Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts

Abstract

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells. Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. CD24(+) Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24(+) cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.

Figure 1. Geometric distribution pattern of Paneth cells and Lgr5 stem cells

a,b: Confocal cross section of Lgr5-EGFP-ires-CreERT2 intestine. E-cadherin (a: white) demarcates cell borders. Lgr5 stem cells (green) and Paneth cells (a, black; b, lysozyme: red). c: Contact area of either Paneth cells or Lgr5 stem cells was quantified with Image J. The values are depicted as mean ± standard deviation from three independent mice. Red columns and green columns indicate contact area with Paneth cells and Lgr5 stem cells, respectively. d: Stills from Suppl Movie 1. Time course of crypt organoid growth. Differential interference contrast image reveals granule-containing Paneth cells (red arrowheads) at the site of budding where a new crypt forms. Lgr5-GFP (green) stem cells expand at crypt base in close proximity to Paneth cells. *: autofluorescence. Scale bar: 50 μm

Figure 2. Paneth cells express CD24 and support growth of Lgr5 stem cells

a: Isolated small intestinal crypt. CD24 (red) is expressed by Paneth cells in which granules are visualized by differential interference contrast. Lgr5-GFP⁺ stem cells (green). Counterstain: DAPI (blue). b: Isolated colonic crypt. CD24⁺ cells (red) are in intimate contact with Lgr5 stem cells (green). Top: longitudinal crypt section; bottom: section through crypt bottom c: FACS plot of dissociated single cells from small intestinal crypts. Two CD24 bright populations differ by side-scatter (SSC) pattern. Sorted CD24^hi/SSC^low and CD24^hi/SSC^hi cells are subsequently stained. CD24^hi/SSC^low cells are positive for the enteroendocrine marker Chromogranin A (top right), while CD24hi/SSChi cells are positive for the Paneth marker Lysozyme (bottom right). d-g: Single sorted Lgr5 stem cells from Lgr5-EGFP-ires-CreERT2 small intestine (d), Sorted single Paneth cells (e) from wild type small intestine and a combination of the two cell types (f) were seeded in round-bottom wells and cultured for 2 days. g: Lgr5 stem cells form expanding Lgr5-GFP⁺ (green) organoids only when reassociated with Paneth cells. h: Stills from Suppl Movie 2. Time course of the reassociation culture with RFP⁺ Lgr5-GFP stem cells (red) and YFP⁺ Paneth cells (yellow). Scale bar: 50 μm.

Figure 3. Paneth cells produce Wnt3 and other essential niche signals for Lgr5 stem cells

a: Heatmap of two independent microarray expression experiments (1/2 and 3/4) performed with dye-swap (1 vs 2 and 3 vs 4) from sorted Paneth cells vs Lgr5 stem cells. b: Wnt3 is expressed by Paneth cells at crypt bottoms as analyzed by in situ hybridization, c-h: Localized Wnt production regulates crypt-villus morphogenesis in culture. c: Freshly isolated crypts from Axin2-lacZ mouse were cultured in standard EGF/Noggin/Rspondin1 medium (ENR) medium for 4 days. LacZ response is only seen near the bottoms of the two crypts. d: Intestinal adenoma samples from Apc^min mice were cultured in ENR medium in the absence of R-spondin for 7 days. e: Axin2-LacZ crypts grown in ENR medium. f: As in e) with the addition of porcupine inhibitor IWP1 at 1 μM. g: Crypts from Axin2-LacZ mouse cultured in ENR medium plus Wnt3a h: same as g) with the addition of IWP1. Insets in e-h depict Axin2-LacZ expression (blue). e, g, h: 6 days culture. f: 3 days culture after which the organoids disintegrate. See also Suppl Fig 2. i: Plating efficiency of Lgr5 stem cell-Paneth doublets, Lgr5 stem cell doublets, single Paneth cells and single Lgr5 stem cells with (grey) or without (black) Wnt-3A at 100 ng ml^-1. Assays were read out as budding organoids at 14 days after sorting. The values are depicted as mean±standard error of the mean (SEM) from three independent experiments. N.D.; not detected. See also Suppl Fig 3 and online methods for detail of doublet isolation and culture.

Figure 4. Paneth cells regulate numbers of intestinal stem cells in vivo

a-k: Paneth cells and stem cells in constitutive models of Paneth cell decrease. a-c: lysozyme stain (brown arrowheads indicate positive cells) and d-f: Olfm4 staining of crypts of adult (6-7 week-old) mice of the indicated genotypes. g-j: double stain, lysozyme (brown), Olfm4 (blue) of a representative crypt of the indicated genotype. Brown and blue arrowheads indicate Paneth cells and stem cells, respectively. k: Quantification of stem and Paneth cell numbers in both models. For each bar, 100 crypts were scored for each of three mice. Mutant mice (red) and their control mice (blue). *: p<0.01. l-t: Paneth cells and stem cells in inducible Paneth cells depletion model. Mice indicated genotypes were injected with β-napthoflavone to induce Cre and were analyzed 54 or 67 days after Cre induction by staining for Sox9 protein (brown), Olfm4 or Cryptdin mRNA (blue). Serial sections: i,o,r; m,p,s; and n,q,t. See text for experimental detail. Note the absence of Paneth cells (s) and stem cells (p) in Sox9-/- crypts (m). Scale bar: 50 μm