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

Abstract

BackgroundA 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.ObjectiveTo investigate the regulation of stem cell-driven tissue renewal by canonical Wnt and TGFβ/bone morphogenetic protein (BMP) pathways in the native human colonic epithelium.DesignIntact 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.ResultsNative human colonic crypts exhibited distinct activation profiles for canonical Wnt, TGFβ 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 TGFβ 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.ConclusionsSteady-state tissue renewal in the native human colonic epithelium is dependent on canonical Wnt signals combined with suppressed TGFβ/BMP pathways. Stem/progenitor cell proliferation is uncoupled from crypt cell migration and shedding, and is required to constantly replenish the crypt cell population.

A Human Colonic Crypt Culture System to Study Regulation of Stem Cell-Driven Tissue Renewal and Physiological Function

“…This 'mini-gut' culture system formed the basis for the development of human organoid cultures from the intestinal epithelium [13,14,1] and from (inducible) pluripotent stem cells [15], both of which hold the potential for therapeutic transplantation [16,17]. For the study of native intact human colonic crypts, we adopted a similar approach whereby crypts are embedded in a basement membrane-derived matrix, Matrigel [18], instead of collagen I [7], and serum-free culture medium is supplemented with the intestinotrophic factors IGF-1 (activator of RAS signalling pathway) and R-spondin (activator of Wnt signals) in combination with inhibitors of BMP (Noggin or Gremlin proteins) and TGF (SB431542 or A83-01 small molecules) signalling pathways [18]. The established human colonic crypt culture system provides a near-native model of a self-renewing tissue unit whereby epithelial cell polarity, tissue topology and the hierarchy of stem cell-driven tissue renewal are maintained.…”

“…Development of a validated near-native colonic crypt culture system ex vivo provides the opportunity to gain insights into the regulation of (patho)physiological function. For example, assessment of signalling pathway status in immediately fixed clinical human colorectal tissue samples, together with assessment of the effects of pathway activation/inhibition on the processes of crypt (stem) cell proliferation, migration and shedding in cultured crypts, demonstrated that activation of canonical Wnt signals in combination with suppressed TGF/BMP pathways promotes renewal of the native human colonic epithelium [18,19]. Similarly, comparison of polarised expression patterns for NKCC1 in immediately fixed clinical human colorectal tissue samples with near-native cultured human colonic crypts proved invaluable when studying the functional regulation of fluid secretion by calcium-dependent internalisation of NKCC1 in native polarised colonic crypt cells [7].…”

“…4. To monitor crypt cell volume [7] or viability [18], load crypts with calcein-AM (5 M) for one hour, wash twice with HBS (2.4.3). Excite at 488 nm and visualise emission at 520 nm.…”

“…Scrambled probes should be used as a negative control. A positive control probe for a ubiquitously expressed gene such as beta actin should also be used [18].…”

Tissue Engineering Functional Gastrointestinal Regions: The Importance of Stem and Progenitor Cells

“…Challenges with identifying the low levels of Lgr5 as a marker lead to the discovery of Olfm4-a specific marker for Lgr5-positive stem cells that is more highly expressed in the small intestine, but not in other murine gastrointestinal (GI) tissues or bone marrow (Schuijers et al 2014). Olfm4 is highly expressed in human colon stem cells (Reynolds et al 2014). Ascl2, a neural notch pathway target, functions in the intestine as a critical Wnt target allowing crypt proliferation (van der Flier et al 2009).…”