Paediatric gastric organoids as a tool for disease modelling and clinical translation

Purpose: Knowledge of gastric epithelial homeostasis remains incomplete, lacking human-specific models for study. This study establishes a protocol for deriving gastric epithelial organoids from paediatric gastric biopsies, providing a platform for modelling disease and developing translational therapies. Methods: Full-thickness surgical samples and endoscopic mucosal biopsies were obtained from six patients. Gastric glands were isolated by a chemical chelation protocol and then plated in 3D culture in Matrigel® droplets in chemically defined medium. After formation, organoids were passaged by single cell dissociation or manual disaggregation. Cell composition and epithelial polarity of organoids were assessed by bright field microscopy and immunofluorescence analysis, comparing them to native paediatric gastric tissue. Results: Gastric glands were successfully isolated from all six patients who were aged 4 months to 16 years. Gastric glands from all patients sealed to form spherical gastric organoids. These organoids could be passaged by manual disaggregation or single cell dissociation, remaining proliferative up to 1 year in culture. Organoids retained normal epithelial cell polarity, with the apical surface orientated towards the central lumen. Organoids expressed markers of mature gastric epithelial cell types, except for parietal cells. Conclusion: Gastric organoids can be reliably generated from paediatric biopsies and are a representative in vitro model for studying gastric epithelium

Primary human organoids models: Current progress and key milestones

During the past 10 years the world has experienced enormous progress in the organoids field. Human organoids have shown huge potential to study organ development, homeostasis and to model diseases in vitro. The organoid technology has been widely and increasingly applied to generate patient-specific in vitro 3D cultures, starting from both primary and reprogrammed stem/progenitor cells. This has consequently fostered the development of innovative disease models and new regenerative therapies. Human primary, or adult stem/progenitor cell-derived, organoids can be derived from both healthy and pathological primary tissue samples spanning from fetal to adult age. The resulting 3D culture can be maintained for several months and even years, while retaining and resembling its original tissue’s properties. As the potential of this technology expands, new approaches are emerging to further improve organoid applications in biology and medicine. This review discusses the main organs and tissues which, as of today, have been modelled in vitro using primary organoid culture systems. Moreover, we also discuss the advantages, limitations, and future perspectives of primary human organoids in the fields of developmental biology, disease modelling, drug testing and regenerative medicine

Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes

Duchenne muscular dystrophy (DMD)–associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients' somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient–derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD

Il‐17 promotes nitric oxide production in non‐small‐cell lung cancer

Introduction: Lung cancer is the second most frequent malignancy worldwide, but its aetiology is still unclear. Inflammatory cytokines and Th cells, including Th17, are now emerging as being involved in NSCLC pathways, thus postulating a role of IL‐17 in tumour angiogenesis by stimulating the vascular endothelial growth factor and the release of nitric oxide. Despite the fact that many biomarkers are used for chest malignancy diagnosis, data on FeNO levels and inflammatory cytokines in NSCLC are still few. Our study aimed to evaluate the relationship between pulmonary nitric oxide production and VEGF and Th17‐related cytokines in the EBC of patients affected by early‐stage NSCLC. Methods: FeNO measurement and lung function tests were performed in both patients affected by NCSLC and controls; EBC samples were also taken, and Th1 (IL‐1, IL‐6, IL‐12, IFN‐g, TNF‐a), Th17 (IL‐17, IL‐23) and Th2 (IL‐4, IL‐5, IL‐13) related cytokines were measured. Results: Th1 and Th17‐related cytokines in EBC, except for IFN‐gamma and TNF-alpha, were significantly higher in patients than in healthy controls, whereas no differences were seen for Th2‐related cytokines. FeNO at the flow rate of 50 mL/s, JawNO and CalvNO levels were significantly higher in patients affected by NSCLC compared to controls. Significant correlations were found between FeNO 50 mL/s and IL‐17, IL‐1 and VEGF. JawNO levels positively correlated with IL‐6, IL‐17 and VEGF. No correlations were found between FeNO and Th2‐related cytokines. Conclusion: This is the first report assessing a relationship between FeNO levels and Th17‐related cytokines in the EBC of patients affected by early‐stage NSCLC. IL‐17, which could promote angiogenesis through the VEGF pathway, might be indirectly responsible for the increased lung production of NO in patients with NSCLC

Intravital three-dimensional bioprinting

Fabrication of three-dimensional (3D) structures and functional tissues directly in live animals would enable minimally invasive surgical techniques for organ repair or reconstruction. Here, we show that 3D cell-laden photosensitive polymer hydrogels can be bioprinted across and within tissues of live mice, using bio-orthogonal two-photon cycloaddition and crosslinking of the polymers at wavelengths longer than 850 nm. Such intravital 3D bioprinting\u2014which does not create by-products and takes advantage of commonly available multiphoton microscopes for the accurate positioning and orientation of the bioprinted structures into specific anatomical sites\u2014enables the fabrication of complex structures inside tissues of live mice, including the dermis, skeletal muscle and brain. We also show that intravital 3D bioprinting of donor-muscle-derived stem cells under the epimysium of hindlimb muscle in mice leads to the de novo formation of myofibres in the mice. Intravital 3D bioprinting could serve as an in vivo alternative to conventional bioprinting

Intravital three-dimensional bioprinting

Fabrication of three-dimensional (3D) structures and functional tissues directly in live animals would enable minimally invasive surgical techniques for organ repair or reconstruction. Here, we show that 3D cell-laden photosensitive polymer hydrogels can be bioprinted across and within tissues of live mice, using bio-orthogonal two-photon cycloaddition and crosslinking of the polymers at wavelengths longer than 850 nm. Such intravital 3D bioprinting—which does not create by-products and takes advantage of commonly available multiphoton microscopes for the accurate positioning and orientation of the bioprinted structures into specific anatomical sites—enables the fabrication of complex structures inside tissues of live mice, including the dermis, skeletal muscle and brain. We also show that intravital 3D bioprinting of donor-muscle-derived stem cells under the epimysium of hindlimb muscle in mice leads to the de novo formation of myofibres in the mice. Intravital 3D bioprinting could serve as an in vivo alternative to conventional bioprinting

Synchronisation of apical constriction and cell cycle progression is a conserved behaviour of pseudostratified neuroepithelia informed by their tissue geometry

Neuroepithelial cells balance tissue growth requirement with the morphogenetic imperative of closing the neural tube. They apically constrict to generate mechanical forces which elevate the neural folds, but are thought to apically dilate during mitosis. However, we previously reported that mitotic neuroepithelial cells in the mouse posterior neuropore have smaller apical surfaces than non-mitotic cells. Here, we document progressive apical enrichment of non-muscle myosin-II in mitotic, but not non-mitotic, neuroepithelial cells with smaller apical areas. Live-imaging of the chick posterior neuropore confirms apical constriction synchronised with mitosis, reaching maximal constriction by anaphase, before division and re-dilation. Mitotic apical constriction amplitude is significantly greater than interphase constrictions. To investigate conservation in humans, we characterised early stages of iPSC differentiation through dual SMAD-inhibition to robustly produce pseudostratified neuroepithelia with apically enriched actomyosin. These cultured neuroepithelial cells achieve an equivalent apical area to those in mouse embryos. iPSC-derived neuroepithelial cells have large apical areas in G2 which constrict in M phase and retain this constriction in G1/S. Given that this differentiation method produces anterior neural identities, we studied the anterior neuroepithelium of the elevating mouse mid-brain neural tube. Instead of constricting, mid-brain mitotic neuroepithelial cells have larger apical areas than interphase cells. Tissue geometry differs between the apically convex early midbrain and flat posterior neuropore. Culturing human neuroepithelia on equivalently convex surfaces prevents mitotic apical constriction. Thus, neuroepithelial cells undergo high-amplitude apical constriction synchronised with cell cycle progression but the timing of their constriction if influenced by tissue geometry

SARS-CoV-2 infection and replication in human gastric organoids

COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission

A soil fungus confers plant resistance against a phytophagous insect by disrupting the symbiotic role of its gut microbiota

Plants generate energy flows through natural food webs, driven by competition for resources among organisms, which are part of a complex network of multitrophic interactions. Here, we demonstrate that the interaction between tomato plants and a phytophagous insect is driven by a hidden interplay between their respective microbiotas. Tomato plants colonized by the soil fungus Trichoderma afroharzianum, a beneficial microorganism widely used in agriculture as a biocontrol agent, negatively affects the development and survival of the lepidopteran pest Spodoptera littoralis by altering the larval gut microbiota and its nutritional support to the host. Indeed, experiments aimed to restore the functional microbial community in the gut allow a complete rescue. Our results shed light on a novel role played by a soil microorganism in the modulation of plant-insect interaction, setting the stage for a more comprehensive analysis of the impact that biocontrol agents may have on ecological sustainability of agricultural systems