Esophageal atresia and tracheoesophageal fistula (EA/TEF) are congenital malformations of the foregut. We identified two distinct variants in the BMP/TGFβ repressor SMAD6 in two individuals exhibiting EA/TEF. We investigated the function of SMAD6 in tracheoesophageal development using two orthogonal approaches in Xenopus embryos, both resulting in foregut malformations similar to those observed in EA/TEF. We then used human pluripotent stem cell-derived foregut epithelium and mesenchyme to explore the separate roles of SMAD6 in these germ layers. CRISPR-mediated disruption of human SMAD6 caused an increase in BMP signaling in foregut epithelium and mesenchyme consistent with its role as a BMP repressor. Loss of SMAD6 caused patterning defects in both tissue types; SMAD6-/- endoderm showed increased expression of distal gut tube markers and SMAD6-/- mesenchyme showed increased expression of ventral and posterior markers, including markers of cardiac and liver mesenchyme lineages. Furthermore, SMAD6-/- mesenchyme had decreased ability to form CD31-positive endothelial cells. Our results demonstrate that SMAD6 is required for foregut development and that rare variants in this gene are likely causative for foregut malformations in EA/TEF.

The goal of engineering increasingly complex human organoid models is to more accurately model human organogenesis and disease. Recently, human antral gastric organoids (hAGOs) were engineered to contain splanchnic mesenchyme (SM) and enteric neural crest cells (NCCs), resulting in functional three germ layer gastric organoids. To determine the robustness of hAGOs and how additional germ layers impact development, we benchmarked these hAGOs with the developing human stomach. Human AGOs in vitro were most similar to 7 week fetal stomach with the epithelium comprising primarily mucous precursors. The SM and NCCs added to hAGOs formed fetal gastric-like mesenchymal or neuroglial precursors, respectively. Incorporation of SM and NCCs did not drastically alter the cellular diversity of the epithelium in vitro. Following transplantation, hAGOs with SM matured into tissue more like the 2nd trimester stomach. Bioinformatic inference confirmed known signaling crosstalk between germ layers and identified new signaling candidates that may regulate tissue assembly. Together, three germ layer hAGOs faithfully model the multilayer complexity of the fetal stomach at the single-cell transcriptomic level and provide insight into human stomach development.

Key limitations of current human gastrointestinal organoids include incomplete physiological maturation and the need for complex, time-consuming assembloid approaches to integrate a functional nervous system for transplantation. Here we present a confined culture system (CCS) method that generates large-scale, elongated and functional human small intestinal, colonic and gastric tissues with a de novo enteric nervous system (ENS). We use a 3D-printed scaffolding tray to restrict spheroid fusion and growth, promoting the spontaneous co-development of a functional ENS. Transcriptomic and electrophysiological data demonstrate selective neuromuscular function and the presence of excitatory and inhibitory neurons within the tissues. When compared to traditional methods, the CCS expedites tissue maturation for transplantation, yielding organoids that are up to ten times larger, reaching widths of 8 cm after 10 weeks, and exhibit enhanced engraftment rate. CCS organoids integrate and adapt to murine luminal environment while maintaining barrier integrity and functional capacity. The CCS methodology simplifies current protocols while accelerating the production of complex, functional and clinically relevant gut tissues.

Multiple signaling pathways and transcription factors (TFs) establish organ domains in the developing gastrointestinal tract. How these are integrated into spatial-temporal networks to regulate organogenesis and how disruptions to those networks lead to congenital syndromes remain poorly understood. Using human pluripotent stem cell cultures and Xenopus embryos, we demonstrate that retinoic acid (RA) from the lateral plate mesoderm directly activates expression of the TF rfx6 in posterior foregut endoderm. Rfx6 subsequently promotes posterior foregut identity while suppressing Wnt-dependent hindgut and Bmp-dependent pharyngeal fates through direct and indirect mechanisms. Rfx6 can directly activate the expression of several key foregut TFs (onecut1 and pdx1) and Wnt antagonists (sfrp2/5) while indirectly restricting expression of Wnt and Bmp ligands. Rfx6 also directly suppresses transcription of the Wnt-dependent hindgut TF cdx2 and the Bmp-dependent pharyngeal TFs nkx2-5 and nkx2-6. Thus, Rfx6 acts at multiple levels to integrate RA, Wnt, and Bmp activity into a network with lineage-promoting TFs to control gut tube patterning. These results provide insight into the molecular basis of Mitchell-Riley Syndrome congenital anomalies, which are caused by RFX6 mutations.

Wnt Family Member 2B (WNT2B) mutations result in Diarrhea-9, a congenital diarrhea syndrome with an extreme phenotype and unique histological defects. Attempts to model Diarrhea-9 in rodents and study patient epithelial tissue have not been able to fully reproduce the human phenotype, making understanding this condition challenging. Here, we aimed to interrogate the mechanisms and the specific cellular compartment contributing to Diarrhea-9 using a human intestinal organoid model. Live and histological imaging revealed partial epithelial delamination in human intestinal organoids with WNT2B loss, which was absent in controls. A significant number of crypts in human intestinal organoids with WNT2B loss lacked olfactomedin 4 (OLFM4), a surrogate marker of stem cell activity. Key transcriptomic pathways altered between groups included trafficking of apical digestion proteins, which was confirmed via immunofluorescence. Patient-derived enteroid proteomic analysis revealed similar results. Recombination experiments in human intestinal organoids suggested compartment-specific effects of WNT2B loss, with WNT2B-deficient mesenchyme producing a more pronounced disruption of epithelial architecture and organization. These findings suggest compartment-specific roles for WNT2B in human intestinal development and function, with WNT2B-deficient mesenchyme having a particularly strong influence on epithelial architecture and organization. This study further highlights human intestinal organoids as a useful model for investigating human-specific intestinal disorders that are not fully recapitulated in murine models.

Principles of developmental biology have inspired efforts for directed differentiation of human pluripotent stem cells (hPSCs), leading to the first generation of organoids that are now well established as models of human development and disease. However, first-generation organoid models were missing many cell types that would be needed to study normal and pathological processes. Here, we discuss how designing next-generation organoids with increased cellular complexity has been possible by better reproducing developmental processes in play during organogenesis in vivo. We focus on recent conceptual and technical advances in reconstructing appropriate cellular diversity in organoids, dissecting the importance of tissue-tissue interactions and specialized cell addition, and how engineering technologies can further enhance our ability to control how cells are brought together to mimic human development in vitro.

Rationale:Alpha-1 antitrypsin deficiency (AATD) is a genetically inherited condition that can result in serious lung and liver disease. It is unclear whether testing for common alleles is sufficient or if testing for rare variants is helpful in identifying clinically significant disease. Methods:A retrospective review was performed on adult patients who had alpha-1 antitrypsin phenotyping from January 2016 through December 2021. We recorded clinical characteristics and then grouped patients based on Pi*types: normal, Pi*Z-heterozygotes (Het), Pi*ZZ, Pi*S-Hets, and defined a Pi*Non-S/Non-Z group to include other identified Pi*types. Chi-square and Kruskal-Wallis tests were used for bivariate analyses, generalized linear models for modeling forced expiratory volume in 1-second (FEV1), and logistic regression modeling for health care utilization outcomes adjusted for race, age, and sex. Results:A total of 1777 tests were ordered from January 2016 through December 2021. Testing identified 79.5% Pi*MM, 8.4% Pi*Z-Het, 8.4% Pi*S-Het, 3.3% Pi*Non-S/Non-Z, and 0.5% Pi*ZZ. FEV1 to forced vital capacity and FEV1 percentage predicted were lowest in the Pi*Non-S/Non-Z group compared to the other groups. The Pi*Non-S/Non-Z group had higher mean neutrophil lymphocyte ratio compared to the other groups (p=0.021), higher hospitalization for acute respiratory events (27.6%; p=0.019), intensive care unit utilization (15.3%, p=0.011), and death (25.4%, p=0.041) compared to the other groups. Conclusion:AATD Pi*typing identified several allelic combinations not previously linked with clinical disease. Compared to other Pi*type groups, the Pi*Non-S/Non-Z group had impairments in pulmonary function, elevated inflammatory markers, higher health care utilization, and death. Our results underpin the need to explore relationships between rare allelic combinations and clinical outcomes.

Pancreas organogenesis relies on sequential interactions between the pancreatic epithelium and surrounding mesodermal cell types that initiate epithelial budding and branching to form a complex ductal network with terminal acini. Despite recent advances with pluripotent stem cell-based approaches, there are no models that robustly recapitulate pancreas morphogenesis or the spatial organization of ductal, exocrine, endocrine and mesenchymal cells seen in the native organ. Here, we introduce a new pluripotent stem cell-based pancreatic organoid that captures the complexity seen during pancreatic development, with budding and stratification of multipotent progenitors followed by formation of a ductal network that give rise to peripheral acini. We identify a critical role for mesenchyme-derived factors to robustly promote pancreatic organoid formation and morphogenesis. Human pancreatic organoids are correctly patterned, with functional exocrine acini secreting digestive enzymes into a ductal network. Comparative analysis confirms that the pancreatic organoids are similar to early second trimester human pancreas, with potential to further mature upon transplantation in mice. Finally, we show that endocrinogenesis can be reproduced in organoids, generating functional islet-like clusters interspersed within the ductal network. Together, this represents an exciting new platform to study human pancreas development and a broad array of pancreatic diseases.

BACKGROUND & AIMS: Organs of the gastrointestinal tract contain tissue-resident immune cells that function during tissue development, homeostasis, and disease. However, most published human organoid model systems lack resident immune cells, thus limiting their potential as disease avatars. For example, human intestinal organoids (HIOs) derived from pluripotent stem cells contain epithelial and various mesenchymal cell types but lack immune cells. In this study, we aimed to develop an HIO model with functional tissue-resident macrophages. METHODS: HIOs and macrophages were generated separately through the directed differentiation of human pluripotent stem cells and combined in vitro. Following 2 weeks of coculture, the organoids were used for transcriptional profiling, functional analysis of macrophages, or transplanted into immunocompromised mice and matured in vivo for an additional 10-12 weeks. RESULTS: Macrophages were incorporated into developing HIOs and persisted for 2 weeks in vitro HIOs and for at least 12 weeks in HIOs in vivo. These cocultured macrophages had a transcriptional signature that resembled those in the human fetal intestine, indicating that they were acquiring the features of tissue-resident macrophages. HIO macrophages could phagocytose bacteria and produced inflammatory cytokines in response to proinflammatory signals, such as lipopolysaccharide, which could be reversed with interleukin-10. CONCLUSIONS: We generated an HIO system containing functional tissue-resident macrophages for an extended period. This new organoid system can be used to investigate the molecular mechanisms involved in inflammatory bowel disease. (Cell Mol Gastroenterol Hepatol 2025;19:101444; https://doi.org/ 10.1016/j.jcmgh.2024.101444)

Human pancreas development remains incompletely characterized due to restricted sample access. We investigate whether pigs resemble humans in pancreas development, offering a complementary large-animal model. As pig pancreas organogenesis is unexplored, we first annotate developmental hallmarks throughout its 114-day gestation. Building on this, we construct a pig single-cell multiome pancreas atlas across all trimesters. Cross-species comparisons reveal pigs resemble humans more closely than mice in developmental tempo, epigenetic and transcriptional regulation, and gene regulatory networks. This further extends to progenitor dynamics and endocrine fate acquisition. Transcription factors regulated by NEUROG3, the endocrine master regulator, are over 50% conserved between pig and human, many being validated in human stem cell models. Notably, we uncover that during embryonic development, emerging beta-cell heterogeneity coincides with a species-conserved primed endocrine cell (PEC) population alongside NEUROG3-expressing cells. Overall, our work lays the foundation for comparative investigations and offers unprecedented insights into evolutionarily conserved pancreas organogenesis mechanisms across animal models.

Metaplasia is an adaptative response to injury and inflammation and can be a precursor to dysplasia and cancer. Metaplasia in the esophagus, termed Barrett's esophagus, is the replacement of the stratified squamous epithelium by glandular tissue comprising gastric and/or intestinal cell lineages. Metaplasia in the stomach can be divided further into pyloric metaplasia, in which corpus glands become more antral-like, or gastric intestinal metaplasia (GIM), in which gastric cells are replaced by intestinal cell lineages, with the latter subdivided into complete and incomplete. The routine diagnosis of metaplasia and dysplasia is performed by examining hematoxylin and eosin-stained sections and mucin immunohistochemistry. However, these methods fail to capture the cellular diversity across glands and the molecular changes in cells that can predict possible progression to dysplasia or cancer. The use of immunohistochemistry- or immunofluorescence-based biomarkers can improve our understanding of gland phenotypes and aid the differentiation of metaplastic and dysplastic transitions. Here, we provide an overview of the pathophysiology of metaplasia in the esophagus and stomach and detail the current understanding of biomarker expression across metaplastic transitions. We suggest a cohort of biomarkers that can differentiate between metaplastic phenotypes in the esophagus (gastric-type and intestinal-type) and the stomach (pyloric metaplasia, incomplete GIM, and complete GIM) that might be used in research and clinical settings. Importantly, we detail the status of dysplasia biomarkers in both the esophagus and stomach, which may have clinical relevance in stratification of high-risk patients.

A confined culture system (CCS) establishes methods to generate complex functional human gastrointestinal tissues. This approach yields large-scale innervated small intestinal, colonic and gastric organoids with an elongated tubular shape for both in vitro and in vivo studies. Transcriptomic and electrophysiological data demonstrate the co-development of a functional de novo enteric nervous system, which is absent from conventional organoids. When compared to traditional methods, CCS derived small intestinal, colonic and gastric organoids reached maturation supporting transplantation in half of the time, resulting in enhanced engraftment rates and sizes. Murine luminal content exposure within CCS organoids in vivo further augmented function, supporting the potential translational benefits required to model complex intestinal diseases. In summary, the CCS methodology simplifies current protocols while adding complexity and expediting the generation of clinically relevant functional gut tissues.

The embryonic development of the liver is initiated by the emergence of hepatoblasts, originating from the ventral foregut endoderm adjacent to the heart. Here, we identify and characterize a previously unrecognized population of early hepatoblasts at the ventroposterior part of the emerging liver bud, traced from Cdx2-positive endoderm progenitors, which we term primitive hepatoblasts. Mouse and human single-cell transcriptomics reveals the expression of both canonical hepatoblast markers TBX3, FGB, and KRT8/18 and primitive-specific mesenchymal markers ID3, VIM, and GATA4. Lineage tracing revealed the notable contribution up to 12.6% of LIV2+ hepatoblasts at E11.5 but diminishes in late fetal and postnatal development. Epigenetic and functional perturbation studies further uncover that primitive hepatoblast emergence is primed by WNT-suppression on RA-permissive CDX2+FOXA2+ progenitors. Furthermore, human pluripotent stem cell-derived primitive hepatoblast-like cells secrete pleiotrophin and midkine to amplify hepatoblast populations and develop epithelial-mesenchymal hybrid tissues in vivo. Our results provide a new framework for understanding lineage heterogeneity during early hepatogenesis and offer revised insights into strategies to model normal and abnormal liver development.

The vasculature and mesenchyme exhibit distinct organ-specific characteristics adapted to local physiological needs, shaped by microenvironmental and cell-cell interactions from early development. To recapitulate this entire process, we co-differentiated mesoderm and endoderm within the same spheroid to vascularize lung and intestinal organoids from induced pluripotent stem cells (iPSCs). Bone morphogenetic protein (BMP) signaling fine-tuned the endoderm-to-mesoderm ratio, a critical step in generating appropriate proportions of endothelial and epithelial progenitors with tissue specificity. Single-cell RNA sequencing (scRNA-seq) revealed organ-specific gene signatures of endothelium and mesenchyme and identified key ligands driving endothelial specification. The endothelium exhibited tissue-specific barrier function, enhanced organoid maturation, cellular diversity, and alveolar formation on the engineered lung scaffold. Upon transplantation into mice, the organoid vasculature integrated with the host circulation while preserving organ specificity, further promoting organoid maturation. Leveraging these vascularized organoids, we uncovered abnormal endothelial-epithelial crosstalk in patients with forkhead box F1 (FOXF1) mutations. Multilineage organoids provide an advanced platform to study intricate cell-to-cell communications in human organogenesis and disease.

Background and Aims:WNT2B mutations result in Diarrhea-9 (DIAR9), a congenital diarrhea syndrome with an extreme phenotype and unique histological defects. Attempts to model DIAR9 in rodents and study patient epithelial tissue have not been able to fully reproduce the human phenotype, making understanding this condition challenging. Here, we aimed to interrogate the mechanisms and the specific cellular compartment contributing to DIAR9 using a human intestinal organoid model. Methods:Human intestinal organoids (HIOs) generated from both a patient-derived WNT2B R69* iPSC line and a control line were transplanted into immunocompromised mice for 10 weeks. Grafts were harvested and histologically compared. Bulk RNA sequencing was performed on both organoid groups and on patient-biopsy derived enteroids. In vitro recombination experiments were performed to describe the causative cellular compartment. Results:Live and histological imaging revealed partial epithelial delamination in WNT2B R69* HIOs, which was absent in controls. A significant number of crypts in WNT2B R69* HIOs lacked OLFM4, a surrogate marker of stem cell activity. Key transcriptomic pathways altered between groups included trafficking of apical digestion proteins, which was confirmed via immunofluorescence. Patient derived enteroid proteomic analysis revealed similar results. Recombination experiments in HIOs revealed that while both epithelial and mesenchymal WNT2B are important for stem cell function, lack of mesenchymal WNT2B was sufficient to elicit the phenotype. Conclusion:We demonstrated that mesenchymal WNT2B is critical for supporting human intestinal epithelial development and function.

Introduction: Alpha-1 antitrypsin deficiency (AATD) is the most common genetic etiology of chronic obstructive pulmonary disease (COPD), with considerable phenotypic variability among affected individuals sharing disease-causing variants. Therefore, a multi-center longitudinal cohort study of participants with PiZZ AATD was established to examine disease heterogeneity and the natural history of disease through a deep phenotyping strategy including quantitative computed tomography (CT) imaging, serum, and airway biomarkers. Methods: In 2019, the Alpha-1 Foundation established a research registry (A1F Registry), containing extensive patient-reported clinical data. All 18 years or older self-reported PiZZ AATD participants in the A1F Registry were invited to join the A1BC across nine participating centers. Inclusion and exclusion criteria were designed to capture PiZZ individuals experiencing a broad range of AATD-associated pulmonary and liver manifestations. Children and pregnant females were excluded from enrollment, as were those listed for transplant, with clinically severe bronchiectasis, recent pulmonary exacerbations, and past major thoracic surgery. Upon enrollment, clinical questionnaires, spirometry, and quantitative CT imaging was performed. The Jonchkeere's trend test was applied to the 15th percentile in Hounsfield units (PERC15) and GOLD stages using R software. Study design was approved by the Western Institutional Review Board-Copernicus Group (WCG). Results: Of the 275 participants enrolled, 107 were male (38.9%). 46.2% were originally tested for AATD due to lung symptoms, 11.3% for liver symptoms, 25.1% via familial testing, 10.9% through direct-to-consumer DNA testing, and 3.3% for other reasons. One participant was a current smoker (0.36%), 93 (33.8 %) were former smokers, and 181 (65.8 %) were non-smokers. Those who smoked had an average 14.1 pack-years (SD=14.3). Among the cohort, 43 patients (15.6%) reported two or more acute exacerbations of COPD in the year prior to enrollment. More than half of participants (56.0%) had airflow obstruction (post-BD FEV1/FVC <0.7). 15th percentile in Hounsfield units (PERC15) significantly decreased as GOLD stage increased among the cohort (Figure). 146 (53.1%) participants were actively receiving augmentation therapy, with 72.6% of those receiving 60mg/kg/week infusions. Conclusion: A1BC is a robust cohort of PiZZ AATD participants with detailed metrics of disease severity. The cohort reveals a decrease in PERC15 with increased GOLD stage amongst participants recruited in an era of widely available genetic screening, familial testing, advancements in COPD care, and reduced smoking rates. With longitudinal data collection and follow-up, the A1BC has the potential to systematically and precisely evaluate disease heterogeneity and identify factors associated with severity and progression.

Human organoid model systems have changed the landscape of developmental biology and basic science. They serve as a great tool for human-specific interrogation. In order to advance our organoid technology, we aimed to test the compatibility of a piezoelectric material with organoid generation, because it will create a new platform with the potential for sensing and actuating organoids in physiologically relevant ways. We differentiated human pluripotent stem cells into spheroids following the traditional human intestinal organoid (HIO) protocol atop a piezoelectric nanofiber scaffold. We observed that exposure to the biocompatible piezoelectric nanofibers promoted spheroid morphology 3 days sooner than with the conventional methodology. At day 28 of culture, HIOs grown on the scaffold appeared similar. Both groups were readily transplantable and developed well-organized laminated structures. Graft sizes between groups were similar. Upon characterizing the tissue further, we found no detrimental effects of the piezoelectric nanofibers on intestinal patterning or maturation. Furthermore, to test the practical feasibility of the material, HIOs were also matured on the nanofiber scaffolds and treated with ultrasound, which lead to increased cellular proliferation which is critical for organoid development and tissue maintenance. This study establishes a proof of concept for integrating piezoelectric materials as a customizable platform for on-demand electrical stimulation of cells using remote ultrasonic waveforms in regenerative medicine.

BACKGROUND & AIMS:Intercellular orchestration across hepatic and immune lineages governs critical liver development and disease processes. Existing in vitro human liver models limit immune lineage outputs partly due to the lack of an endogenous niche for blood co-development. By modeling a developmental niche, we developed human fetal liver-like organoids (FLOs) harboring a multipotent hematopoietic system to model and study the complex multilineage interaction in liver development and injury. METHODS:We generated FLOs from human pluripotent stem cells to study cross-lineage self-organization by co-developing a hemogenic mesoderm and hepatic endoderm. Hematopoietic progenitor cell and epithelial lineage potential and dynamics were assessed through single-cell and bulk transcriptomics, immunophenotyping, and functional assays. RESULTS:FLOs established a bona fide niche that supports the simultaneous emergence of hepatobiliary, endothelial, and mesenchymal lineages, along with multipotent hematopoietic progenitor cells exhibiting predominant myeloid lineage commitment while retaining the potential for fetal B and T cell differentiation. Within the FLO microenvironment, hepatic and hematopoietic lineages continued to mature in the absence of extrinsic differentiation factors. Enriching hematopoietic derivatives with a small molecule-cytokine cocktail generated divergent immune cell populations, including granulocytes and polarized macrophages equipped with immunoreactive and lineage-specific function. Finally, FLOs mechanistically revealed an IL-8-mediated neutrophil-driven injury response upon steatotic-lipotoxic injury, highlighting key liver-intrinsic immune mechanisms. CONCLUSIONS:FLOs provide a physiologically relevant model for multi-lineage hemato-hepatogenesis and innate immunity in the liver. By recapitulating critical fetal liver tissue functions, FLOs offer a translational platform for studying human fetal liver development, immune-mediated hepatic injury, and regenerative therapies. IMPACT AND IMPLICATIONS:This study establishes a human pluripotent stem cell-derived fetal liver-like organoid (FLO) system that reconstitutes hepato-hematopoietic co-development and innate immune function. FLOs offer a human platform to dissect cross-lineage signaling during liver development and immune-mediated injury. Clinically and translationally, FLOs enable mechanistic modeling of pediatric liver diseases and regenerative interventions, offering a tractable in vitro system for preclinical screening and precision-medicine approaches.