Abstract
Despite being exposed to microbial and food antigens, the human gastrointestinal tract does not exist in a state of inflammation. This homeostasis is critically dependent on the colonic epithelial barrier, which when dysfunctional is associated with inflammatory bowel disease (IBD), obesity and type two diabetes. Limitations in current models of the colonic epithelial barrier have driven the development of human colonic organoids, organotypic 3D models of the colonic epithelium derived from colonic stem cells. While colonic organoids are an exciting new model, they contain only 2-5% goblet cells, a critical secretory component of the barrier, in contrast to the ~50% expression seen in native colonic tissue.
High Wnt and Notch signalling promotes the differentiation of intestinal epithelial stem cells into the absorptive lineage, at the expense of the secretory lineage. The maintenance of colonic organoids requires high Wnt and Notch signalling, which we hypothesised was suppressing the differentiation of colonic stem cells into goblet cells. Our aim was to determine if reducing Wnt and Notch signalling in colonic organoids increases the expression of goblet cells. Additionally, this study characterised the growth of organoids in high Wnt and Notch conditions, quantifying any changes observed in low Wnt and Notch conditions.
Human colonic organoids derived from biopsy were grown in low Wnt3a conditioned media (Wnt3a-CM, 0, 5, 10, 25 and 50%) and Notch signalling was inhibited with DAPT (5 µM, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). The expression of goblet cells was determined by labelling with alcian blue and MUC2 staining, while quantitative polymerase chain reaction (qPCR) was used to quantify MUC2 transcript expression. Proliferation was quantified with Ki67 staining. The number and mean diameter, as an index of organoid size, were also recorded to assess growth.
Organoids grown in reduced Wnt3a-CM exhibited no changes in the number of goblet cells or organoid differentiation, survival and proliferation, but were significantly smaller (P<0.05). To mitigate against endogenous epithelial Wnt production, organoids were grown in 0% Wnt3a-CM + reduced Rspondin conditioned media (Rspo-CM, 0, 3 and 12%), which is critical for Wnt signalling. Reducing Rspo in the absence of Wnt3a significantly increased the expression of MUC2 positive cells (P<0.001), but only increased the expression of alcian blue positive cells (P<0.0001) in 0% Wnt3a-CM + 12% Rspo-CM. Organoids had significantly less Ki67 positive nuclei in the absence of Wnt and Rspo (P<0.01), although their size was unchanged.
DAPT significantly increased the number of alcian blue positive cells (P<0.0001), but not MUC2 positive cells or MUC2 transcript expression, suggesting DAPT alters non-MUC2 mucin expression. DAPT treated organoids were significantly smaller (P<0.0001), although the number of Ki67 positive nuclei was only decreased in 5% Wnt3a-CM + DAPT (P<0.05). Notch inhibition had no effect on organoid survival.
Importantly, a majority of the alcian blue positive and MUC2 positive cells observed in this study appeared to be immature, lacking the goblet morphology, basal nuclei and dense apical staining observed in mature goblet cells.
These findings demonstrate the modulation of Wnt and Notch signalling can alter the expression of goblet cell markers in colonic organoids but is insufficient to drive the development of mature goblet cells. It is likely the addition, or exclusion, of factors such as lipopolysaccharide (LPS) or SB202190 are required for full goblet cell maturation. The growth of human colonic organoids in high Wnt3a-CM supports current hypotheses on organoid growth kinetics and demonstrate that the modulation of Wnt and Notch signalling had varied, and often minimal effects on colonic organoid growth.