ABSTRACT – PROJECT 2 The central purpose of Project 2 is to utilize a primary human tissue model to investigate the features of gastric stem cells and how the cells interact with Helicobacter pylori (Hp). Hp commonly triggers an inflammatory process that leads to gastric intestinal metaplasia (GIM), a condition that can evolve into invasive carcinoma. However, not all people with GIM develop gastric cancer. This project proposes an experimental platform to investigate the features of high-risk GIM. Its hypothesis is that high-risk GIM lesions possess epithelial stem cells with unique properties predisposed to neoplastic progression, especially in the setting of Hp infection. Project 2 has three aims to investigate this hypothesis: (1) Characterizing the epithelial progenitor cells of high-risk GIM. (2) Modeling genomic alterations from high-risk GIM. (3) Elucidating interactions between Hp and gastric organoids from high-risk GIM. For Aim 1, Dr. Amieva’s group will develop a library of stem-cell rich, apical-out gastric organoids representing the gastric neoplastic spectrum across various topographical locations in the stomach. The characteristics of these stem cell organoids will be identifying using next generation sequencing and advanced microscopy. In Aim 2, the organoids will be perturbed with select genetic alterations (identified in Project 1) through CRISPR gene editing. Subsequent changes to gene expression may provide insight into the biology of high-risk versus low-risk GIM tissues. Lastly in Aim 3, the investigators will infect apical-out gastric organoids (from Aim 1) with human-derived Hp strains of known genotypes and phenotypes. Single-cell genomics will allow investigators to distinguish the unique transcriptional profiles of organoids at different time points, states of infection and inflammation, and enable studies into bacterial genes that are crucial for colonization and host perturbations. While Project 2 will be conducted in a basic research laboratory, its aims and results are directly related to improving overall understanding of Hp behavior in the human stomach, and molecular changes that prime tissue for carcinogenesis.