Nina Bertaux-Skeirik

CD44 plays a functional role in Helicobacter pylori-induced epithelial cell proliferation.

The cytotoxin-associated gene (Cag) pathogenicity island is a strain-specific constituent of Helicobacter pylori (H. pylori) that augments cancer risk. CagA translocates into the cytoplasm where it stimulates cell signaling through the interaction with tyrosine kinase c-Met receptor, leading cellular proliferation. Identified as a potential gastric stem cell marker, cluster-of-differentiation (CD) CD44 also acts as a co-receptor for c-Met, but whether it plays a functional role in H. pylori-induced epithelial proliferation is unknown. We tested the hypothesis that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation. To assay changes in gastric epithelial cell proliferation in relation to the direct interaction with H. pylori, human- and mouse-derived gastric organoids were infected with the G27 H. pylori strain or a mutant G27 strain bearing cagA deletion (∆CagA::cat). Epithelial proliferation was quantified by EdU immunostaining. Phosphorylation of c-Met was analyzed by immunoprecipitation followed by Western blot analysis for expression of CD44 and CagA. H. pylori infection of both mouse- and human-derived gastric organoids induced epithelial proliferation that correlated with c-Met phosphorylation. CagA and CD44 co-immunoprecipitated with phosphorylated c-Met. The formation of this complex did not occur in organoids infected with ∆CagA::cat. Epithelial proliferation in response to H. pylori infection was lost in infected organoids derived from CD44-deficient mouse stomachs. Human-derived fundic gastric organoids exhibited an induction in proliferation when infected with H. pylorithat was not seen in organoids pre-treated with a peptide inhibitor specific to CD44. In the well-established Mongolian gerbil model of gastric cancer, animals treated with CD44 peptide inhibitor Pep1, resulted in the inhibition of H. pylori-induced proliferation and associated atrophic gastritis. The current study reports a unique approach to study H. pylori interaction with the human gastric epithelium. Here, we show that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation.

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

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.

Hedgehog signaling in the stomach

The Hedgehog (Hh) signaling pathway not only plays a key part in controlling embryonic development, but in the adult stomach governs important cellular events such as epithelial cell differentiation, proliferation, gastric disease, and regeneration. In particular, Sonic Hedgehog (Shh) signaling has been well studied for its role in gastric physiology and pathophysiology. Shh is secreted from the gastric parietal cells and contributes to the regeneration of the epithelium in response to injury, or the development of gastritis during Helicobacter pylori infection. Dysregulation of the Shh signaling pathway leads to the disruption of gastric differentiation, loss of gastric acid secretion and the development of cancer. In this chapter, we will review the most recent findings that reveal the role of Shh as a regulator of gastric physiology, regeneration, and disease.

CD44 variant isoform 9 emerges in response to injury and contributes to the regeneration of the gastric epithelium

The CD44 gene encodes several protein isoforms due to alternative splicing and post translational modifications. Given that CD44 variant isoform 9 (CD44v9) is expressed within Spasmolytic Polypeptide/TFF2‐Expressing Metaplasia (SPEM) glands during repair, CD44v9 may be play a funcitonal role during the process of regeneration of the gastric epithelium. Here we hypothesize that CD44v9 marks a regenerative cell lineage responsive to infiltrating macrophages during regeneration of the gastric epithelium. Ulcers were induced in CD44‐deficient (CD44KO) and C57BL/6 (BL6) mice by a localized application of acetic acid to the serosal surface of the stomach. Gastric organoids expressing CD44v9 were derived from mouse stomachs and transplanted at the ulcer site of CD44KO mice. Ulcers, CD44v9 expression, proliferation and histology were measured 1, 3, 5 and 7‐days post‐injury. Human‐derived gastric organoids were generated from stomach tissue collected from elderly (>55 years) or young (14–20 years) patients. Organoids were transplanted into the stomachs of NOD scid gamma (NSG) mice at the site of injury. Gastric injury was induced in NRG‐SGM3 (NRGS) mice harboring human‐derived immune cells (hnNRGS) and the immune profile anlayzed by CyTOF. CD44v9 expression emerged within regenerating glands the ulcer margin in response to injury. While ulcers in BL6 mice healed within 7‐days post‐injury, CD44KO mice exhibited loss of repair and epithelial regeneration. Ulcer healing was promoted in CD44KO mice by transplanted CD55v9‐expressing gastric organoids. NSG mice exhibited loss of CD44v9 expression and gastric repair. Transplantation of human‐derived gastric organoids from young, but not aged stomachs promoted repair in NSG mouse stomachs in response to injury. Finally, compared to NRGS mice, huNRGS animals exhibited reduced ulcer sizes, an infiltration of human CD162+ macrophages and an emergence of CD44v9 expression in SPEM. Thus, during repair of the gastic epithelium CD44v9 emerges within a regenerative cell lineage that coincides with macrophage inflitration within the injured mucosa. Copyright

Co-culture of Gastric Organoids and Immortalized Stomach Mesenchymal Cells

Three-dimensional primary epithelial-derived gastric organoids have recently been established as an important tool to study gastric development, physiology, and disease. Specifically, mouse-derived fundic gastric organoids (mFGOs) co-cultured with Immortalized Stomach Mesenchymal Cells (ISMCs) reflect expression patterns of mature fundic cell types seen in vivo, thus allowing for long-term in vitro studies of gastric epithelial cell physiology, regeneration, and bacterial-host interactions. Here, we describe the development and culture of mFGOs, co-cultured with ISMCs.

Oncogenic Transformation of Human-Derived Gastric Organoids

The culture of organoids has represented a significant advancement in the gastrointestinal research field. Previous research studies have described the oncogenic transformation of human intestinal and mouse gastric organoids. Here we detail the protocol for the oncogenic transformation and orthotopic transplantation of human-derived gastric organoids.

Establishment of Human- and Mouse-Derived Gastric Primary Epithelial Cell Monolayers from Organoids

Organoid cultures generated from gastrointestinal tissues have been an invaluable advancement for in vitro studies of physiological function and disease. Here we present a comprehensive protocol for the establishment and culture of human- and mouse-derived 3-dimensional gastric organoids transferred to 2-dimensional gastric epithelial cell monolayers. We introduce two methods that include the establishment of monolayers from: (1) intact organoids, and (2) single cells dissociated from intact organoids.