor cell markers Sox9 and EphB2 was significantly increased in Vill-Grem1 animals (n = 10 villi for each strain, P < 0.01, t-test) and emergent spheroids had a further increase in both Wnt target and progenitor marker genes. (c) Villus spheroid immunostain showing nuclear β–catenin staining, membranous EphB2 and nuclear Sox9 stain. (d) Selection of somatically mutant cells on the villus of Vill-Grem1/Apc^Min/+ mice. Extracted non-dysplastic villi entering into culture retained a residual wild-type Apc allele, whilst emergent spheroids had lost this allele. (e) There was an increase in the percentage of villi transforming into clonogenic spheroids with increasing age of Vill-Grem1/Apc^Min/+ mice. Histology review showed that this increase correlated with the emergence of villus ectopic crypts (black arrowheads). Scale bars are 100 μm.