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

Abstract

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 TGFβ/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, 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.

Conclusions: Steady-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.

Keywords: Epithelial Differentiation; Epithelial Kinetics; Epithelial Proliferation; Imaging; Stem Cells.

Figure 1

Hierarchy of stem/progenitor cell proliferation along the native human colonic crypt-axis. (A) Classical profile for Ki-67 immunolabelling (red) of microdissected (ie, native) human colonic crypts; B-base, SB supra-base, M-mid, T-top. (B) Ki67-positive cell nuclei predominate at the crypt-base and the mid-crypt region (N=13 subjects, n=58 crypts). (C) Dual in situ hybridisation and immunolabelling of lgr5-mRNA (red) and OLFM4 protein (white) identifies a population of lgr5-mRNA+/OLFM4+cells at the base of native human colonic crypts; E-CAD (green) demarks crypt cell membranes; filled white arrows indicate cells exhibiting intense fluorescent labelling for lgr5-mRNA and OLFM4; open arrow denotes nucleus of pericryptal myofibroblast; asterisk signifies a goblet cell with nucleus in the confocal image plane; scale bar—30 mm. (D) Double immunolabelling of LGR5 protein (red) and OLFM4 (white) confirms congruent expression of both intestinal stem cell markers by individual crypt cells within the crypt-base; annotations as above; see online supplementary figure S1 for schematic representation. (E) Quantification of cell types according to stem cell marker expression and cell morphology along the crypt-axis (see online supplementary figure S1 for an example, of lgr5-mRNA/MUC-2 double labelling); the congruence of OLFM4 and either lgr5-mRNA or LGR5 protein expression was 96%±4% (mean±SD, n=20 microdissected crypts from N=4 subjects). (F) Analysis of crypt stem/progenitor cell proliferation (n=10 crypts from N=5 subjects; see online supplementary figure S1 for a crypt image of double OLFM4/Ki-67 immunolabelling). Arrows indicate examples of intense labelling for lgr5+/OLFM4+ stem cells. *Denotes an example of a goblet cell with nucleus in plane of focus. DIC, differential interference contrast; E-CAD, E-cadherin.

Figure 2

Wnt/β catenin and SMAD signalling profiles along the native human colonic crypt-axis. (Ai) Immunolabelling of total β catenin performed on native human colonic crypts reveals intense membrane and nuclear localisation at the colonic crypt-base. (Aii) The fluorescence intensity of nuclear β catenin predominates at the crypt-base and diminishes progressively towards the top of the crypt (Spearman Rank, r=−0.47, p<0.001). (B) A similar gradient exists for the immunofluorescence intensity of nuclear Axin-2 labelling (r=−0.48, p<0.001). Conversely, the immunofluorescence for nuclear phospho-SMAD 2,3 (C) predominates in the mid-crypt region, while nuclear phospho-SMAD 1,5,8 (D) exhibits a retrogradient that is more intense at the crypt opening (r=0.84, p<0.04). All values in each bar chart were normalised to the intensity value at the crypt-base. For each antibody, data were collated from n≥10 crypts microdissected from N≥3 patients. Filled arrowheads indicate intense nuclear labelling; open arrowheads mark nuclei of lower fluorescence intensity.

Figure 3

A combination of Wnt pathway activators and TGFβ/BMP pathway inhibitors is required for maintenance of cultured human colonic crypts ex vivo (A) Overview of human colonic crypts cultured within a Matrigel droplet under optimised conditions described in panel D; the bright field image was created by stitching together an array of 12 adjacent fields of view taken with a ×4 objective lens; scale bar=0.5 mm. (B) Enlargement of insert depicted in (A) representing a typical field of view (×4 objective lens); example crypt-base and shedding domains are denoted by open and closed arrowheads, respectively; *dead crypt fragments; scale bar=0.5 mm. (C) Example paired differential interference contrast images (×20 objective) of human colonic crypts cultured under optimised conditions for 0–4 and 0–7 days; d1=day 1, d4=day 4, d7=day 7; scale bar=100 μm. (D) Quantification of crypt length at day 4 or day 7 (with respect to the initial crypt length 4 h post-isolation, Day 0) following culture in the presence of the indicated combination of recombinant human growth factors, recombinant human BMP binding protein and/or small molecule ALK 4/5/7 inhibitor: IGF-1 (50 ng/mL), Gremlin-1 (200 ng/mL), Noggin (100 ng/mL), Wnt3A (100 ng/mL), R-Spondin-1 (500 ng/mL), A83-01 (0.5 μM); n≥6 crypts derived from N≥3 subjects.

Figure 4

Exogenous and crypt-autonomous Wnt ligand promotes canonical Wnt/β catenin signals in cultured human colonic crypts. (A) Confocal images of dephospho β catenin immunolabelling following treatment with exogenous Wnt-3A (100 ng/mL, 30 min), in the presence or absence of Dikkopf-1 (DKK-1; 800 ng/mL); bar chart illustrates image analysis of nuclear immunofluorescence intensity. (B) Visualisation and analysis of nuclear Axin-2 3 days post-transduction with adenoviral GFP (Control: green Ad-GFP; red—Axin2) or dominant negative-TCF4. Effects of IWP2 (2 μM) on lentiviral (LV)-TOP-GFP expression (C) and nuclear axin-2 (D) immunofluorescence following 3 days culture. (E) Immunolabelling of human Wnt-3A (arrows indicate intense labelling basal membranes) and expression of Wnt-3A mRNA by RT-PCR using cDNA from freshly isolated human colonic crypts; expected Wnt 3A PCR product is 404 bp and the arrow denotes a 500 bp marker. All values in (A)–(D) bar charts were normalised to the control value in the crypt-base region. Control media: for A, C and D=IGF-1 (50 ng/mL)/Noggin (100 ng/mL)/R-spondin-1 (500 ng/mL); Wnt-3A (100 ng/mL) where indicated; for B=IGF-1 (50 ng/mL)/Noggin (100 ng/mL)/R-spondin-1 (500 ng/mL)/Wnt-3A (100 ng/mL). Statistical significance assessed by ANOVA followed by Tukey's post-hoc analysis; significant differences between pairs of mean values are indicated by linked dashed lines, *p<0.01; n≥4 crypts for each experimental group and the data are representative of at least three independent experiments in each case.

Figure 5

TGFβ and BMP pathway activation inhibits canonical Wnt signalling along the cultured human colonic crypt-axis. (A) Confocal images of phospho-SMAD2,3 immunolabelling following treatment with TGFβ (20 ng/mL, 2 days), in the presence or absence of A83-01 (0.5 μM); bar chart illustrates image analysis of nuclear immunofluorescence intensity. (B) Effects of BMP (100 ng/mL, 2 days) and/or noggin (100 ng/mL) on nuclear phospho-SMAD1,5,8 immunofluorescence intensity levels. (C) TGFβ and (D) BMP suppression of nuclear Axin-2 immunofluorescence, and rescue by pretreatment with noggin or A83-01, respectively. All values in (A–D) were normalised to the control value in the crypt-base region. Culture conditions: (A and C)—IGF-1 (50 ng/mL)/R-spondin-1 (500 ng/mL)/Wnt-3A (100 ng/mL)/Noggin(100 ng/mL) and TGFβ (20 ng/mL) and/or A83-01 (0.5 μM) where indicated; (B and D)—IGF-1 (50 ng/mL)/R-spondin-1 (500 ng/mL)/Wnt 3A (100 ng/mL)/A83-01 (0.5 μM) and BMP (100 ng/mL) and/or noggin (100 ng/mL) where indicated. Significant differences were assessed by ANOVA followed by Tukey's post-hoc analysis; significant differences between pairs of mean values are indicated by linked dashed lines; ^#p<0.01, *p<0.02, ^&p<0.05; n≥4 crypts for each experimental group and the data are representative of at least three independent experiments in each case. Scale bars=75 μm.

Figure 6

Canonical Wnt signals maintain cultured human colonic crypt stem/progenitor cell proliferation. (A) Effects of exogenous Wnt-3A (100 ng/mL) and/or DKK-1 (800 ng/mL) on nuclear BrdU uptake and Ki67 labelling after 3 days culture. (B) Dominant-negative TCF4 abrogates crypt cell proliferation 3 days post-transduction. (C) Coexpression of OLFM4 and LGR5 by a population of slender cells (arrowheads) interspersed between goblet-like cells (asterisk) located at the base of human colonic crypts cultured for 4 days. (D) The relative effects of Wnt-3A (100 ng/mL) and DKK-1 (800 ng/mL) on the percentage of OLFM4-positive cells following 3 days in culture. (E) Confocal images and (F) image analysis of LGR5 immunolabelling following 4 days in culture: suppression by IWP2 (2 μM) and rescue by exogeneous Wnt-3A (100 ng/mL). (G) Wnt pathway activators promote BrdU incorporation into the nuclei of LGR5-positive colonic crypt cells. (H) Immunolabelling of differentiated cell types in cultured colonic crypts: distinct labelling of cells positive for (i) MUC-2 or OLFM4 (arrows), (ii) chromogrannin A or OLFM4, and (iii) COX-1; all shown at the base of human colonic crypts; (iv, v) intense FABP1 labelling at the crypt opening (asterisk and bracket indicate crypt-base). The effects of the Notch inhibitor, DBZ (1 mM) on goblet cell number and OLFM4-positive cell number illustrated in (H) are quantified in (I and J), respectively. Significant differences were assessed by ANOVA followed by Tukey's post-hoc analysis; significant differences between pairs of mean values are indicated by linked dashed lines; *p<0.001, ^$p<0.002, ^øp<0.02, ^#p<0.01, ^&p<0.05. Scale bars=50 μm. Control media: I=IGF-1 (50 ng/mL), N=Noggin (100 ng/mL), R=R-spondin-1 (500 ng/mL), A83-01 (0.5 μM); W3A or W=Wnt-3A (100 ng/mL), DKK-1 (Dikkopf-1; 800 ng/mL) and DBZ=dibenzazepine (1 mM) where indicated.

Figure 7

Activation of TGFβ or BMP pathways suppress cultured human colonic crypt stem/progenitor cell proliferation. (A) Effects of treatment with TGFβ (20 ng/mL, 2 days) and/or the ALK4/5/7 inhibitior A83-01 (0.5 μM) on nuclear incorporation of BrdU incorporation into human colonic crypt cells. (B) A pan-specific monoclonal TGFβ antibody (10 μg/mL) mimicks the effects of A83-01 (0.5 μM) on crypt cell proliferation; the irrelevant monoclonal anti-COX2 (10 μg/mL) was included as an IgG1 control. (C) BMP (100 ng/mL) abolishes human colonic crypt cell proliferation. Noggin (100 ng/mL) promotes crypt cell proliferation and prevents the inhibitory effects of BMP4. (D) The BMPR1 (ALK2/3) inhibitor DMH-1 (1 μM) mimics the stimulatory effects of noggin on crypt cell proliferation. (E) BMP pathway or TGFβ pathway activation suppress LGR5 immunolabelling. Significant differences were assessed by ANOVA followed by Tukey's post-hoc analysis; significant differences between pairs of mean values are indicated by linked dashed lines; *p<0.001, ^&p<0.05; n≥4 crypts for each experimental group and the data are representative of at least three independent experiments in each case. Control media: (A and B) W/I/N/R; (C and D) W/I/R/A83-01; (E) W/I/N/R/A83-01. W=Wnt 3A, I=IGF-1, ‘N’ or ‘Nog’=noggin, R=R-spondin-1.

Figure 8

Crypt cell migration and shedding complete tissue renewal ex vivo. (A) A BrdU pulse-chase experiment demonstrating the relative upward migration of cells from the crypt-base along the crypt-axis in the absence (IGF-1, 50 ng/mL and Noggin, 100 ng/mL) or presence of Wnt stimulation (Wnt-3A, 100 ng/mL and R-spondin-1, 500 ng/mL); for each condition, images are shown 1 h following the BrdU pulse and 2 days after the ‘cold’ chase. (B) Analysis of BrdU pulse-chase images illustrating the respective decrease and increase in BrdU-positive crypt cells in the lower and upper half of the crypt in the presence of Wnt stimulation. (C) Analysis of time-lapse data acquired during days 2–3 in culture under different degrees of Wnt and TGFβ signalling pathway activation; relative changes in crypt length (Ci) and crypt cell mitoses (Cii) versus constant crypt cell migration (Ciii) are illustrated. (Di) hierarchy of calcein-labelled live cells and propidium iodide (PI)-positive dead cells; (Dii) cells at the crypt opening are positive for activated caspase 3 and (Diii) the number of PI-positive shed cells increases over time in culture (see online supplementary movie S4). Significant differences were assessed by ANOVA followed by Tukey's post-hoc analysis; significant differences between pairs of mean values are indicated by linked dashed lines; *p<0.01. W=Wnt 3A, I=IGF-1, N=noggin, R=R-spondin-1, DKK-1=dikkopf-1.