Fig 1.
Mouse multi-layered glandular structures (MLGS) show homology to human esophageal submucosal glands ducts (SMGD) and human multi layered epithelium (MLE).
[a] Human esophageal submucosal gland (SMG) duct (black dotted box) and mouse MLGS at the SCJ (green dotted box) upon bile stimulation. IF staining for squamous markers K14 (red) and K5 (green) and columnar markers K8 (green) and K7 (red). [b] PAS and Alcian blue staining of multi-layered glandular structures (MLGS) at the SCJ. [c] Serial sections of a MLGS at the SCJ in mice with bile reflux, which extends towards the surface (arrow). [d] IHC for p63, K5, K7 and Alcian blue staining of human early non-goblet cell columnar metaplasia (red dotted line) arising from submucosal multi-layered glandular structures (black dotted box), human esophageal SMGD and murine MLGS. (Scale bars 100μm).
Fig 2.
Human BE refluxates mainly contains conjugated bile acids and stimulates expansion of multi-layered gland at the SCJ in mice.
[a] HPLC analysis of the bile composition of the refluxates of 16 BE patients. [b] Concentration of bile acids in BE patient’s refluxates determined by HPLC. [c,d,e,f,g and h] Analyses of mice after 8 weeks of treatment with human refluxates via oral gavage combined with acidified drinking water pH2.0 (n = 50), acidified drinking water alone (n = 21), or normal tap water (n = 12). [c] Glands (encircled) at the SCJ (line) in mice after treatment. [d] Single staining for K14 and K19 using IHC and double staining for K14 (red) and K19 (green) using IF of MLGS at the SCJ in mice. DAPI (blue) was used as a nuclear counterstain. [e] Percentage of mice showing glands at their SCJ after treatment. [f] Average number of glands at the SCJ per mouse after treatment. Data are represented as (±SD), Unpaired t test *p<0.05, *p<0.01, ***p<0.001, ****p<0.0001. [g] Percentage of mice showing glands at the SCJ, which secrete acidic mucins after treatment. [h] Secretion of acidic mucins in the glandular structures per mouse after treatment, represented by the expression level of Alcian blue staining (S2C Fig). Data are represented as (±SD), Unpaired t test *p<0.05, *p<0.01, ***p<0.001, ****p<0.0001. (Scale bars 100μm).
Fig 3.
Glycine-conjugated BAs are most effective in inducing the development of multi-layered glandular structures and an intestinal type of metaplasia in mice.
[a] H&E and Alcian blue staining of multi-layered glands (MLGS) at the SCJ of mice after 16 weeks of treatment with individual bile components (n = 6 per bile component). [b] Quantification of number of glands and [c] expression level of Alcian blue staining inside the glandular structures (S2C Fig) at the SCJ in mice treated with single BAs for 16 weeks. Treatment with single BAs was compared to treatment with acidified water (pH 2.0). Data are represented as mean±SD. Unpaired t test *p<0.05, *p<0.01, ***p<0.001, ***p<0.0001. Taurocholic (TC), Taurodeoxycholic (TDC), Taurochenodeoxycholic (TCDC), Glycocholic (GC), Glyco-deoxycholic (GDC) and Glycochenodeoxycholic acid (GCDC). [d] Alcian blue staining of MLGS at the SCJ in mice. Goblet cell like structures (encircled) after 16 weeks of glycine-conjugated bile acid treatment. [e] Mucin2 (MUC2) staining of MLGS at the SCJ in mice after treatment with glycine-conjugated bile acids. (Scale bars 100μm).
Fig 4.
Mice MLGS are a stem cell niche of distinct populations of columnar and squamous stem cells, where K5 positive cells give rise to squamous lineages.
[a] Expression of SOX2, K14 (squamous stem cell markers) and DCLK1, Lgr5-lacZ, SOX9 and Olfm4 (in situ) (gastric or intestinal stem cell markers) in MLGS.[b] Lineage tracing of Lgr5+ cells (i), Dclk1+ cells (ii) and K5+ cells (iii) after Tamoxifen injection in Lgr5-cre (n = 10), Dclk1-cre (n = 10) and K5-cre mice (n = 10) crossed with Rosa26-Tomato-GFP mice. Results of n = 3 mice are shown per cell lineage. All mice were treated with a patient’s refluxate for 8 weeks. Outer squamous (green) layer (closed arrow), inner (red) columnar layer (open arrow). [c] MLGS at the SCJ in mice treated with patient’s refluxate for 8 weeks showing DCLK1 (IHC, brown) and Lgr5-lacZ (blue) positive cells. [d] IF staining for Ki67 (green) and K14 (red) of human SMG ducts and MLGS at the SCJ in mice treated for 8 weeks with patient’s refluxate. Ki67 expression (green), K14 (red) expression in human SMGD and murine MLGS as found at the SCJ. Squamous cells at the upper part towards the luminal surface are indicated by closed arrows. The lower part of the duct and glands are indicated by open arrows. DCLK1 (green) and BrdU (red) expression in the murine MLGS. DAPI (blue) is used as a counterstain. (Scale bars 100μm).
Fig 5.
Glycine-conjugated BAs cause budding and branching of MLGS followed by crypt fission.
[a] K19+ cuboidal cells in the proximal two-thirds (green), and K14+ stratified squamous epithelium (red) towards the luminal surface in the MLGS at the SCJ. After glycine-conjugated BA treatment the MLGS extend towards the surface and align with the columnar surface epithelium (closed arrows). Budding of the inner layer after bile treatment (open arrows). [b] H&E staining of MLGS at the SCJ show budding of the inner layer projecting in the lumen of the gland (arrows). [c] Serial sections of the SCJ in mice treated with bile shows budding, which are projecting into the lumen of the MLGS, resulting in the formation of two separate glands by the process of crypt fission. [d] MLGs at the SCJ showing sparse ki67+ proliferating cells. (Scale bars 100μm).
Table 1.
Antibodies used for IHC and IF.
Table 2.
Tamoxifen concentrations used for lineage tracing in mouse models.