American Journal of Physiology-Gastrointestinal and Liver Physiology

Background: Intestinal fibroblasts are extensively remodeled in inflammatory bowel disease (IBD), yet the soluble stromal signals that directly influence epithelial maturation remain incompletely understood. We examined whether fibroblasts derived from inflamed IBD colon display an osteopontin (OPN; SPP1)-enriched secretory phenotype and whether extracellular OPN directly modifies non-neoplastic human colonic epithelium. Methods: Conditioned media from 5 noninflamed-associated fibroblast (NAF) and 4 inflammatory-associated fibroblast (IAF) cultures were analyzed in the validated multi-donor cytokine-array matrix, with orthogonal SPP1 RT-qPCR validation in a complementary fibroblast cohort. Recombinant OPN was then tested in human colonic organoids from 3 donors using donor-resolved molecular and functional analyses under standard, fibroblast-conditioned, and WNT-modified culture conditions. Donor identity defined biological replication. Results: OPN showed the strongest positive rank-based separation between IAF and NAF cultures: all 4 IAF values were higher than all 5 NAF values (Cliff's delta=1.00; exact Mann-Whitney P=0.0159; median ratio=3.64; Benjamini-Hochberg q=.19). Fibroblast RT-qPCR showed approximately 10-fold higher mean SPP1 expression in IAF than NAF cultures (P<.05). In organoids, OPN consistently reduced KRT20, FABP1, CA2, and MUC2 from Day 5 to Day 9. SOX9, HES1, and NOTCH1 increased at Day 9, whereas LGR5 and ALDH provided no evidence of canonical stem-cell expansion. Organoid-area and EdU responses were modest and donor dependent. Conclusions: IBD-derived colonic fibroblasts can display an OPN-enriched secretory phenotype. In human colonic organoids, OPN is sufficient to impair epithelial maturation, whereas its effects on growth and proliferation are variable and depend on the surrounding niche.

Pediatric endoscopy is performed only when clinically indicated, limiting access to healthy duodenal tissue. Biopsies with duodenal no pathologic abnormality (NPA) are often used as controls despite the presence of symptoms or inflammatory disease found elsewhere in the gastrointestinal (GI) tract. We characterized pediatric duodenal NPA tissue across clinical, histologic, cellular, and transcriptomic domains. Methods Archival duodenal NPA biopsies were obtained with clinical metadata and hematoxylin-and-eosin whole-slide images (WSIs). Duodenal mRNA-seq data were analyzed from a subset of patients with duodenal NPA. Clinical metadata and WSIs underwent machine-learning analysis, cell populations were quantified from WSIs, and RNA-seq data underwent differential expression and pathway-enrichment analyses. Results The primary cohort included 195 patients with duodenal NPA. Comparisons between patients with non-duodenal GI disease and those with no GI disease showed differences in inflammatory biomarkers and follow-up utilization. Unsupervised clinical clustering identified three clusters with partial enrichment for IBD with colonic inflammation and Eosinophilic Esophagitis (EoE) with esophageal inflammation. Supervised clinical classification showed modest discrimination. WSI clustering showed limited disease-status discrimination, and cell quantification showed no significant group differences. In the separate RNA-seq cohort of 43 patients, differential-expression and pathway-enrichment analyses identified transcriptional and pathway-level differences between disease-status groups. Conclusions This multi-level characterization indicates that pediatric duodenal NPA tissue should not be treated as a uniform control category. Clinical metadata and transcriptomics revealed clinical and molecular heterogeneity, while histologic and cell analyses showed limited disease-status separation, supporting a refined definition of control tissue.

Intestinal epithelial cells (IECs) undergo rapid and continuous renewal to maintain gut epithelial homeostasis and barrier integrity at the interface with luminal microbiota, dietary antigens, and enteric pathogens. IEC proliferation is tightly regulated by a complex machinery of cell cycle regulators and effectors, and genetic or transcriptional dysregulation of cell-cycle regulatory pathways is a hallmark of colorectal cancer development and progression. Previously, JDP2 (Jun dimerization protein 2) has been shown to be involved in cell cycle control and is associated with an array of cancers. JDP2 functions as a transcription factor or epigenetic regulator depending on cellular context, disease, and cancer types. However, the role of JDP2 in CRC, cell cycle homeostasis of colon cancer cells, and their fate remains poorly defined. In this study, we found that patients with higher JDP2 expression have significantly worse survival than patients with lower JDP2 expression. Our functional assays showed that JDP2 depletion increased EdU incorporation during S phase and PHH3 abundance, which is consistent with increased proliferative and mitotic activity. Our single-cell RNA sequencing and transcriptomic profiling further revealed reprogramming of cell-cycle-associated transcriptional states following JDP2 depletion. Differential-expression and pathway analysis identified changes in mitotic and cell-cycle regulatory genes, including programs involving cyclins, cyclin-dependent kinases, chromosome segregation, and mitotic progression. We also found that lower residual JDP2 expression was associated with escalated G2/M representation and a change toward later states along a G1 - S - G2/M-associated transcriptional trajectory. Furthermore, low-JDP2 cells showed significantly greater late-state occupancy than High-JDP2 cells (33.6% versus 24.0%; paired P=0.048). Among the JDP2-siRNA-treated cells, analysis of residual JDP2-expressing cells also revealed heterogeneous associations of JDP2 with cell-cycle, survival, apoptotic, and stress-response programs, consistent with JDP2's context-dependent transcriptional functions. Together, these data identify JDP2 as an important component of intestinal epithelial cell-cycle homeostasis and transcriptional landscape and suggest that JDP2 depletion perturbs the balance of proliferative cell states.

Inflammatory bowel disease (IBD) often involves disrupted intestinal epithelial barrier, but therapies specifically targeting this barrier are limited. We found that downregulated AUF1 (HNRNPD) contributes to defective barrier integrity in ulcerative colitis (UC). Compared to controls, its expression level was decreased and inversely correlated with clinical severity. Knocking down AUF1 in human and mouse colonic organoids led to impaired barrier function, with reduced Occludin and upregulated Claudin-2, mimicking characteristic IBD-associated mucosal alterations. Distinct RNA-binding activity of AUF1 protein isoforms contributed to these changes: p37 stabilized Occludin mRNA and blocked microRNA-122/Ago2-mediated repression, whereas p40 promoted Claudin-2 mRNA degradation via ubiquitin-proteasome pathway. Restoring AUF1 expression in organoids enhanced epithelial properties and, when transplanted into mice with established colitis, accelerated mucosal healing and epithelial regeneration in recipient mice and decreased fibrosis. Our study unravelled a post-transcriptional mechanism important for intestinal homeostasis and demonstrated a concept of using engineered organoids for treating IBD.

The murine intestine reactivates developmental gene programs following various forms of damage in vivo and in vitro; however, injury response mechanisms used by the human intestine remain unclear. Using adult human small intestinal epithelium-only organoids ("enteroids"), we characterized the early response to eight injury conditions and injury-associated signaling pathways (P53, PGE2, YAP, TGFB) to interrogate whether human developmental genes were activated. P53 activation and decreased proliferation were common features across treatments. Most (7/8) injuries did not activate human development genes. Butyrate is a notable exception given it inhibited P53 and promoted a human developmental transcriptional signature. We observe that P53 induces a human adult gene signature while TGFB and YAP promote a developmental signature. Together our data characterizes various transcriptional responses to injury, supports injury-associated signaling pathways as regulators of human adult and developmental genes, and highlights how our data can be mined to predict injury-specific interventions for epithelial protection.

Background & Aims Dynamic cell cycle control is critical for intestinal crypt maintenance and injury responses, yet genetic regulators driving these changes remain poorly defined. As reserve intestinal stem cells (rISCs) are often considered to be slowly-cycling and can resist replication-dependent injury, factors that restrain proliferation may confer cytoprotection. Here, we define SOX9 as a regulator of intestinal stem cell (ISC) cycling and injury resistance. Methods Primary human ISCs were engineered to tune SOX9 levels, visualize cell cycle state, and manipulate cell cycle regulators. Using this system, we tested how SOX9 dosage impacts stemness, differentiation, proliferative recovery after SOX9 washout, and survival after 5-FU-mediated injury. Transcriptional analyses identified candidate links between SOX9 levels and cell cycle control, which were functionally tested using inducible INK4A (CDKN2A) and Cyclin D2 (CCND2) ISC lines. Results SOX9 induction lengthens the cell cycle in a dose-dependent manner largely by elongating G1 phase through the INK4A-Rb pathway. The effects of high SOX9 levels repressing proliferation and stem cell activity are reversible. SOX9 induction protects against 5-FU toxicity. This protection is mimicked by INK4A overexpression or pharmacological G1 phase arrest and repressed by CCND2 induction. Conclusions These findings identify SOX9-mediated G1 elongation as a reversible cytoprotective program that confers key functional properties associated with rISCs: proliferative restraint, retained stem cell potential, and resistance to replication-dependent injury. This positions G1 length as a potential determinant of which crypt cells survive injury to act as reserve stem cells.

Background and aims: Cyclin E1 is a canonical regulator of cell cycle progression known to promote liver fibrosis and hepatocarcinogenesis. However, its precise contribution to hepatic immune responses remains poorly understood. We investigated whether Cyclin E1 regulates macrophage-associated inflammation and acute immune-mediated liver injury. Methods: Hepatic immune populations were analyzed in untreated and concanavalin A-treated wildtype and constitutive Ccne1-/- mice by flow cytometry. Liver injury was assessed by survival, histopathology, and caspase activation. Inflammatory mediators were quantified by qPCR and ELISA. Bone marrow-derived macrophages were examined during differentiation and pro-inflammatory polarization using flow cytometry, cytokine measurements, and RNA-sequencing. Lipid nanoparticle-mediated delivery of Ccne1-specific siRNA was evaluated in J774A.1 macrophage-like cells. Results: Under homeostatic conditions, Ccne1-/- mice exhibited reduced hepatic immune cell numbers, including Kupffer cells and CD4+ and CD8+ lymphoid cells. Following concanavalin A administration, Ccne1-/- mice showed improved survival, reduced hepatic necrosis, and diminished caspase-3 and caspase-8 activation. Protection was associated with reduced Kupffer and CD8+ lymphoid cell abundance and attenuated inflammatory mediator expression. Cyclin E1 deficiency did not substantially alter bone marrow-derived macrophage differentiation or cell cycle distribution. However, pro-inflammatory Ccne1-/- macrophages showed reduced interleukin-6 expression, attenuated inflammatory gene programs, and lower inferred IRF1 and NF-{kappa}B1 activity. Ccne1 knockdown similarly reduced interleukin-6 expression in pro-inflammatory J774A.1 cells. Conclusions: Cyclin E1 contributes to hepatic immune homeostasis and promotes macrophage-associated inflammatory signaling during immune-mediated hepatitis. Its inflammatory function is uncoupled from overt changes in macrophage cell cycle progression and may involve regulation of IRF1- and NF-{kappa}B-associated transcriptional programs. Targeting Cyclin E1 may therefore provide a strategy to attenuate selected inflammatory responses in liver disease.

Gastrointestinal dysfunction is a prominent feature of neurodevelopmental disorders and contributes significantly to visceral discomfort, but the specific etiology is unclear. In particular, it is unknown which cell types within central, autonomic or enteric nervous systems, gut muscle, epithelium or immune system are the principal drivers of gut dysfunction. We report that the Mecp2 mouse model of Rett syndrome, a neurodevelopmental disorder with profound gut dysmotility, exhibits impaired whole gut transit times and constipation in vivo as well as striking alterations to propagating neurogenic contractions in ex vivo colons. Selective elimination of Mecp2 in neurons, but not glia or smooth muscle, recapitulates these dysmotility phenotypes. Bulk RNA sequencing of colonic external muscle, containing the myenteric compartment of the enteric nervous system, showed significant expression changes in specific neurochemical signaling pathways. Pharmacological modulation of one, the M1 acetylcholine receptor, modified the aberrant neurogenic contractile phenotype in mutant mice and reproduced it in wild-type mice. These results identify disruptions to neuronal muscarinic signaling in the enteric nervous system in a mouse model of Rett syndrome and provide an experimental strategy to identify translationally relevant cellular and molecular pathways underlying gastrointestinal dysmotility in neurodevelopmental disorders.

Primary sclerosing cholangitis (PSC) is a rare, progressive liver disease characterized by biliary inflammation and bile duct strictures and no approved medical therapy. Despite its clinical severity, the pathological mechanisms underlying PSC remain poorly understood, largely due to early diagnostic challenges. Here we provide complementary evidence in human PSC samples, transcriptomic data, mouse models, and 3D cholangiocyte cultures that underscore the importance of hyaluronic acid (HA) and its cognate receptor CD44 in PSC pathogenesis. HA is a glycosaminoglycan abundant in the extracellular matrix in inflammatory disorders, yet its role in PSC has not been well characterized. We demonstrate that in early-stage PSC, cholangiocytes aberrantly produce high molecular weight HA that accumulates in the peribiliary matrix, increasing local tissue stiffness. This mechanical signal is transduced by a CD44/Integrin {beta}1 receptor complex in cholangiocytes, driving cell proliferation, YAP mechanosignaling, pro-inflammatory cytokine production with a transition to a ductular reactive phenotype. CD44 knockdown in cholangiocyte cell lines and mouse models significantly lowered stiffness, and attenuated inflammation. Together, these findings reveal a mechano-inflammatory axis in which HA-driven matrix stiffening perpetuates biliary inflammation and disease progression, identifying HA targeting and CD44 as promising therapeutic strategies. One Sentence SummaryHyaluronan and CD44 mediate matrix changes and progressive fibrosis in primary sclerosing cholangitis.

Environmental enteropathy (EE) is a chronic, subclinical disorder of the small intestine common in low- and middle-income countries (LMICs), where access to sanitation and exposure to enteric pathogens vary greatly by socioeconomic status (SES). Systemic immune cell activation by enteric microbial exposure is a suspected but poorly characterized driver of EE severity. We hypothesised that adults from Low-SES communities would have more severe EE than adults from High-SES communities and that this would be associated with distinct circulating immune cell phenotypes. We enrolled clinically healthy adults from High- (n=26) and Low-SES (n=76) communities in Lusaka, Zambia. Duodenal biopsies from these adults were used for microscopic morphometry assessments, while plasma and stool biomarkers of epithelial damage, intestinal inflammation, microbial translocation, and systemic inflammation were measured by ELISA. Circulating monocyte, neutrophil and T cell phenotypes were characterised in buffy coat cells by flow cytometry. Compared with the High-SES group, adults from Low-SES communities had higher duodenal villus width and crypt depth and lower epithelial surface area, indicative of more severe EE pathology, and higher levels of plasma biomarkers associated with microbial translocation and systemic inflammation. The Low-SES group also had higher expression of activation markers (CD86 and TLR4) and lower expression of HLA-DR on circulating classical monocytes and neutrophils, higher percentages of gut-homing (4{beta}7+) and activated/exhausted (PD-1+) T cells, including gut-homing (4{beta}7+) regulatory T cells. Principal Component Analysis identified key patterns of immune cell phenotypes across SES groups. Confounder-adjusted linear regression models showed that Principal Component 1 (monocyte/neutrophil activation) was inversely associated with duodenal villus height and epithelial surface area across SES groups. These findings indicate that EE severity varies by SES within LMIC and suggest that monocyte and neutrophil activation is linked to greater duodenal remodelling in adults with EE.

Mitochondria are increasingly recognized as regulators of cellular differentiation, but their role in esophageal epithelial homeostasis remains poorly understood. Here, we investigated mitochondrial remodeling during esophageal epithelial differentiation and whether mitochondrial depletion contributes to the acquisition of the differentiated phenotype. Mitochondrial abundance was assessed across normal human esophageal epithelium and in non-transformed immortalized human esophageal epithelial cells (EPC2-hTERT) using two differentiation models. Mitochondrial architecture was quantified, and the functional role of mitochondrial abundance was examined using doxycycline-inducible depletion of transcription factor A, mitochondrial (TFAM). Mitochondrial abundance progressively decreased from basal to superficial compartments of normal human esophageal epithelium and during in vitro differentiation, accompanied by fragmentation and remodeling of the mitochondrial network. TFAM depletion reduced mitochondrial abundance and increased squamous differentiation markers, indicating that mitochondrial depletion was sufficient to promote differentiation. Analysis of candidate mitochondrial clearance pathways identified Bcl2-interacting protein 3-like (BNIP3L)/NIX (NIP3-like protein X) as preferentially associated with differentiated epithelial cells and induced during differentiation. NIX was associated with mitochondria and increased as mitochondrial abundance declined. NIX depletion prevented differentiation-associated mitochondrial depletion and attenuated differentiation marker induction. In human esophageal epithelium, NIX expression increased across the basal-to-suprabasal compartment before declining superficially. BNIP3L expression was reduced in active eosinophilic esophagitis (EoE) and increased following corticosteroid-associated remission. Interleukin-13 similarly reduced BNIP3L expression and suppressed epithelial differentiation in EPC2-hTERT cells. These findings identify NIX-associated mitochondrial remodeling as an important component of esophageal epithelial differentiation and suggest that disruption of mitochondrial quality control may contribute to impaired epithelial differentiation in EoE.

Intestinal epithelial barrier is a key feature of the healthy gut, and disruption of this barrier is a driver of mucosal inflammation. Integrity of the intestinal epithelial barrier is controlled by tight junctions (TJs) and adherens junctions (AJs) associated with the underlying actin cytoskeleton. Posttranslational modifications of actins affect the cytoskeletal assembly and dynamics, however whether such modifications regulate epithelial barriers remains unknown. SETD3 methyltransferase methylates actins at the His73 residue, thereby enhancing filamentous (F)-actin polymerization and turnover. This study examines the role of SETD3 in regulating structure and function of normal intestinal epithelial barrier, as well as its involvement in barrier disruption during mucosal inflammation. siRNA mediated knockdown of SETD3 in DLD-1 and HCA-7 human intestinal epithelial cells (IEC) resulted in several functional and morphological cell abnormalities. These include increased permeability of the IEC barrier, diminished assembly of the perijunctional F-actin belt, increased cell size, and delayed TJ reassembly during calcium switch. Consistently, SETD3 knockout (KO) mice displayed increased intestinal permeability and decreased labeling intensity of apical {beta}-actin as compared to wildtype controls. Furthermore, SETD3 KO mice showed increased cell stemness and proliferation in colonic epithelium without defects in AJ/TJ assembly and secretory cell differentiation. In addition, SETD3 KO mice developed more severe disease symptoms during dextran sodium sulfate (DSS)-induced colitis, which were accompanied by accelerated mucosal erosion and increased macrophage, T cell and neutrophil infiltration into colonic tissue. Overall, our data suggests that SETD3 acts as a novel positive regulator of the intestinal epithelial barrier and plays a protective role during intestinal mucosal damage and inflammation.

Background and Aims: Fibrostenotic stricture is a severe complication of Crohn's disease (CD) that affect up to 50% of patients over their lifetime, causing irreversible narrowing of the intestinal lumen and bowel obstruction, but the molecular programs that distinguish stenotic from non-stenotic inflammation and the relative contributions of fibrosis and smooth-muscle remodeling remain incompletely characterized. We aimed to dissect bowel-wall compartment-specific and histology-defined remodeling programs in fibrostenotic CD. Methods: Matched full-thickness terminal ileal tissues from 4 patients with fibrostenotic CD were sampled from stenotic stricture (SS), non-stenotic inflamed bowel (NS), and non-inflamed bowel (NI). Histomorphology-guided NanoString GeoMx whole-transcriptome profiling was performed across mucosa, muscularis mucosae, submucosa, and muscularis propria. GeoMx data were processed by standard software provided by GeoMx platform. DESeq2 models included patient identity as a blocking factor, and the analysis was performed in 74 histologically valid regions of interest (ROIs) representing full spectrum of CD histopathologies. DEGs were defined as adjusted P < .05 and |log2FC| >= 1.0. Results: Out of 18,677 genes analyzed in each of the ROIs, direct SS-versus-NS comparison identified 0 significant DEGs in mucosa, 18 in muscularis mucosae, 43 in submucosa, and 0 in muscularis propria. NS-versus-NI submucosa showed 132 DEGs (131 increased, 1 decreased), dominated by humoral/plasma-cell genes with a modest stromal-remodeling component. SS-versus-NI submucosa showed 144 DEGs (138 increased, 6 decreased) with coordinated contractile, extracellular-matrix/remodeling and cell-matrix adhesion programs. Muscularized versus fibrotic SS submucosa showed 219 DEGs, with marked enrichment of contractile and remodeling genes in muscularized regions. Excluding 5 follicle-containing submucosal ROIs markedly altered the NS comparison but not the core SS fibromuscular program. Exploratory follicular-niche analyses showed that lymphoid follicle-rich (LFR) submucosa was lymphoid/B-cell enriched relative to lymphoid follicle-null (LFN) areas; SS-LFR nevertheless acquired a stronger fibroblast/ECM program relative to NS-LFR, while CCL19 was the only significant LFR-versus-LFN DEG within SS. Conclusions: Fibrostenotic CD is characterized by spatially concentrated submucosal fibromuscular remodeled inflammatory program. Non-stenotic submucosa is heterogeneous and may already show regional stromal remodeling, whereas established stenosis displays a broader and more robust ECM-contractile-adhesion phenotype with molecularly distinct fibrotic and muscularized niches.

Human intestinal diseases, including inflammatory bowel disease (IBD), are difficult to model because existing animal and in vitro systems do not capture the integrated vascular, immune, and metabolic complexity of the intestine. Here, we establish a custom normothermic machine perfusion platform that maintains surgically resected human intestinal tissue for up to 72 hours under near-physiological conditions. Discarded specimens from patients with nonmalignant intestinal diseases, including IBD and diverticulitis, were continuously perfused at 37{degrees}C with real-time hemodynamic and metabolic monitoring. Perfusion preserved tissue viability, mucosal architecture, epithelial integrity, oxygen consumption, metabolic activity, and vascular patency. Daily perfusate sampling enabled longitudinal profiling and experimental modulation of inflammatory mediators, revealing patient-specific immune states. This long-duration platform bridges reductionist in vitro models and clinical disease, enabling mechanistic studies of intestinal inflammation and future testing of personalized therapies in intact patient-derived tissue.

Developmental programs establish tissue identity and organization, but how their reactivation contributes to adult tissue responses remains poorly understood. Here, we identify a functional contribution of apolipoprotein A-I (APOA1), which marks a late fetal colonic epithelial state that reappears after injury. Spatial and temporal profiling localized Apoa1 transcription predominantly to epithelial cells at the tips of developing proximal colonic folds. Apoa1 loss delayed fold maturation and altered the abundance and composition of developing enteroendocrine populations, linking this apolipoprotein to mucosal architecture and epithelial differentiation. APOA1 protein closely interacted with WNT5A during development and injury, while Apoa1 deficiency increased Wnt5a expression and altered its epithelial protein distribution. Following colonic injury, single-cell and spatial profiling identified genotype-associated transcriptional changes involving stress responses, barrier and immune functions, and metabolism, with a shared component across males and females. These changes accompanied reduced enteroendocrine representation and increased representation of inflammation-associated macrophages. Finally, regional and compartmental expression analyses, together with hindgut explant perturbation, identified HOXB7 transcription factor as a regulator of developmental Apoa1 expression. Together, these findings establish functional relevance for a component of a reactivated late fetal epithelial program, connecting APOA1 to colonic maturation and the epithelial and inflammatory organization of injured tissue.

Background: Inflammatory bowel disease (IBD) has been linked to a two- to eightfold increase in colorectal cancer (CRC) risk. However, particular genes that bridge intestinal dysbiosis and oncogenic transcriptional reprogramming remain unclear. The Hippo/YAP pathway is a major regulator of intestinal epithelial homeostasis whose effector YAP is hyperactivated in CRC, but its transcriptional engagement in IBD mucosa and its link to specific cancer-risk genes has not been thoroughly mapped. Methods: Bulk RNA-seq data were integrated from two independent cohorts: GSE235236 (IBD colon mucosa; UC/CD vs. healthy controls; n = 56) and TCGA-COAD/READ (CRC tumors stratified by YAP activity; n = 592). Differential expression analysis was conducted separately within each cohort using Welch's t-test with Benjamini-Hochberg FDR correction. YAP transcriptional activity was estimated per sample as the mean row-wise Z-score of seven canonical YAP target genes, and TCGA tumors were stratified into Hippo-High (Q4) and Hippo-Low (Q1) groups. Genes showing consistent dysregulation across both cohorts were identified and ranked to define a Dysbiosis-Hippo Response (DHR) module, which was further organized into functional gene archetypes. Results: IBD mucosa showed 2,848 significantly dysregulated genes (FDR < 0.05). Comparison with the CRC cohort identified 120 genes showing concordant dysregulation, forming the DHR module. These genes were significantly enriched in TNF-alpha signaling via NF-kB (adjusted p = 0.006), inflammatory response (adjusted p = 0.013), and Myogenesis (adjusted p = 0.013). DHR gene classification revealed three functional archetypes: (i) innate immune activation (SPI1, OSM, IL11, LILRA2, SLC11A1) reflecting convergent myeloid reprogramming; (ii) epithelial-mesenchymal transition (TWIST1, SNAI1, PRRX2, MMP9) priming invasive potential before malignancy; and (iii) tumor microenvironment immune regulation (TN-FRSF4, TNFRSF18, PDCD1, VASN, SPON2) shaping immune tolerance. Interestingly, the canonical YAP target genes (CTGF, CYR61, ANKRD1, AMOTL2, and FSTL1), sharply upregulated in Hippo-High CRC, were essentially unchanged in IBD mucosa. This signifies that the shared DHR programme operates through non-canonical YAP effectors. And yet, YAP target scores are positively correlated with DHR module expression across IBD samples (Spearman rho = 0.726, p = 2.5 x 10-10). Conclusions: Our findings define a three-archetype DHR module that links gut dysbiosis to YAP-driven oncogenesis at single-gene resolution. The programme shared between IBD and CRC is driven by innate immune activation, EMT priming, and remodeling of the immune microenvironment, not by the canonical CTGF/CYR61 YAP target axis, which appears only at the CRC stage. This gene-level framework provides tractable mechanistic targets for chemoprevention and biomarker development at the inflammation-to-cancer transition.

Human pluripotent stem cell-derived intestinal organoids hold great promise for disease modeling, drug screening, and regenerative medicine. However, conventional intestinal organoids are predominantly epithelial, small in scale, and lack the multicellular complexity required to recapitulate the pathophysiology of intestinal disorders such as inflammatory bowel disease (IBD) and colorectal cancer (CRC). Here, we report the development of Centimeter-Scale, purely 3D self-organized human intestinal organoids (IOs) from induced pluripotent stem cells (iPSCs) that encompass multiple tissue lineages, including epithelium, mesenchyme, smooth muscle, neurons, immune cells, and vasculature. These organoids achieve functional maturation by day 100+, exhibiting rhythmic peristaltic-like contractions, and by day 147 they display histological structures including lumens, crypt-like architecture, goblet cells, and smooth muscle. Importantly, for the first time, the neuro-muscle lineages arise spontaneously and autonomously in a purely 3D culture system, without any external stimulation (e.g., electrical, chemical, or mechanical), and mature to form functional neuromuscular junctions, driving macroscopically visible peristaltic-like contractions that mimic intestinal motility entirely through in vitro culture, without any xenotransplantation. Single-cell RNA sequencing at day 115 identified 12 cell subtypes across four major lineages, recapitulating the cellular diversity of the developing human intestine. Using this platform, we established an LPS/IFN-{gamma}-induced IBD model that recapitulated key pathological features, including epithelial disruption, immune cell infiltration, and IL-6 elevation. Transcriptomic analysis confirmed activation of the NF-{kappa}B and JAK2-STAT3 pathways, multi-modal cell death, and immune recruitment machinery, all consistent with clinical IBD pathology. Furthermore, we developed intestinal cancer models at 7 and 21 days showing abnormal hyperplasia, and a probiotic co-culture system demonstrating anti-inflammatory efficacy. Together, these results establish Centimeter-Scale intestinal organoids as a physiologically relevant, multicellular platform for modeling intestinal diseases and evaluating therapeutic interventions.

Notch signaling is essential for maintaining intestinal stem cell activity and directing epithelial cell fate. Paneth cells have been proposed to be niche cells, providing Notch signal to neighboring stem cells through expression of the key Notch ligands DLL1 and DLL4. However, intestinal stem cells persist in the absence of Paneth cells. Here, we used genetic mouse models to clarify the role of Paneth cell-derived Notch ligands for stem cell function. Paneth cell-specific deletion of Dll1 and Dll4 resulted in loss of crypt base stem cells, with no effect on overall crypt cell proliferation. Organoid growth was reduced in knockout mice, suggesting reduced stem cell function. Upon irradiation injury, stem cell return and crypt regeneration were significantly impaired in the Notch ligand-deleted mice. These findings suggest that Paneth cell-derived Notch ligands DLL1 and DLL4 are crucial for crypt base stem cell maintenance and for crypt regeneration after injury.

The role of aberrant glycosylation in pancreatitis remains an under-investigated area of research. Here, we determined that the ST6GAL1 sialyltransferase, which adds 2-6-linked sialic acids to N-glycoproteins, was upregulated in pancreatic tissues from patients with acute (AP) and chronic (CP) pancreatitis, and in mice with experimental AP. Within these tissues, ST6GAL1 was selectively expressed in acinar cells undergoing acinar to ductal metaplasia (ADM), a process whereby injured acinar cells de-differentiate and re-enter the cell cycle to enable tissue repair. To study the functional role of ST6GAL1 in pancreatitis, we used HPNE pancreatic epithelial cells with modulated ST6GAL1 expression, along with pancreatic organoids from mice with transgenic expression of ST6GAL1 (SC mice). In these models, ST6GAL1 activity promoted the activation of EGFR (a well-known ADM-driver), ERK, and AKT. ST6GAL1-expressing cells also displayed an ERK-dependent upregulation of the anti-apoptotic proteins, Mcl-1 and Bcl-xL, and impaired apoptotic signaling by the TNFR1 and Fas death receptors. Unbiased kinomics profiling revealed that ST6GAL1 induced the activation of many receptor tyrosine kinases associated with cell survival and proliferation (e.g., EGFR, PDGFR, MET). Furthermore, ST6GAL1 enhanced the survival and proliferation of cells exposed to stress-inducing conditions such as serum withdrawal. Based on these findings, we hypothesized that the pro-survival phenotype imparted by ST6GAL1 would facilitate tissue healing following a bout of pancreatitis. Accordingly, we induced AP in wild type and SC mice via L-arginine injection and found that SC mice had more rapid and efficient tissue repair, evidenced by accelerated restoration of the acini, diminished acinar apoptosis, and reduced immune cell infiltration. Together, these findings highlight a novel glycosylation-dependent mechanism that drives cell survival, positioning ST6GAL1 as a key adaptive mediator of pancreatic regeneration.

Introduction: Adipose tissue fibrosis has been associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and suboptimal weight loss after bariatric surgery. The objective was to characterize changes in adipose tissue fibrosis and extracellular matrix (ECM) components in adipose tissues from two abdominal fat compartments, subcutaneous and omental, in response to bariatric surgery; and determine the effect of weight loss on the association between adipose tissue fibrosis and MASLD feature changes with weight loss. Methods: Liver and adipose tissue biopsy samples were obtained before and after weight loss from patients who underwent two-stage bariatric surgery. Adipose tissue pericellular fibrosis was evaluated histologically with picrosirius red staining for collagen quantification and normalized to adipocyte count per microscope field. Transcript levels of ECM remodeling and inflammatory genes were measured by RNA sequencing in subcutaneous adipose tissue pre- and post-surgery. Results: At baseline, adipose tissue pericellular fibrosis did not associate with MASLD features, metabolic dysfunction-associated steatohepatitis or hepatic fibrosis in patients with severe obesity. Changes in liver histology after surgery were unrelated to changes in adipose tissue fibrosis. Transcripts for most genes involved in ECM remodeling and inflammation were downregulated in subcutaneous fat one year after bariatric surgery. Omental, but not subcutaneous, adipose tissue fibrosis decreased after surgery when adjusted for cell number (mean {+/-} SD: 0.41 {+/-} 0.12 vs 0.32 {+/-} 0.08 {micro}m2/cell, p<0.005). Higher subcutaneous adipose tissue fibrosis correlated with reduced weight loss (estimate {+/-} SE: -2.04 {+/-} 0.99, p<0.05) and BMI loss (-0.73 {+/-} 0.35, p<0.05), while increased omental adipose tissue fibrosis after weight loss was associated with worsening hepatic fibrosis (0.31 {+/-} 0.14, p<0.05). Conclusion: In individuals with severe obesity undergoing bariatric surgery, adipose tissue fibrosis is poorly correlated with MASLD features. Notably, pericellular fibrosis in subcutaneous abdominal, but not omental, adipose tissue was associated with a reduced weight-loss response to surgery.