Prospective identification of a multilineage progenitor in murine stomach epithelium

Abstract

Background & aims: Epithelial stem cells in the stomach are responsible for constant renewal of the epithelium through generation of multiple gastric cell lineages that populate the gastric glands. However, gastric stem or progenitor cells have not been well-characterized because of the lack of specific markers that permit their prospective recognition. We identified an intestinal promoter that is active in a rare subpopulation of gastric epithelial cells and investigated whether these cells possess multilineage potential.

Methods: A marked allele of the endogenous mouse villin locus was used to visualize single beta-galactosidase-positive cells located in the lower third of antral glands. A 12.4-kb villin promoter/enhancer fragment drives several transgenes (EGFP, beta-galactosidase, and Cre recombinase) in these cells in a pattern similar to that of the marked villin allele. Reporter gene activity was used to track these cells during development and to examine cell number in the context of inflammatory challenge while Cre activity allowed lineage tracing in vivo.

Results: We show that these rare epithelial cells are normally quiescent, but multiply in response to interferon gamma. Lineage tracing studies confirm that these cells give rise to all gastric lineages of the antral glands. In the embryo, these cells are located basally in the stomach epithelium before completion of gastric gland morphogenesis.

Conclusions: We have identified a rare subpopulation of gastric progenitors with multilineage potential. The ability to prospectively identify and manipulate such progenitors in situ represents a major step forward in gastric stem cell biology and has potential implications for gastric cancer.

Figure 1. The villin promoter is active in a rare sub-population cells in the adult gastric antrum

A) In the antrum of a control wild type mouse stained with X-gal, no β-gal positive cells can be detected. B) Widely spaced β-gal positive cells are detected in a section from the antrum of an untreated Villin^β-gal/+ mouse. The inset reveals an oval shaped cell with large nucleus to cytoplasm ratio. C) Cells are located in the lower one third of the gland. D) Rarely, two β-gal positive cells are observed in a single sectioned gland. E) Schematic views of gastric glands from corpus and antrum are shown. Yellow cells indicate parietal cells, diagnostic for the corpus. The regions designated as pit, isthmus and base are shown for each gland. F) The relative distribution of β-gal cells within one entire mouse stomach is plotted as the percent of cells at various positions along the gland axis in corpus and antrum. This distribution was similar in the three control animals examined. In A and B, Bar = 50μm; in B, Bar = 20μm; in C, bar = 10μm.

Figure 2. Behavior of β-gal positive cells in mouse models of metaplasia

A, B) One year old Villin^β-gal/+/Cdx2 mice. A) Widespread intestinal metaplasia in the antrum is detected by alkaline phosphatase staining (red). B) X-gal staining in the same mouse reveals β-gal positive cells at the surface, within the metaplastic region. The number and location of deep gland β-gal positive cells is similar to wild type littermates. C–E) Six month old Villin^β-gal/+/Ctox-7 mice. C) Alkaline phosphatase staining (red) shows focally positive antral glands. D) The number of β-gal positive cells is greatly expanded. One gland contains two β-gal positive cells distributed along the long axis of the gland (arrow). E) Numerous β-gal positive cells are detectable in the lower region of the antral glands. F) IFNγ treated mice. The number of β-gal positive cells is increased in this model after only two weeks of treatment (compare to Figure 1B). In A–C, Bar = 50 μm; in D–F, Bar = 20μm.

Figure 3. β-gal positive cells are located at or below the isthmus in isolated antral glands

A–E) Note the distribution of β-gal marked cells at variable distances in relation to the gland base. F) Two β-gal positive cells appear to be completing mitosis along the long axis of the gland. G,H) Two glands from INFγ-treated mice; β-gal positive cells are distributed to two opposite sides of the gland tip. I,J) Labeled cells from a 12.4KVil- EGFP mouse. The two labeled cells in the gland in (J) both possess long cytoplasmic processes that are visible with the EGFP label.

Figure 4. Lineage tracing studies - single β-gal positive cells clonally populate the gland with multiple gastric cell types

A) Antral gland in a Villin^β-gal/+/ROSA26R control mouse treated with PBS. Few such fully stained glands are seen in control animals. B,C) Fully labeled antral glands from Villin^β−gal/+/ROSA26R mice treated with INFγ. Labelled glands tend to cluster. D,E) Whole-mount β-gal staining of the antrum in Villin^β-gal/+/ROSA26R mice treated with PBS (D) or IFN γ (E) and sacrificed after 12 weeks. The PBS-treated antrum shows no β-gal staining (D). The β-gal positive region at the bottom is duodenum. In an INFγ–treated mouse (E), large patches of contiguous β-gal positive glands are visible on the ventral side of the antrum. F–J) Fully stained glands are expressing gastric markers. For (F), anti-β-gal = red; FITC-conjugated GSII lectin = green (neck/gland cell marker). In (G), anti-β-gal = red; FITC-conjugated UAEI lectin = green (surface pit cell marker). H, I) The section is stained with anti-β-gal (green) and anti-serotonin (red), marking a common subtype of enteroendocrine cells. The image in (I) is a higher magnification of the region that is boxed in (H). J) A section from the junction of the corpus and antrum, co-stained with anti-β-gal (green) and anti-H⁺,K⁺ ATPase (red), a parietal cell marker. For D and E, Bar = 100μm; for F, Bar = 50μm; for all other panels, Bar = 20μm.