Wnt Signals and Imaging Tissue Renewal in the Human Colonic Epithelium
Background The human intestinal epithelium is one of the most rapidly renewing tissues in the mammalian body, with 10 billion cells being shed from its surface every day. Renewal of the intestinal epithelium takes place at the base of flask-like epithelial invaginations known as crypts. Within these crypts reside a stem cell population that continuously repopulates the epithelium with all of its specialised cell lineages. Many signalling pathways are involved in the regulation of these activities, the predominant one being the canonical Wnt signalling pathway. The Wnt signalling pathway is known to be dysregulated in many cancers and has been posed as a potential therapeutic target in such conditions. Objective To investigate the status of canonical Wnt signalling in human colonic crypts and assess the regulation of crypt cell proliferation and migration in the context of tissue renewal. Design In the first instance, the status of the canonical Wnt signalling pathway in relation to the proliferation hierarchy was characterised in native human colonic crypts. The functional link between canonical Wnt signals and regulation of human colonic crypt cell renewal was investigated using near-native, cultured human colonic crypts. Live intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture in which conditions were optimised for steady-state tissue renewal. Crypts were observed either in control conditions, stimulated with Wnt3a or denied Wnt exposure due to the presence of its antagonist Dkk1. From these observations, it was possible to determine the effect of Wnt signalling on crypt cell kinetics. Results Cell proliferation and canonical Wnt signalling pathway activity predominated at the base of native human colonic crypts. Colonic crypt morphology was preserved in culture, demonstrated by the presence of goblet cells, enteroendocrine cells and cell polarity. Proliferation in the crypt-base was significantly higher in crypts stimulated with Wnt3a compared to control conditions, although the rate of cell migration between the two groups was not significantly different. The absence of Wnt ligand or the presence of the Wnt inhibitor Dkk1 caused crypts to shrink in length compared to Wnt3a and control groups without affected crypt cell migration. Conclusions Canonical Wnt signalling is required for steady-state tissue renewal in the human colonic epithelium. Proliferation and migration within the crypt are regulated independently of each other, thus appropriate regulation of cell Cproliferation is constantly required to maintain the crypt cell population.
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