Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche - PubMed (original) (raw)

Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche

Akifumi Ootani et al. Nat Med. 2009 Jun.

Abstract

The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.

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Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/.

Figures

Figure 1

Long-term intestinal culture. (a) Time-course analysis of short-term air-liquid interface culture of neonatal small intestine. Stereomicroscopy shows the progressive growth of intestinal cultures, forming cyst-like structures in the collagen gel. Arrowheads indicate growth of individual spheres. Scale bars, 1 mm. (b) Long-term stereomicroscopy of air-liquid interface cultures of neonatal small intestine in collagen gel. The same field is visualized. Scale bars, 5 mm. (c) Histological analysis of small intestinal cultures (day 10). The wall of the intestinal spheres consists of a polarized epithelial monolayer and outer lining myofibroblasts (arrowheads). Tall-columnar absorptive enterocytes are visible on H&E staining. Alcian blue (Al-B) staining shows goblet cells with secreted mucus in the lumen. Chromogranin A immunohistochemistry reveals the presence of enteroendocrine cells. Lysozyme immunohistochemistry shows Paneth cells. PCNA staining reveals active proliferation of jejunal cultures at day 217. Scale bars, 10 μm. (d) Stereomicroscopy of long-term colon culture. Scale bars, 1 mm. The same field of view is depicted in both photographs. (e) Histology of long-term colonic cultures. Differentiated colonic epithelial cells are visible on H&E staining (top; day 92, middle; day 357). PCNA staining (bottom) shows continued active proliferation of cultured colonic mucosa at day 357. (f) Electron micrographs of jejunal culture (day 14). Goblet cells (G) contain mucus granules (M) in their apical cytoplasm. The epithelium is lined by myofibroblasts (MF). The apical surface of absorptive cells (A) shows microvilli (MV) ending on the terminal web (TW). Note the well organized tight junction (arrowhead) and desmosome (arrow) at the basolateral domain anchoring adjacent epithelial cells. Enteroendocrine cells (EE) contain secretory granules (SG) in their basal cytoplasm.

Figure 2

Intestinal cultures from juvenile and adult mice. (a–h) Histology of jejunal culture at day 7 from 3-week-old (a–f) or 26-week-old mice (g,h). Staining for H&E (a,b,g,h), PCNA (c,d) or CD44 (e,f) is depicted. (i,j) RSpo1-Fc treatment permitted longer-term jejunal culture (day 28) from 8-week-old adult intestine. Stereomicroscopy (i) and H&E staining (j) is depicted. Arrows indicate highly proliferative, PCNA+ crypt-like structures, which invaginated from the sphere wall into the surrounding collagen matrix. Arrowheads indicate quiescent PCNA– villus-like protrusions. Numerous sludged or dead cells are present in the sphere lumen, indicated by the asterisk.

Figure 3

Wnt signaling regulates proliferation of cultured intestinal epithelium. (a–c) Dkk1 inhibits intestinal epithelial growth. (a) Stereomicroscopy and histology of Dkk1-treated jejunal cultures. Cultures were maintained with Dkk1 (50 μg ml−1) for 7 d followed by incubation without Dkk1 for an additional 28 d. (b) Dose-dependent inhibition of jejunal sphere growth by Dkk1. Cultures were treated with various concentrations of Dkk1 on days 0–7. The number of spheres was counted. Error bars indicate s.e.m.; n ≥ 7. *P < 0.05 versus vehicle control (VC); **P < 0.01 versus VC. (c) H&E and PCNA staining after Dkk1 treatment of pre-established intestinal cultures. Cultures were preestablished for 28 d followed by Dkk1 treatment for 5 d. (d–h) RSpo1-Fc treatment of intestinal epithelial cultures. (d) Stereomicroscopic visualization and PCNA staining of the jejunal cultures. RSpo1-Fc was included once weekly until harvest at day 29. (e) H&E and PCNA staining of colon cultures that were preestablished for 28 d followed by RSpo1-Fc treatment for 5 d. (f–h) Proliferative activity (f), number (g) and size (h) of jejunal and colon spheres. RSpo1-Fc was included once weekly for 5 weeks (f) or from days 0–7 (g,h). Error bars indicate s.e.m.; n ≥ 7. *P < 0.05 versus VC; **P < 0.01 versus VC.

Figure 4

Notch and Neurogenin-3 regulate intestinal cell fate in vitro. (a) Treatment with Notch inhibitor dibenzazepine (DBZ) in neonatal jejunal cultures. H&E and PAS staining show morphological differentiation and mucus secretion of the epithelial cells. PCNA staining reveals active proliferation of the cultured cells. (b) Adenoviral expression of Neurogenin3 (Ad Ngn3) or a control immunoglobulin Fc fragment (Ad Fc) in jejunal cultures. Immunohistochemistry of chromogranin A demonstrates enteroendocrine cell differentiation in vitro (top). Percentages of enteroendocrine cells per sphere were analyzed (bottom). Error bars represent s.e.m.; n ≥ 8 per condition. **P < 0.01 versus Ad Fc.

Figure 5

Putative intestinal stem cell populations with or without R-spondin1 treatment in culture. (a,b) Jejunal cultures contain Bmi1 lineage–derived cells. (a) Whole-mount LacZ staining of intestinal spheres. Bmi1+ cells and their progeny are identified as blue LacZ+ patches in RSpo1-Fc treatment versus vehicle control. (b) Frozen section of LacZ-stained intestinal spheres showing LacZ+ Bmi1 lineage–derived cells and their progeny in the epithelial layer of RSpo1-Fc-treated cultures. (c) Fluorescent in situ hybridization (FISH) for Lgr5 (orange) with simultaneous DAPI nuclear stain (blue) at culture day 35. (d) Percentage of Lgr5+ cells as a fraction of epithelial cells after RSpo1-Fc treatment compared to vehicle control at day 35. RSpo1-Fc was added once weekly for 5 weeks. Error bars indicate s.e.m.; n ≥ 4. *P < 0.05 versus vehicle control. (e) In situ hybridization (ISH) for Lgr5 in vivo from mice 7 d after single intravenous administration of adenoviruses encoding RSpo1-Fc (Ad RSpo1-Fc) or antibody Fc control (Ad Fc). (f) Histology of jejunum and colon from Ad RSpo1-Fc–treated– or Ad Fc–treated mice. H&E and PCNA staining of intestine 7 d after single intravenous injection of Ad RSpo1-Fc or Ad Fc is depicted (e,f). Scale bars, 100 μm.

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Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche - PubMed (original) (raw)