Gastrointestinal (GI) organoids are now widely used for disease modeling, drug discovery, regenerative-, and precision medicine. As publications using GI organoids have increased exponentially, methodological reporting across GI organoid studies remains inconsistent, making results difficult to interpret, compare, and reproduce. To address this, we present community-informed guidance for reporting key experimental details in GI organoid research. Our recommendations were developed by integrating established biomedical reporting frameworks, organoid-focused resources from international organizations, and structured input from the GI organoid community through an expert panel survey. These inputs informed a core set of reporting items, including stem cell source and donor context, media and supplement composition, extracellular matrix type, culture configuration, and other parameters essential for replication. We provide an “Organoid Reporting Toolkit” containing practical checklists and templates intended to help authors, reviewers, editors, and journals. Together, these resources aim to improve transparency and reproducibility, facilitate cross-study comparison, and streamline communication in the GI organoid field while preserving flexibility for methodological innovation.

Patient-derived intestinal enteroids are valuable models for studying gastrointestinal physiology and disease, but their dependence on Matrigel limits reproducibility and clinical translation. Here, we developed a fully synthetic four-armed poly(ethylene glycol)-maleimide (PEG-4MAL) hydrogel platform to support human intestinal enteroid culture. Guided by enteroid transcriptomics revealing high expression of α2β1 integrin and matrix metalloproteinases (MMPs), we systematically evaluated collagen-mimetic GFOGER and fibronectin-derived RGD (Arg-Gly-Asp) peptides combined with four MMP-sensitive crosslinkers. GFOGER-functionalized hydrogels significantly outperformed RGD formulations across all metrics. The optimized PEG-4MAL-GFOGER formulation demonstrated enteroid formation efficiency and viability approaching Matrigel performance. Bulk RNA sequencing across multiple patient-derived lines demonstrated transcriptomic similarity between synthetic and Matrigel hydrogels, with preservation of stem cell, differentiation, and regional identity markers. This rationally designed synthetic platform overcomes key limitations of biological matrices while supporting robust enteroid growth and functionality comparable to standard biological matrices.

Clostridioides difficile causes severe disease in adults but commonly colonizes infants asymptomatically. The consequences of early-life colonization on host development remain unknown. In a neonatal mouse model, C. difficile colonization drove proinflammatory and tissue repair responses in the intestinal epithelium, enriching injury-associated intestinal stem cell populations and skewing differentiation toward secretory lineages. Despite transient colonization, exposure to C. difficile early in life led to persistent changes into adulthood. Epithelial responses were toxin dependent, as colonization with nontoxigenic strains or maternal vaccination with a C. difficile-targeted messenger RNA-lipid nanoparticle vaccine protected neonates. Human infant intestinal epithelial cells were sensitive to C. difficile toxins, and biopsies from colonized infants exhibited altered intestinal stem cell behavior. This study redefines C. difficile as an underappreciated early-life pathogen with lasting effects on host development.

An accumulating body of evidence suggests that carriers of a pathogenic germline variant (PGV) in BRCA1 or BRCA2 have an increased gastric cancer risk. BRCA1 and BRCA2 are tumor suppressor genes involved in promoting homologous recombination to repair double-stranded DNA breaks. The aim of this investigation was to identify differences within the gastric epithelium and in patient-derived gastric organoids (PDGO) between BRCA1 and BRCA2 carriers and noncarriers to determine if evidence of early gastric carcinogenesis exists among these carriers. First, using gastric epithelial biopsies, BRCA2 carriers were found to harbor higher expression of the proliferative marker Ki-67 within the antral gastric epithelium, and strikingly, biopsies from both BRCA1 and BRCA2 carriers displayed a marked increase in double-stranded DNA damage. These results were further explored using PDGOs, in which a growth advantage was observed for both BRCA1 and BRCA2 PDGOs compared with noncarrier PDGOs. Furthermore, both BRCA1 and BRCA2 PDGOs displayed a more pronounced enhancement of Ki-67 expression as well as increased double-stranded DNA damage compared with noncarrier PDGOs. Importantly, none of the PDGOs showed signs of BRCA1 or BRCA2 loss of heterozygosity, potentially indicating a haploinsufficient phenotype. Taken together, these novel findings suggest that haploinsufficiency in BRCA1 and BRCA2 carriers may lead to DNA damage in the gastric epithelium, which may serve as an early event contributing to gastric cancer development. IMPLICATIONS:The elevated risk of gastric cancer for BRCA1 and BRCA2 PGV carriers may be due to haploinsufficiency, which warrants further investigation into mechanisms of BRCA1- and BRCA2-associated gastric cancer.

Post-transcriptional gene regulation-particularly through RNA modifications-plays an essential but understudied role in development, homeostasis, and regeneration of rapidly changing tissues like the mammalian intestinal epithelium. RNA modifications such as N6-methyladenosine (m⁶A) represent a burgeoning area of research in posttranscriptional regulation, with m⁶A being the most abundant modification found in approximately 25% of all mRNA transcripts. Multiple groups have begun to report m⁶A and associated regulation of mRNA fate as critical to key process in the intestinal epithelium. In this review, we synthesize key findings to date into the following 3 categories: m⁶A changes in response to the homeostatic luminal environment, m⁶A as a mediator of stemness in the crypt, and m⁶A as a tool for reacting to inflammation and injury. Over the course of this review, we will demonstrate how m⁶A is uniquely positioned to regulate homeostasis and disease states in the challenging and dynamic environment of the intestinal epithelium.

Cytokines mediating epithelial and immune cell interactions modulate mucosal healing-a process that goes awry with chronic inflammation as in inflammatory bowel disease. TNFSF13 is a cytokine important for B cell maturation and function, but roles for epithelial TNFSF13 and putative contribution to inflammatory bowel disease are poorly understood. We evaluated functional consequences of a novel monoallelic TNFSF13 variant using biopsies, tissue-derived colonoids and induced pluripotent stem cell (iPSC)-derived colon organoids. TNFSF13variant colonoids exhibited a >50% reduction in secreted TNFSF13, increased epithelial proliferation, and reduced apoptosis, which was confirmed in iPSC-derived colon organoids. Single cell RNA-sequencing and flow cytometry suggested FAS as the predominant colonic epithelial receptor for TNFSF13, which was confirmed by co-immunoprecipitation and binding assays. Imaging mass cytometry revealed an increase in epithelial-associated B cells in TNFSF13 variant colon tissue sections. Finally, TNFSF13 variant colonoids co-cultured with memory B cells demonstrated a reduction in immunoglobulin-producing plasma cells compared to control colonoid cocultures. Our findings support a role for epithelial TNFSF13 as a regulator of colonic epithelial growth and epithelial crosstalk with B cells.

BACKGROUND & AIMS:Defining consequential differences in intestinal epithelial stem cells in healthy humans vs those with inflammatory bowel disease (Crohn's disease and ulcerative colitis) is essential for the development of much needed therapies to restore the epithelial barrier and maintain its fidelity. METHODS:We used single-cell transcriptomic and epigenomic approaches in matched patient tissues and organoids to investigate epithelial gene expression and function in children with no pathological diagnosis in the lower gastrointestinal tract and healthy adults compared with those with Crohn's disease. RESULTS:We identify an inflammatory secretory progenitor (ISP) cell state present almost exclusively in patients with Crohn's disease compared with healthy subjects. ISPs exhibit gene expression profiles consistent with normal secretory progenitor cells but concomitantly express a suite of distinguishing pro-inflammatory genes. Mechanistically, ISPs exhibit open chromatin at ISP gene loci. Although ISP-specific genes are not expressed in intestinal stem cells, their chromatin is accessible in Crohn's disease stem cells, suggesting that ISP genes are epigenetically poised in stem cells and subsequently transcriptionally activated in ISPs in the presence of inflammatory stimuli. Consistently, Crohn's disease colonoids exhibit sustained ISP gene expression that can be elicited further with pro-inflammatory cytokines or via co-culture with pro-inflammatory macrophages. CONCLUSIONS:We have defined differences in the epithelial stem and progenitor compartment of patients with Crohn's disease that suggest aberrant stem cell differentiation and inflammatory gene expression arise and persist during disease.

Background: The intestinal epithelium is a single but heterogeneous layer of cells that acts as a barrier between the luminal contents of the gastrointestinal tract and underlying mucosa. In patients with inflammatory bowel disease (IBD), dysregulated immune response and impaired mucosal healing lead to chronic inflammation. Our lab has identified a unique cell state, termed inflammatory secretory progenitor (ISP) cell state, in the intestinal epithelium of pediatric patients with Crohn’s disease. ISP gene expression is found in OLFM4-/REG1A+ secretory progenitors that maintain an inflammatory profile with the expression of immune-related and antimicrobial peptides, and antigen presentation proteins. We posit that the presence of cells in an ISP state may modulate epithelial response to injury and sustained inflammation, such as impaired repair that poises the epithelium for exacerbated disease pathogenesis or oncogenesis. Consistent with previous findings that show it is upregulated in Crohn’s, one of the ISP markers is lipocalin-2 (LCN2). First identified for its role in innate immunity, LCN2 is a secreted molecule expressed by multiple cell types. LCN2 can facilitate cellular uptake of different small weight molecules, including iron-binding compounds. Epithelial cell expression of LCN2 could therefore modify local iron stores during intestinal inflammation and repair. Mice with whole body deletion of Lcn2 are more sensitive to bacterial infection, but also appear to be less susceptible to acute colitis. No established role for epithelial expression of LCN2 in patients with IBD exists. The functional consequence of ISPs has yet to be determined, and thus the contribution of individual markers like LCN2 to disease development is not clear. We hypothesize: that ISPs give rise to an inflamed secretory environment in part via the upregulation of LCN2 which contributes to epithelial barrier damage. Methods: Stem cell derived 3D organoids allow us to examine epithelial-intrinsic defects that may contribute to disease. Given LCN2’s role in iron modulation, we generated Lcn2 knockdown colonoids—organoids derived from colonic epithelium— to probe sensitivity to iron-mediated cell death (ferroptosis) and inflammatory cytokines using organoid morphology, western blot, and immunostaining. We also evaluated single cell RNA-sequencing data for LCN2 expression in ISP and other cell types and confirmed findings using immunostaining in control and Crohn’s colonoids with or without cytokine treatment. Results & conclusions: Preliminary data show that Lcn2 KD murine colonoids show no morphological differences to control colonoids but exhibited reduced expression of ferroptosis marker TFRC at homeostasis and under cytokine stimulation via western blot. Cytokine-stimulated control colonoids exhibited increased membrane localization of TFRC compared to Lcn2 KD colonoids via immunostaining. Given goblet cells are the primary secretory cell type in the colon, our human data at the single cell and protein level (immunostaining) also support that goblet cell expression of LCN2 is altered in Crohn’s patients and under cytokine stimulation in Crohn’s disease colonoids. Taken together, our data suggest that LCN2 expression in ISPs may promote enhanced susceptibility of secretory progenitors and goblet cells to ferroptosis. Thus, the presence of ISPs may promote an aberrant secretory phenotype in the intestinal epithelium by altering the antimicrobial peptide content and susceptibility to cell death. Ongoing and future studies will evaluate whether this altered secretory phenotype contributes directly to alterations in mucus secretion and composition, and immune-epithelial crosstalk. University of Pennsylvania School of Veterinary Medicine; Institute for Infectious & Zoonotic Disease Martin and Pamela Winter Infectious Disease Pre-Doctoral Fellowship This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Cytokines mediating epithelial and immune cell interactions modulate mucosal healing- a process that goes awry with chronic inflammation as in inflammatory bowel disease. TNFSF13 is a cytokine important for B cell maturation and function, but roles for epithelial TNFSF13 and putative contribution to inflammatory bowel disease are poorly understood. We evaluated functional consequences of a novel monoallelic TNFSF13 variant using biopsies, tissue-derived colonoids and induced pluripotent stem cell (iPSC)-derived colon organoids. TNFSF13 variant colonoids exhibited a >50% reduction in secreted TNFSF13, increased epithelial proliferation, and reduced apoptosis, which was confirmed in iPSC-derived colon organoids. Single cell RNA-sequencing, flow cytometry, and co-immunoprecipitation identified FAS as the predominant colonic epithelial receptor for TNFSF13. Imaging mass cytometry revealed an increase in epithelial-associated B cells in TNFSF13 variant colon tissue sections. Finally, TNFSF13 variant colonoids co-cultured with memory B cells demonstrated a reduction in the production of IgA+ plasma cells compared to control colonoid co-cultures. Our findings support a role for epithelial TNFSF13 as a regulator of colonic epithelial growth and epithelial crosstalk with B cells.