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Congenital diarrheas and enteropathies (CODEs) constitute a heterogeneous group of individually rare disorders manifesting with infantile-onset chronic diarrhea. Genomic deletions in chromosome 16, encompassing a sequence termed the ‘intestine-critical region (ICR)’, were recently identified as the cause of an autosomal recessive congenital enteropathy. The regulatory sequence within the ICR is flanked by an unannotated open reading frame termed PERCC1, which plays a role in enteroendocrine cell (EEC) function. We investigated two unrelated children with idiopathic congenital diarrhea requiring home parenteral nutrition attending the Irish Intestinal Failure Program. Currently 12 and 19-years old, these Irish male patients presented with watery diarrhea and hypernatremic dehydration in infancy. Probands were phenotyped by comprehensive clinical investigations, including endoscopic biopsies and serum gastrin level measurements. Following negative exome sequencing, PCR and Sanger sequencing of the entire coding region and intron boundaries of PERCC1 were performed for each proband and their parents. In both patients, serum gastrin levels were low and failed to increase following a meal challenge. While no deletions involving the ICR were detected, targeted sequencing of the PERCC1 gene revealed a shared homozygous c.390C > G stop gain variant. We report clinical and molecular findings in two unrelated patients harboring a shared homozygous variant in PERCC1, comprising the first description of a point mutation in this gene in association with CODE. That both parenteral nutrition dependent children with unexplained diarrhea at our institution harbored a PERCC1 mutation underscores the importance of its inclusion in exome sequencing interpretation.

A major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

Human organoids have potential to revolutionizein vitrodisease modeling by providing multicellular architecture and function that are similar to thosein vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, we overcome these challenges by engineering “microarray three-dimensional (3D) bioprinting” technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into liver and intestine organoids forin situfunctional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

Background Small airways are known to be affected early in the course of chronic obstructive pulmonary disease (COPD); however, traditional spirometric indices may not accurately identify small airways disease. Research Question Can FEV3/FEV6 identify early airflow abnormalities and predict future clinically important respiratory-related outcomes, including development of COPD? Study Design and Methods We included eight hundred thirty-two current and former smokers with post-bronchodilator FEV1/FVC ≥0.7 from the SPIROMICS cohort. Participants were classified as having a reduced pre-bronchodilator FEV3/FEV6 based on lower limit of normal (LLN) values. Repeatability analysis was performed for FEV3 and FEV6. Regression modeling was used to evaluate the relationship between baseline FEV3/FEV6 and outcome measures including functional small airways disease on thoracic imaging and respiratory exacerbations. Interval censored analysis was used to assess progression to COPD. Results FEV3/FEV6 Interpretation FEV3/FEV6 is a routinely available and repeatable spirometric index which can be useful in the evaluation of early airflow obstruction in current and former smokers without COPD. A reduced FEV3/FEV6 can identify those at risk for future development of COPD and respiratory exacerbations.

PURPOSE OF REVIEW: The intestinal enteroendocrine cells (EECs) are specialized hormone secreting cells that respond to both circulating and luminal cues. Collectively, EECs constitute the largest endocrine organ of the body and signal to a multitude of targets including locally to neighboring intestinal cells, enteric neurons, as well as systemically to other organs such as the pancreas and brain. To accomplish their wide range of downstream signaling effects, EECs secrete multiple hormones; however, the mechanisms that influence EEC development in the embryo and differentiation in adults are not well-defined. RECENT FINDINGS: This review highlights the recent discoveries in EEC differentiation and function while also discussing newly revealed roles of transcription factors and signaling networks involved in the allocation of EEC subtypes that were discovered using a combination of novel intestinal model systems and genetic sequencing. We also discuss the potential of these new experimental models that study the mechanisms regulating EEC function and development both to uncover novel therapeutic targets. SUMMARY: Several EEC hormones are being used to treat various metabolic disorders such as type 2 diabetes and obesity. Therefore, understanding the signaling pathways and gene regulatory networks that facilitate EEC formation is paramount to the development of novel therapies.

Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal–immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.

Complex three-dimensional in vitro organ-like models, or organoids, offer a unique biological tool with distinct advantages over two-dimensional cell culture systems, which can be too simplistic, and animal models, which can be too complex and may fail to recapitulate human physiology and pathology. Significant progress has been made in driving stem cells to differentiate into different organoid types, though several challenges remain. For example, many organoid models suffer from high heterogeneity, and it can be difficult to fully incorporate the complexity of in vivo tissue and organ development to faithfully reproduce human biology. Successfully addressing such limitations would increase the viability of organoids as models for drug development and preclinical testing. On April 3-6, 2022, experts in organoid development and biology convened at the Keystone Symposium "Organoids as Tools for Fundamental Discovery and Translation" to discuss recent advances and insights from this relatively new model system into human development and disease.

Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the endoderm and adjacent mesoderm cells in the fetal foregut. While the recent successes in recapitulating developmental signaling in vitro has enabled the differentiation of human pluripotent stem cells (hPSCs) into various types of organ-specific endodermal epithelium, the generation of organ-specific mesenchyme has received much less attention. This is a major limitation in ongoing efforts to engineer complex human tissue. Here, we describe protocols to differentiate hPSCs into different types of organ-specific mesoderm leveraging signaling networks and molecular markers elucidated from single-cell transcriptomics of mouse foregut organogenesis. Building on established methods, hPSC-derived lateral plate mesoderm (LPM) treated with either retinoic acid (RA) or RA together with a Hedgehog (HH) agonist generates posterior or anterior foregut splanchnic mesoderm respectively (pFG-SpM or aFG-SpM) after 4 days cultures. These are directed into organ-specific mesenchyme lineages by the combinatorial activation or inhibition of WNT, BMP, RA or HH pathways from days 4 to 7 in cultures. By day 7 the cultures are enriched for different types of mesoderm with distinct molecular signatures: 60–90% pure liver septum transversum/mesothelium-like (STM/mesothelium), 70–80% pure liver-like fibroblasts (LF), and populations of ~35% respiratory-like mesoderm (RM), gastric-like mesoderm (GM), or esophageal-like mesoderm (EM). This protocol can be performed by anyone with moderate experience differentiating hPSCs and provides a novel platform to study human mesoderm development and can be used to engineer more complex foregut tissue for disease modeling and regenerative medicine.

Long-term impacts of diet have been well studied; however, the immediate response of the intestinal epithelium to a change in nutrients remains poorly understood. We use physiological metrics and single-cell transcriptomics to interrogate the intestinal epithelial cell response to a high-fat diet (HFD). Within 1 day of HFD exposure, mice exhibit altered whole-body physiology and increased intestinal epithelial proliferation. Single-cell transcriptional analysis on day 1 reveals a cell-stress response in intestinal crypts and a shift toward fatty acid metabolism. By 3 days of HFD, computational trajectory analysis suggests an emergence of progenitors, with a transcriptional profile shifting from secretory populations toward enterocytes. Furthermore, enterocytes upregulate lipid absorption genes and show increased lipid absorption in vivo over 7 days of HFD. These findings demonstrate the rapid intestinal epithelial response to a dietary change and help illustrate the essential ability of animals to adapt to shifting nutritional environments.

SummaryWhile the systemic impacts of overnutrition are well-known to cause obesity and metabolic syndrome over the course of months, the immediate adaptive response of the intestinal epithelium to dietary changes remains poorly understood. Here we used physiological metrics and single cell analyses to interrogate the adaptive response of intestinal epithelial cells when moved to a high fat diet (HFD). Within 1 day of HFD exposure, mice exhibited altered feeding behavior, an increase in energy expenditure and an increase in intestinal epithelial proliferation. Single cell transcriptional analysis demonstrated several acute cellular changes on day 1, including a cell-stress response in intestinal crypts, de-granularization of Paneth cells, and a shift towards fatty acid cellular metabolism. By 3 days of HFD, there was an emergence of uncommitted progenitors with a transcriptional profile indicative of a shift from secretory populations and towards an absorptive fate. In enterocytes, genes regulating lipid transport and absorption increased over the first 3 days which paralleled a functional increase in lipid absorption in vivo over the course of 7 days on HFD. These findings demonstrate that intestinal epithelial cell populations respond rapidly to changes in diet through initial changes in cellular function followed by a shift in cellular composition.

Background & Aims Shiga toxin (Stx)-producing Escherichia coli (eg, O157:H7) infection produces bloody diarrhea, while Stx inhibits protein synthesis and causes the life-threatening systemic complication of hemolytic uremic syndrome. The murine intestinal tract is resistant to O157:H7 and Stx, and human cells in culture fail to model the complex tissue responses to intestinal injury. We used genetically identical, human stem cell–derived intestinal tissues of varying complexity to study Stx toxicity in vitro and in vivo. Methods In vitro susceptibility to apical or basolateral exposure to Stx was assessed using human intestinal organoids (HIOs) derived from embryonic stem cells, or enteroids derived from multipotent intestinal stem cells. HIOs contain a lumen, with a single layer of differentiated epithelium surrounded by mesenchymal cells. Enteroids only contain epithelium. In vivo susceptibility was assessed using HIOs, with or without an enteric nervous system, transplanted into mice. Results Stx induced necrosis and apoptotic death in both epithelial and mesenchymal cells. Responses that require protein synthesis (cellular proliferation and wound repair) also were observed. Epithelial barrier function was maintained even after epithelial cell death was seen, and apical to basolateral translocation of Stx was seen. Tissue cross-talk, in which mesenchymal cell damage caused epithelial cell damage, was observed. Stx induced mesenchymal expression of the epithelial marker E-cadherin, the initial step in mesenchymal–epithelial transition. In vivo responses of HIO transplants injected with Stx mirrored those seen in vitro. Conclusions Intestinal tissue responses to protein synthesis inhibition by Stx are complex. Organoid models allow for an unprecedented examination of human tissue responses to a deadly toxin., Graphical abstract

Background: Forced expiratory volume in 1 second (FEV(1)) is central to the diagnosis of chronic obstructive pulmonary disease (COPD) but is imprecise in classifying disease burden. We examined the potential of the maximal mid-expiratory flow rate (forced expiratory flow rate between 25% and 75% [FEF(25%-75%)]) as an additional tool for characterizing pathophysiology in COPD. Objective: To determine whether FEF(25%-75%) helps predict clinical and radiographic abnormalities in COPD. Study Design and Methods: The SubPopulations and InteRediate Outcome Measures In COPD Study (SPIROMICS) enrolled a prospective cohort of 2978 nonsmokers and ever-smokers, with and without COPD, to identify phenotypes and intermediate markers of disease progression. We used baseline data from 2771 ever-smokers from the SPIROMICS cohort to identify associations between percent predicted FEF(25%-75%) (%predFEF(25%-75%)) and both clinical markers and computed tomography (CT) findings of smoking-related lung disease. Results: Lower %predFEF(25-75%) was associated with more severe disease, manifested radiographically by increased functional small airways disease, emphysema (most notably with homogeneous distribution), CT-measured residual volume, total lung capacity (TLC), and airway wall thickness, and clinically by increased symptoms, decreased 6-minute walk distance, and increased bronchodilator responsiveness (BDR). A lower %predFEF(25-75%) remained significantly associated with increased emphysema, functional small airways disease, TLC, and BDR after adjustment for FEV(1) or forced vital capacity (FVC). Interpretation: The %predFEF(25-75%) provides additional information about disease manifestation beyond FEV(1). These associations may reflect loss of elastic recoil and air trapping from emphysema and intrinsic small airways disease. Thus, %predFEF(25-75%) helps link the anatomic pathology and deranged physiology of COPD.

Introduction: Patients with chronic obstructive pulmonary disease (COPD) are at increased risk of coronary artery disease (CAD). Proline-glycine-proline (PGP) is an extracellular matrix-derived chemokine ( matrikine ) that is increased in COPD and known to promote neutrophil chemotaxis, endothelial dysfunction, and vascular inflammation through activation of the CXC Chemokine Receptor 2 (CXCR2). Despite these observations, no study has examined PGP as a marker of CAD within COPD patients. Hypothesis: We hypothesized that circulating PGP is associated with comorbid CAD amongst COPD patients. Methods: Current and former smokers with COPD enrolled in the UAB P rospective R epository for coupling EVE nts to N ovel pa T hways in COPD (PREVENT COPD) cohort underwent phlebotomy and spirometry. Cardiovascular diagnoses were adjudicated by two physician reviewers with access to medical records. CAD was defined as a composite of prior myocardial infarction, coronary revascularization, or by physician diagnosis of CAD. Plasma PGP was measured using a solid-phase extraction method coupled with tandem mass spectrometry. Other known CXCR2 ligands (e.g. GRO alpha, IL-8) were measured by multiplex protein assay. SPSSv27 was used for logistic regression models to measure associations between plasma biomarkers and the presence of CAD. Results: The 104 participants were 60±9 years old, 50% male, and 50% Black race. CAD was prevalent in 21 (21%) of the cohort. Plasma PGP was detectable for all participants (0.05 - 15.1 ng/mL). Participants with CAD had higher PGP concentrations compared to participants without CAD (1.26 vs. 0.52 ng/mL, p=0.042). Similarly, PGP was increased among participants with prior coronary revascularization (p Conclusions: Our results highlight PGP as a potentially novel biomarker of CAD among individuals with COPD. Future studies are needed to assess whether plasma PGP is predictive of CAD outcomes in COPD, as well as determine the significance of endothelial CXCR2 activation on the development of atherosclerosis.

Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell‐derived human intestinal organoids engrafted into mice and patient‐derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase‐dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti‐phasic necrotic cell death responses to TcdB. RNA‐Seq analysis shows that ~3–10% of the detectable transcripts are rhythmically expressed in mouse and human enteroids. Remarkably, we observe anti‐phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids, and disruption of Rac1 abolishes clock‐dependent necrotic cell death responses. Our findings uncover robust functions of circadian rhythms regulating clock‐controlled genes in both mouse and human enteroids governing organism‐specific, circadian phase‐dependent necrotic cell death responses, and lay a foundation for human organ‐ and disease‐specific investigation of clock functions using human organoids for translational applications., Mouse and human 3D in vitro models show tissue maturation to determine species‐specific circadian rhytms in the intestine.

Recently in Nature, using biologically inspired design principles to recapitulate native crypt-villi topography and luminal flow, Nikolaev et al. engineered an intestinal organotypic culture system that is capable of long-term epithelial homeostasis. Mini-gut tubes were used in month-long studies of parasite-host interactions and completely regenerated following injury.

Two patients with homozygous mutations in PDX1 presented with pancreatic agenesis, chronic diarrhea, and poor weight gain, the causes of which were not identified through routine clinical testing. We aimed to perform a deep analysis of the stomach and intestine using organoids derived from induced pluripotent stem cells from PDX1Gastric fundic, antral, and duodenal organoids were generated using induced pluripotent stem cell lines from a PDX1Patient-derived PDX1These patients will now be monitored for the progression of metaplasia and gastrointestinal complications that might be related to the reduced gastric and intestinal endocrine cells. This study demonstrates the utility of organoids in diagnosing uncovered pathologies.

SUMMARYThe development of human organoid model systems has provided new avenues for patient-specific clinical care and disease modeling. However, all organoid systems are missing important cell types that, in the embryo, get incorporated into organ tissues during development. Based on the concept of how embryonic organs are assembled, we developed an organoid assembly approach starting with cells from the three primary germ layers; enteric neuroglial, mesenchymal, and epithelial precursors, all separately derived from human pluripotent stem cells. From these we generated human gastric tissue containing differentiated glands, surrounded by layers of smooth muscle containing functional enteric neurons that controlled contractions of the engineered tissue. We used this highly tractable system to identify essential roles for the enteric nervous system in the growth and regional identity of the gastric epithelium and mesenchyme and for glandular morphogenesis of the antral stomach. This approach of starting with separately-derived germ layer components was applied to building more complex fundic and esophageal tissue, suggesting this as a new paradigm for tissue engineering.

Chronic obstructive pulmonary disease (COPD) is a systemic disease strongly associated with cigarette smoking, airway inflammation, and acute disease exacerbations. Changes in terminal sialylation and fucosylation of asparagine (N)-linked glycans have been documented in COPD, but the role that glycosyltransferases may play in the regulation of N-linked glycans in COPD has not been fully elucidated. Recent studies suggest that modulation of ST6GAL1 (ST6 beta-galactoside alpha-2,6-sialyltransferase-1), which catalyzes terminal α2-6 sialylation of cellular proteins, may regulate inflammation and contribute to COPD phenotype(s). Interestingly, it has been previously demonstrated that ST6GAL1, a Golgi resident protein, can be proteolytically processed by BACE1 (beta-site amyloid precursor protein cleaving enzyme-1) to a circulating form that retains activity. In this study, we showed that loss of ST6GAL1 expression increased interleukin (IL)-6 expression and secretion in human bronchial epithelial cells (HBECs). Furthermore, exposure to cigarette smoke medium/extract (CSE) or BACE1 inhibition resulted in decreased ST6GAL1 secretion, reduced α2-6 sialylation, and increased IL-6 production in HBECs. Analysis of plasma ST6GAL1 levels in a small COPD patient cohort demonstrated an inverse association with prospective acute exacerbations of COPD (AECOPD), while IL-6 was positively associated. Altogether, these results suggest that reduced ST6GAL1 and α2-6 sialylation augments IL-6 expression/secretion in HBECs and is associated with poor clinical outcomes in COPD.

In this case report, we describe the clinical course of a neonate who presented initially with respiratory distress and later with choking during feeding. He was subsequently found to have an esophageal bronchus to the right upper lung lobe, a rare communicating bronchopulmonary foregut malformation. Histological and molecular analysis of the fistula and distal tissues revealed that the proximal epithelium from the esophageal bronchus has characteristics of both esophageal and respiratory epithelia. Using whole exome sequencing of the patient's and parent's DNA, we identified gene variants that are predicted to impact protein function and thus could potentially contribute to the phenotype. These will be the subject of future functional analysis.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

SummaryTwo patients with mutations in PDX1 presented with pancreatic agenesis, chronic diarrhea and poor weight gain, the causes of which were not identified through routine clinical testing. We generated patient derived organoids as a novel diagnostic strategy and observed that PDX1188delC/188delC antral organoids convert to an intestinal phenotype, while intestinal organoids undergo gastric metaplasia with significant reduction in enteroendocrine cells. This prompted a re-examination of gastric and intestinal biopsies from both PDX1188delC/188delC patients, which recapitulated organoid phenotypes. Antral biopsies had increased parietal cells and lacked G-cells suggesting loss of antral identity. These patients will now be monitored for the progression of metaplasia. This study demonstrates the utility of organoids for patient diagnoses and treatment.

Trachea-esophageal defects (TEDs), including esophageal atresia (EA), tracheoesophageal fistula (TEF), and laryngeal-tracheoesophageal clefts (LTEC), are a spectrum of life-threatening congenital anomalies in which the trachea and esophagus do not form properly. Up until recently, the developmental basis of these conditions and how the trachea and esophagus arise from a common fetal foregut was poorly understood. However, with significant advances in human genetics, organoids, and animal models, and integrating single cell genomics with high resolution imaging, we are revealing the molecular and cellular mechanisms that orchestrate tracheoesophageal morphogenesis and how disruption in these processes leads to birth defects. Here we review the current understanding of the genetic and developmental basis of TEDs. We suggest future opportunities for integrating developmental mechanisms elucidated from animals and organoids with human genetics and clinical data to gain insight into the genotype-phenotype basis of these heterogeneous birth defects. Finally, we envision how this will enhance diagnosis, improve treatment, and perhaps one day, lead to new tissue replacement therapy.

Congenital tracheomalacia, resulting from incomplete tracheal cartilage development, is a relatively common birth defect that severely impairs breathing in neonates. Mutations in the Hedgehog (HH) pathway and downstream Gli transcription factors are associated with tracheomalacia in patients and mouse models; however, the underlying molecular mechanisms are unclear. Using multiple HH/Gli mouse mutants, including one that mimics Pallister–Hall Syndrome, we show that excessive Gli repressor activity prevents specification of tracheal chondrocytes. Lineage-tracing experiments show that Sox9+ chondrocytes arise from HH-responsive splanchnic mesoderm in the fetal foregut that expresses the transcription factor Foxf1. Disrupted HH/Gli signaling results in (1) loss of Foxf1, which in turn is required to support Sox9+ chondrocyte progenitors, and (2) a dramatic reduction in Rspo2, a secreted ligand that potentiates Wnt signaling known to be required for chondrogenesis. These results reveal an HH-Foxf1-Rspo2 signaling axis that governs tracheal cartilage development and informs the etiology of tracheomalacia. This article has an associated First Person interview with the first author of the paper., Summary: Genetic and molecular analyses of mutant mouse embryos reveal a HH-Foxf1-Rspo2 signaling axis that informs the mechanistic basis of tracheomalacia in individuals with Hedgehog pathway mutations.

Perfused three-dimensional (3D) cultures enable long-term in situ growth and monitoring of 3D organoids making them well-suited for investigating organoid development, growth, and function. One of the limitations of this long-term on-chip perfused 3D culture is unintended and disruptive air bubbles. To overcome this obstacle, we invented an imaging platform that integrates an innovative microfluidic bubble pocket for long-term perfused 3D culture of gastrointestinal (GI) organoids. We successfully applied 3D printing technology to create polymer molds that cast polydimethylsiloxane (PDMS) culture chambers in addition to bubble pockets. Our developed platform traps unintended, or induced, air bubbles in an integrated PDMS pocket chamber, where the bubbles diffuse out across the gas permeable PDMS or an outlet tube. We demonstrated that our robust platform integrated with the novel bubble pocket effectively circumvents the development of bubbles into human and mouse GI organoid cultures during long-term perfused time-course imaging. Our platform with the innovative integrated bubble pocket is ideally suited for studies requiring long-term perfusion monitoring of organ growth and morphogenesis as well as function.

Background COPD is a leading cause of mortality. Research Question We hypothesized that applying machine learning to clinical and quantitative CT imaging features would improve mortality prediction in COPD. Study Design and Methods We selected 30 clinical, spirometric, and imaging features as inputs for a random survival forest. We used top features in a Cox regression to create a machine learning mortality prediction (MLMP) in COPD model and also assessed the performance of other statistical and machine learning models. We trained the models in subjects with moderate to severe COPD from a subset of subjects in Genetic Epidemiology of COPD (COPDGene) and tested prediction performance in the remainder of individuals with moderate to severe COPD in COPDGene and Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE). We compared our model with the BMI, airflow obstruction, dyspnea, exercise capacity (BODE) index; BODE modifications; and the age, dyspnea, and airflow obstruction index. Results We included 2,632 participants from COPDGene and 1,268 participants from ECLIPSE. The top predictors of mortality were 6-min walk distance, FEV1 % predicted, and age. The top imaging predictor was pulmonary artery-to-aorta ratio. The MLMP-COPD model resulted in a C index ≥ 0.7 in both COPDGene and ECLIPSE (6.4- and 7.2-year median follow-ups, respectively), significantly better than all tested mortality indexes (P Interpretation An MLMP-COPD model outperformed four existing models for predicting all-cause mortality across two COPD cohorts. Performance of machine learning was similar to that of traditional statistical methods. The model is available online at: https://cdnm.shinyapps.io/cgmortalityapp/.

Visceral organs, such as the lungs, stomach and liver, are derived from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding splanchnic mesoderm (SM). While DE patterning is fairly well studied, the paracrine signaling controlling SM regionalization and how this is coordinated with epithelial identity is obscure. Here, we use single cell transcriptomics to generate a high-resolution cell state map of the embryonic mouse foregut. This identifies a diversity of SM cell types that develop in close register with the organ-specific epithelium. We infer a spatiotemporal signaling network of endoderm-mesoderm interactions that orchestrate foregut organogenesis. We validate key predictions with mouse genetics, showing the importance of endoderm-derived signals in mesoderm patterning. Finally, leveraging these signaling interactions, we generate different SM subtypes from human pluripotent stem cells (hPSCs), which previously have been elusive. The single cell data can be explored at: https://research.cchmc.org/ZornLab-singlecell., The fetal murine foregut develops into visceral organs via interactions between the mesoderm and endoderm, but how is unclear. Here, the authors use single cell RNAseq to show a diversity in organ specific splanchnic mesoderm cell-types, infer a signalling network governing organogenesis and use this to differentiate human pluripotent stem cells.

Gastrointestinal organoids are an exciting new tool for modeling human development, physiology, and disease in human tissue. Derived from pluripotent stem cells, gastrointestinal organoids consist of epithelial and mesenchymal cells organized in an intricate, three-dimensional structure that recapitulates the physiology and microscopic anatomy of the human gastrointestinal (GI) tract. In vitro derivation of gastrointestinal organoids from definitive endoderm has permitted an exploration of the complex signaling pathways required for the initial maturation of each individual gastrointestinal organ. Further maturation beyond an early fetal state currently requires transplantation into an immunocompromised host. Transplantation-induced maturation provides an opportunity to functionally interrogate the key mechanisms underlying development of the human GI tract. Gastrointestinal organoids can also be used to model human diseases and ultimately may serve as the basis for developing novel, personalized therapies for human intestinal diseases.

Congenital tracheomalacia, resulting from incomplete tracheal cartilage development, is a relatively common birth defect that severely impairs breathing in neonates. Mutations in the Hedgehog (HH) pathway and downstream Gli transcription factors are associated with tracheomalacia in patients and mouse models; however, the underlying molecular mechanisms are unclear. Using multipleHH/Glimouse mutants including one that mimics Pallister-Hall Syndrome, we show that excessive Gli repressor activity prevents specification of tracheal chondrocytes. Lineage tracing experiments show that Sox9+ chondrocytes arise from HH-responsive splanchnic mesoderm in the fetal foregut that expresses the transcription factor Foxf1. Disrupted HH/Gli signaling results in 1) loss of Foxf1 which in turn is required to support Sox9+ chondrocyte progenitors and 2) a dramatic reduction inRspo2, a secreted ligand that potentiates Wnt signaling known to be required for chondrogenesis. These results reveal a HH-Foxf1-Rspo2 signaling axis that governs tracheal cartilage development and informs the etiology of tracheomalacia.SUMMARY STATEMENTThis work provides a mechanistic basis for tracheomalacia in patients with Hedgehog pathway mutations.

Our group has developed two transplantation models for the engraftment of Human Intestinal Organoids (HIOs): the renal subcapsular space (RSS) and the mesentery each with specific benefits for study. While engraftment at both sites generates laminated intestinal structures, a direct comparison between models has not yet been performed. Embryonic stem cells were differentiated into HIOs, as previously described. HIOs from the same batch were transplanted on the same day into either the RSS or mesentery. 10 weeks were allowed for engraftment and differentiation, at which time they were harvested and assessed. Metrics for comparison included: mortality, engraftment rate, gross size, number and grade of lumens, and expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism. Mortality was significantly increased when undergoing mesentery transplantation, however engraftment was significantly higher. Graft sizes were similar between groups. Morphometric parameters were similar between groups, however m-tHIOs presented with significantly fewer lumens than k-tHIO. Transcript and protein level expression of markers specific to epithelial differentiation, mesenchymal differentiation, and carbohydrate metabolism were similar between groups. Transplantation into both sites yields viable tissue of similar quality based on our assessments with enhanced engraftment and a dominant lumen for uniform study benefiting the mesenteric site and survival benefiting RSS.