A human colonic crypt culture system to study regulation of stem cell-driven tissue renewal and physiological function.

The intestinal epithelium is one of the most rapidly renewing tissues in the human body and fulfils vital physiological roles such as barrier function and transport of nutrients and fluid. Investigation of gut epithelial physiology in health and disease has been hampered by the lack of ex vivo models of the native human intestinal epithelium. Recently, remarkable progress has been made in defining intestinal stem cells and in generating intestinal organoid cultures. In parallel, we have developed a 3D culture system of the native human colonic epithelium that recapitulates the topological hierarchy of stem cell-driven tissue renewal and permits the physiological study of native polarized epithelial cells. Here we describe methods to establish 3D cultures of intact human colonic crypts and conduct real-time imaging of intestinal tissue renewal, cellular signalling, and physiological function, in conjunction with manipulation of gene expression by lentiviral or adenoviral transduction. Visualization of mRNA- and protein-expression patterns in cultured human colonic crypts, and cross-validation with crypts derived from fixed mucosal biopsies, is also described. Alongside studies using intestinal organoids, the near-native human colonic crypt culture model will help to bridge the gap that exists between investigation of colon cancer cell lines and/or animal (tissue) studies, and progression to clinical trials. To this end, the near native human colonic crypt model provides a platform to aid the development of novel strategies for the prevention of inflammatory bowel disease and cancer.

Luminal microbes promote monocyte-stem cell interactions across a healthy colonic epithelium.

The intestinal epithelium forms a vital barrier between luminal microbes and the underlying mucosal immune system. Epithelial barrier function is maintained by continuous renewal of the epithelium and is pivotal for gut homeostasis. Breaching of the barrier causes mobilization of immune cells to promote epithelial restitution. However, it is not known whether microbes at the luminal surface of a healthy epithelial barrier influence immune cell mobilization to modulate tissue homeostasis. Using a mouse colonic mucosal explant model, we demonstrate that close proximity of luminal microbes to a healthy, intact epithelium results in rapid mucus secretion and movement of Ly6C(+)7/4(+) monocytes closer to epithelial stem cells. These early events are driven by the epithelial MyD88-signaling pathway and result in increased crypt cell proliferation and intestinal stem cell number. Over time, stem cell number and monocyte-crypt stem cell juxtapositioning return to homeostatic levels observed in vivo. We also demonstrate that reduced numbers of tissue Ly6C+ monocytes can suppress Lgr5EGFP+ stem cell expression in vivo and abrogate the response to luminal microbes ex vivo. The functional link between monocyte recruitment and increased crypt cell proliferation was further confirmed using a crypt-monocyte coculture model. This work demonstrates that the healthy gut epithelium mediates communication between luminal bacteria and monocytes, and monocytes can modulate crypt stem cell number and promote crypt cell proliferation to help maintain gut homeostasis.

Canonical Wnt signals combined with suppressed TGFß/BMP pathways promote renewal of the native human colonic epithelium.

BACKGROUND: A defining characteristic of the human intestinal epithelium is that it is the most rapidly renewing tissue in the body. However, the processes underlying tissue renewal and the mechanisms that govern their coordination have proved difficult to study in the human gut. OBJECTIVE: To investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFß/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium. DESIGN: Intact human colonic crypts were isolated from mucosal tissue samples and placed into 3D culture conditions optimised for steady-state tissue renewal. High affinity mRNA in situ hybridisation and immunohistochemistry were complemented by functional genomic and bioimaging techniques. The effects of signalling pathway modulators on the status of intestinal stem cell biology, crypt cell proliferation, migration, differentiation and shedding were determined. RESULTS: Native human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFß and BMP pathways. A population of intestinal LGR5/OLFM4-positive stem/progenitor cells were interspersed between goblet-like cells within the crypt-base. Exogenous and crypt cell-autonomous canonical Wnt signals supported homeostatic intestinal stem/progenitor cell proliferation and were antagonised by TGFß or BMP pathway activation. Reduced Wnt stimulation impeded crypt cell proliferation, but crypt cell migration and shedding from the crypt surface were unaffected and resulted in diminished crypts. CONCLUSIONS: Steady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFß/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population.

Dynamic and differential regulation of NKCC1 by calcium and cAMP in the native human colonic epithelium.

The capacity of the intestine to secrete fluid is dependent on the basolateral Na(+)-K(+)-2Cl(-) co-transporter (NKCC1). Given that cAMP and Ca(2+) signals promote sustained and transient episodes of fluid secretion, respectively, this study investigated the differential regulation of functional NKCC1 membrane expression in the native human colonic epithelium. Tissue sections and colonic crypts were obtained from sigmoid rectal biopsy tissue samples. Cellular location of NKCC1, Na(+)-K(+)-ATPase, M3 muscarinic acetylcholine receptor (M(3)AChR) and lysosomes was examined by immunolabelling techniques. NKCC1 activity (i.e. bumetanide-sensitive uptake), intracellular Ca(2+) and cell volume were assessed by 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), Fura-2 and differential interference contrast/calcein imaging. Unstimulated NKCC1 was expressed on basolateral membranes and exhibited a topological expression gradient, predominant at the crypt base. Cholinergic Ca(2+) signals initiated at the crypt base and spread along the crypt axis. In response, NKCC1 underwent a Ca(2+)-dependent 4 h cycle of recruitment to basolateral membranes, activation, internalization, degradation and re-expression. Internalization was prevented by the epidermal growth factor receptor kinase inhibitor tyrphostin-AG1478, and re-expression was prohibited by the protein synthesis inhibitor cylcoheximide; the lysosome inhibitor chloroquine promoted accumulation of NKCC1 vesicles. NKCC1 internalization and re-expression were accompanied by secretory volume decrease and bumetanide-sensitive regulatory volume increase, respectively. In contrast, forskolin (i.e. cAMP elevation)-stimulated NKCC1 activity was sustained, and membrane expression and cell volume remained constant. Co-stimulation with forskolin and acetylcholine promoted dramatic recruitment of NKCC1 to basolateral membranes and prolonged the cycle of co-transporter activation, internalization and re-expression. In conclusion, persistent NKCC1 activation by cAMP is constrained by a Ca(2+)-dependent cycle of co-transporter internalization, degradation and re-expression; this is a novel mechanism to limit intestinal fluid loss.