The Development of Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) During Gastric Repair Is Absent in the Aged Stomach

Abstract

Background & aims: During aging, physiological changes in the stomach result in more tenuous gastric tissue that is less capable of repairing injury, leading to increased susceptibility to chronic ulceration. Spasmolytic polypeptide/trefoil factor 2-expressing metaplasia (SPEM) is known to emerge after parietal cell loss and during Helicobacter pylori infection, however, its role in gastric ulcer repair is unknown. Therefore, we sought to investigate if SPEM plays a role in epithelial regeneration.

Methods: Acetic acid ulcers were induced in young (2-3 mo) and aged (18-24 mo) C57BL/6 mice to determine the quality of ulcer repair with advancing age. Yellow chameleon 3.0 mice were used to generate yellow fluorescent protein-expressing organoids for transplantation. Yellow fluorescent protein-positive gastric organoids were transplanted into the submucosa and lumen of the stomach immediately after ulcer induction. Gastric tissue was collected and analyzed to determine the engraftment of organoid-derived cells within the regenerating epithelium.

Results: Wound healing in young mice coincided with the emergence of SPEM within the ulcerated region, a response that was absent in the aged stomach. Although aged mice showed less metaplasia surrounding the ulcerated tissue, organoid-transplanted aged mice showed regenerated gastric glands containing organoid-derived cells. Organoid transplantation in the aged mice led to the emergence of SPEM and gastric regeneration.

Conclusions: These data show the development of SPEM during gastric repair in response to injury that is absent in the aged stomach. In addition, gastric organoids in an injury/transplantation mouse model promoted gastric regeneration.

Keywords: CD44v; CD44v, variant isoform of CD44; Cftr, cystic fibrosis transmembrane conductance regulator; CgA, chromagranin A; Clu, Clusterin; Ctss, cathepsin S; DMEM, Dulbecco's modified Eagle medium; DPBS, Dulbecco's phosphate buffered saline; Dmbt1, deleted in malignant brain tumors 1; ES, enrichment score; Epithelial Regeneration; GSEA, gene set enrichment analysis; GSII, Griffonia simplicifolia II; Gastric Cancer; Gpx2, glutathione peroxidase 2 (gastrointestinal); HK, hydrogen potassium adenosine triphosphatase; Human Gastric Organoids; IF, intrinsic factor; Mad2I1, MAD2 mitotic arrest deficient-like 1; Mmp12, matrix metallopeptidase 12 (macrophage elastase); PBS, phosphate-buffered saline; SPEM, spasmolytic polypeptide expressing metaplasia; TFF, trefoil factor; TX, Triton X-100 in PBS; UEA1, ulex europaeus; Wfdc2, WAP 4-disulfide core domain 2; YFP, yellow fluorescent protein; hFGO, human-derived fundic gastric organoid; qRT-PCR, quantitative reverse-transcription polymerase chain reaction.

Figure 1

Histologic changes in gastric tissue of young and aged mice in response to acetic acid–induced injury. (A) H&E staining of gastric tissue from young and aged mice. Uninjured and ulcerated tissue of young and aged mice at (B) 1, (C) 3, (D) 7, and (E) 30 days after injury are shown. Arrows indicate ulcer margin. (F) Ulcer sizes from young (black) and aged (red) mice at 1, 3, and 7 days after injury. *P < .05 compared with the young group, N = 3–6 mice per time point using a 2-way analysis of variance. Scale bars: 500 μm.

Figure 2

Gross morphologic changes in gastric tissue of young and aged mice in response to acetic acid–induced injury. Gross morphology of gastric tissue from young and aged mice at (A and B) 1, (C and D) 3, and (E and F) 7 days after injury. Highlighted areas indicate ulcerated tissue. Representative of N = 4 mice per time point.

Figure 3

Expression of gastric cell lineages within the ulcerated gastric tissue of young and aged mice. Sections of injured gastric tissue collected from (A) young and (B) aged mice were immunostained for UEAI (surface mucous pit cells, green), HK (parietal cells, red), and CgA (endocrine cells, blue) 30 days after injury. Morphometric analysis of UEAI, HK, and CgA-positive cells per field in (C) uninjured stomach tissue, and ulcerated tissue at 1, 3, 7, and 30 days after injury in (D) young and (E) aged mice. (F) Uninjured tissue collected from young and aged mouse stomachs. *P < .05 compared with uninjured tissue (0 days after injury), N = 3–4 mice per time point. Scale bars: 50 μm.

Figure 4

Emergence of SPEM in regenerating gastric tissue after acetic acid–induced injury. RNA sequencing of uninjured and injured gastric tissue from young mice. (A) Gene set enrichment analysis. (B) Heat map depicting average standardized reads per kilobase per million for gene expression changes in SPEM-associated genes. Table shows fold change of up-regulated transcripts associated with the emergence or progression of SPEM. P values were generated by the Fisher exact test as implemented in R. (C) Immunofluorescence staining for SPEM using intrinsic factor (green) co-labeled with GSII-lectin (red) and TFF2 (blue) 7 days after injury. Arrows indicate the ulcer margin. (D) Higher magnification is shown. Immunofluorescence staining for SPEM using IF (green) co-labeled with GSII-lectin (red) and TFF2 (blue) in ulcerated gastric tissue 7 days after injury in aged mice. (E) Immunofluorescence staining for SPEM using tissue collected from an aged stomach 7 days after injury. (F) Higher magnification images of IF, GSII, and TFF2 in epithelium. Scale bars: 50 μm.

Figure 5

Validation of RNA sequencing data. (A) qRT-PCR was performed using RNA isolated from uninjured and ulcerated gastric tissue from young mice 7 days after injury. Shown is the average fold change of SPEM-associated genes Dmbt1, Mmp12, Wfdc2 (HE4), Mad2I1, Cftr, Ctss, Gpx2, and clusterin (Clu). *P < .05 compared with uninjured tissue, N = 3–4 mice per group using 1 way analysis of variance. (B) Heat map depicting average standardized reads per kilobase per million for gene expression changes in SPEM-associated genes. (C) Table showing fold change of transcripts corresponding to the emergence or progression of SPEM associated with inflammation. Data are expressed as the means ± SEM. P < .05 compared with uninjured tissue (0 days after injury) as measured by 1-way analysis of variance.

Figure 6

Emergence of SPEM in regenerating gastric tissue after acetic acid–induced injury. (A) SPEM within injured tissue as indicated by separate GSII (green) and IF (red) granules. Expression of CD44v (green) and TFF2 (red) in (B) uninjured and (C) injured gastric epithelium. Scale bars: 50 μm.

Figure 7

Experimental design and YFP+ gastric organoids. (A) Table comparing mouse and human age. (B) Experimental protocol. Gastric glands were isolated from mice expressing the YFP transgene. YFP gastric organoids were generated from glands. After 7 days in culture, organoids were dissociated and transplanted into the stomach of aged mice immediately after ulcer induction. (C) Gastric organoids were injected within the lumen (LUM), submucosa (SUBM), or both lumen and submucosa of the aged mouse stomach, and ulcer size was measured. *P < .05 compared with PBS control, N = 4 mice per group. (D) Gastric organoids or microspheres (control) were injected within the submucosa of the aged mouse stomach and ulcer size was measured. Data are expressed as means ± SEM. *P < .05 compared to control as assessed by 1-way analysis of variance.

Figure 8

Engraftment of organoids within the mouse gastric epithelium. Immunostaining of YFP+ organoid-derived cells (green) and CD44v (red) within the ulcerated tissue of aged mice transplanted with gastric organoids at (A) 3, (B) 7, (C) 21, and (D) 30 days after injury. Scale bars: 50 μm.

Figure 9

YFP expression in gastric epithelium of organoid transplanted mice. (A) Immunofluorescence staining using an antibody against YFP in untransplanted mice 7 days after injury. Scale bars: 50 μm. (B) YFP expression by RT-PCR using RNA prepared from laser capture microdissection of ulcerated and intact epithelium from stomachs 7 days after injury and gastric organoid transplantation. CAP, captured tissue. (C) Single cells flow sorted from stomach tissue of aged transplanted mice for YFP+ and YFP- grown in culture for 10 days. (D) Fluorescence-activated cell sorting (FACS) histogram depicting YFP+ single-cell population isolated from gastric tissue of aged mice transplanted with gastric organoids. YFP+ single cells were collected from the gastric glands 5 months after injury and gastric organoid transplantation and used to generate YFP+ gastric organoids in culture.

Figure 10

Organoid-derived SPEM cell lineages may contribute to wound healing in the aged stomach. (A) Immunofluorescence staining of ulcerated gastric tissue of aged mice transplanted with gastric organoids 30 days after injury. A lectin specific for surface mucous pit cells (UEAI, green) and antibodies specific for parietal cells (HK, red) and endocrine cells (CgA, blue) were used to identify cell lineages. Scale bars: 50 μm. (B) Quantification of UEAI+, HK+, and CgA+ cells within the damaged epithelium of aged mice transplanted with gastric organoids 1–30 days after injury. Data are presented as means ± SEM, N = 3–4 mice per time point. *P < .05 compared with day 0, determined by 1-way analysis of variance. (C) Ulcer measurements from young (black), aged (red), and aged mice transplanted with gastric organoids (green) at 1, 3, and 7 days after injury. Data are expressed as means ± SEM. *P < .05 compared with aged transplanted with organoids using 1-way analysis of variance, N = 3–10 mice per time point. (D) Ulcer measurements from young mice without (W/O) or with (W/) organoid transplantation at 1, 3, 7, and 30 days after injury. Data are expressed as means ± SEM. *P < .05 compared with young untransplanted mice using 1-way analysis of variance, N = 4 mice per time point. Whole mount immunofluorescence staining to identify parietal cells using antibody specific for H+K+–adenosine triphosphatase in stomach tissue collected from young mice without or with organoid transplantation or uninjured 30 days after injury. (E) Sections of injured gastric tissue collected from young mice were immunostained transplanted with organoids for UEAI (surface mucous pit cells, green), HK (parietal cells, red), and CgA (endocrine cells, blue) 30 days after injury. Arrow indicates area of injury. Scale bar: 50 μm. (F) Morphometric analysis of UEAI, HK, and CgA-positive cells per field in uninjured stomach tissue and ulcerated tissue 30 days after injury. Data are expressed as means ± SEM. *P < .05 compared with young untransplanted mice using 1-way analysis of variance, N = 4 mice per time point.

Figure 11

Expression of SPEM markers in gastric organoid cultures. (A) Heat map of centered reads per kilobase per million values depicting gene expression changes in SPEM-associated genes using RNA isolated from organoid and native fundic tissue. (B) Table showing fold change of transcripts associated with the emergence or progression of SPEM. qRT-PCR was performed using RNA isolated from glands and organoids derived from young and aged mouse stomachs. Shown is the average fold change of SPEM-associated genes (C) TFF2, (D) clusterin (CLU), and (E) HE4. Shown is the average fold change of SPEM-associated genes TFF2, clusterin (CLU), and HE4. *P < .05 compared with glands or normal glands expression. #P < .05 compared with young organoids using either the Student t test or 1-way analysis of variance.

Figure 12

Emergence of SPEM in gastric ulcers of young patients. Immunofluorescence staining using human-specific antibodies for CD44v (hCD44v, green), TFF2 (hTFF2, blue), and GSII (red) in gastric ulcer tissue collected from (A and B) young (age, 27 y) and (C and D) aged (age, 65 y) patients. (B and D) Higher magnification of SPEM in the ulcer margin is shown.

Figure 13

Human-derived gastric organoids. (A) Gastric glands isolated from the fundus of human stomach grew in vitro to form gastric organoids. (B) Expression of genes for the gastric cell lineage markers: parietal cells (ATP4a), chief cells (pepsinogen [PgC]), surface mucous pit cells (Muc5AC), and mucous neck cells (Muc6) in the hFGOs, gastric tissue, human corpus, and antrum. Gastrin (Gast) was not detected in the hFGOs, indicating that the hFGOs are derived from the fundus. (C) hFGOs also expressed SPEM markers Clu, HE4, TFF2, and PgC. N = 4 samples or cultures per group, data are expressed as fold change relative to glands. *P < .05 compared with glands determined by 1-way analysis of variance. (D) Experimental protocol. (E) Ulcer sizes for untransplanted NOD SCID γ mice (control) and hFGO transplanted mice 7 days after injury. *P < .05 compared with control determined by the Student t test, N = 4–8 mice per group.

Figure 14

Engraftment of hFGOs in NOD SCID γ mouse stomach after ulceration. (A–E) Immunofluorescence staining using human-specific antibodies for CD44v (hCD44v, green) and histone (histone) in gastric tissue from NOD SCID γ mice transplanted with hFGOs 7 days after injury. (B, D, and E) Higher magnification of regenerating epithelium in the ulcer margin is shown in insets. (F) Immunofluorescence staining for human-specific histone in gastric tissue from NOD SCID γ mice transplanted with hFGOs 60 days after injury. (G) Higher magnification is shown. (H) Immunofluorescence staining for human-specific histone in gastric tissue from untransplanted NOD SCID γ mice. Arrows indicate area of injury. Scale bars: 50 μm.