Search Results (32)

Despite the progressive extension of CFTR variant eligibility to the triple combination of elexacaftor/tezacaftor/ivacaftor (ETI), most rare CFTR pathogenic variants remain ineligible for CFTR modulators. It is crucial to determine whether unexplored variants are rescuable by clinical modulators and to identify innovative therapeutic strategies for rescuing non-responder variants. The approach known as “theratyping” (in vitro testing of genotypes) has been accepted by the Food and Drug Administration (FDA) for the extension of clinical modulators’ approval for in vitro responding genotypes. We used one of the most advanced models for theratyping: organoids derived from nasal epithelia of people with cystic fibrosis (pwCF). We optimized the forskolin-induced swelling (FIS) of organoids to assess CFTR basal or modulator-restored function. Nasal organoids mimicked the original epithelial tissue, CFTR residual activity, and modulator response. We set up the FIS assay using nasal organoids with reference genotypes and theratyped 38 rare (non-F508del) CFTR genotypes, either eligible or non-eligible for FDA approval, for treatment with ETI or ivacaftor. We found strong correspondence between the in vitro response of CFTR variants to modulators and their FDA approval status. Additionally, some previously uncharacterized CFTR variants have proven responsive to clinical modulators, with significant therapeutic implications. These results suggest that the nasal organoid FIS assay, pending confirmation of the prediction in the corresponding pwCF, might be considered as a powerful in vitro tool to predict modulator efficacy in each pwCF, guiding out-of-label prescription in CF, and to identify uncharacterized variants responsive to modulators. This approach may allow comparison of the efficacy of different therapeutics or the identification of innovative strategies for non-responding genotypes, improving personalized therapy and quality of life for pwCF. Full article

SARS-CoV-2 variants have demonstrated distinct epidemiological patterns and clinical presentations throughout the COVID-19 pandemic. Understanding variant-specific differences at the respiratory epithelium is crucial for understanding their pathogenesis. Here, we utilized human nasal organoid air–liquid interface (HNO-ALI) cell cultures to compare the viral replication kinetics, innate immune response, and epithelial damage of six different strains of SARS-CoV-2 (B.1.2, WA, Alpha, Beta, Delta, and Omicron). All variants replicated efficiently in HNO-ALIs, but with distinct replication kinetic patterns. The Delta variant exhibited delayed replication kinetics, achieving a steady state at 6 days post-infection compared to 3 days for other variants. Cytokine analysis revealed robust pro-inflammatory and chemoattractant responses (IL-6, IL-8, IP-10, CXCL9, and CXCL11) in WA1, Alpha, Beta, and Omicron infections, while Delta significantly dampened the innate immune response, with no significant induction of IL-6, IP-10, CXCL9, or CXCL11. Immunofluorescence and H&E analysis showed that all variants caused significant ciliary damage, though WA1 and Delta demonstrated less destruction at early time points (3 days post-infection). Together, these data show that, in our HNO-ALI model, the Delta variant employs a distinct “stealth” strategy characterized by delayed replication kinetics and epithelial cell innate immune evasion when compared to other variants of SARS-CoV-2, potentially explaining a mechanism that the Delta variant can use for its enhanced transmissibility and virulence observed clinically. Our findings demonstrate that variant-specific differences at the respiratory epithelium could explain some of the distinct clinical presentations and highlight the utility of the HNO-ALI system for the rapid assessment of emerging variants. Full article

Background: The bone morphogenetic protein (BMP) signaling pathway is essential for lung development. BMP4, a key regulator, binds to type I (BMPR1) and type II (BMPR2) receptors to initiate downstream signaling. While the inactivation of Bmpr1a and Bmpr1b leads to tracheoesophageal fistulae, the role of BMPR2 mutations in lung epithelial development remains unclear. Methods: We generated induced pluripotent stem cells (iPSCs) from a patient carrying a BMPR2 mutation (c.631C>T), and gene-corrected isogenic controls were created using CRISPR/Cas9. These iPSCs were differentiated into lung progenitor cells and subsequently cultured to generate alveolar and airway organoids. The differentiation efficiency and epithelial lineage specification were assessed using immunofluorescence, flow cytometry, and qRT-PCR. Results: BMPR2-mutant iPSCs showed no impairment in forming a definitive or anterior foregut endoderm. However, a significant reduction in lung progenitor cell differentiation was observed. Further, while alveolar epithelial differentiation remained largely unaffected, airway organoids derived from BMPR2-mutant cells exhibited impaired goblet and ciliated cell development, with an increase in basal and club cell markers, indicating skewing toward undifferentiated airway cell populations. Conclusions: BMPR2 dysfunction selectively impairs late-stage lung progenitor specification and disrupts airway epithelial maturation, providing new insights into the developmental impacts of BMPR2 mutations. Full article

Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children. Extracellular vesicles (EVs), released by airway epithelial cells, contain proteins and different families of non-coding RNAs (EV cargo) that can modulate the responses of target cells to viral infection. Nasal mucosa is a primary site of viral entry and the source of EVs present in the upper airway secretions. In this study we characterized proteins, including inflammatory mediators and cytokines, and the piwi-interacting RNA (piRNAs) cargo of EVs isolated from pediatric human nose organoids (HNO) and nasopharyngeal secretions (NPS) positive for RSV. Using Proximity Extension Assay (PEA) and Luminex multi-target arrays, we found significant enrichment in several chemokines and other mediators/biomarkers, including CCL2, CCL20, CXCL5, CX3CL1, CXCL6, MMP-1, MMP-10, uPA, Flt3L, ARNT and CD40 in EVs secreted by RSV-infected HNO compared to control mock HNO. Analysis of NPS samples from RSV infected children revealed that CCL3, CCL20, CXCL8, uPA, VEGFA, were concentrated in the NPS-EV fraction. LC-MS/MS and Gene Ontology indicated that RSV positive NPS-EVs originate from different cellular sources, with the most abundant proteins from neutrophils and epithelial cells. A total of 490 piRNAs were detected by NGS sequencing of small RNA libraries obtained from NPS-EVs, which has not been reported prior to this study. Identification of inflammatory mediators and small non-coding RNAs which are compartmentalized in EVs contributes to understanding mechanisms of virus-mediated pathogenesis in RSV infections. Full article

Background: Organoids are a valuable model for studying hereditary diseases such as cystic fibrosis (CF). Recombinant adenoviral (rAdV) and adeno-associated viral (rAAV) vectors are promising tools for CF gene therapy and genome editing. Objective: This study aims to determine the most efficient viral vector (rAdV5, rAAV serotypes 5, 6 and 9) and transduction protocol for delivering transgenes to lung organoids (LOs), providing a foundation for future CF gene therapy development. Methods: Three transduction protocols were used taking into account the specificities of LOs’ cultivation in specific matrices, both with and without organoid extraction from the matrix. This work was carried out on organoids from a healthy donor (LOs-WT) and on a patient with cystic fibrosis (LOs-CF). Results: High transduction efficiency was observed with rAdV5 (30% cells), rAAV6 (>80% cells), and rAAV9 (>40% cells). rAdV5 and rAAV9 transduced basal and secretory cells with >90% efficiency. For rAAV9, Protocol 1 (without extraction of organoids from the matrix) showed lower transduction efficiency (33% for LOs-WT, 9% for LOs-CF), significantly lower than that of Protocols 2 (60% for LOs-WT, 59% for LOs-CF) and 3 (46% for LOs-WT, 35% for LOs-CF) with organoid extraction from the matrix (p < 0.005). Conclusions: rAdV5 and rAAV9 are the most promising vectors for the delivery of transgenes to basal and secretory cells in a lung organoid model, providing a solid foundation for CF gene therapy development. Full article

Studies on human respiratory viral infections and pathogenesis have historically been conducted using immortalized cells and animal models. However, these models are limited in their ability to recapitulate the complex structure of the human airway or the full spectrum of disease symptoms observed in humans. Recently, nose and lung organoids have revolutionized culture complexity in infection biology and have demonstrated potential for research on respiratory virus infections in humans. In this opinion, we review how advances in human nose and lung organoid models, which are able to express all cell types of the respiratory epithelia, i.e., Club, basal, goblet, and ciliated cells, have provided novel insight into the pathogenesis, age-dependent susceptibility, viral attenuation signature, and immune mechanisms of respiratory viruses such as SARS-CoV-2, respiratory syncytial virus, and influenza virus. The models have also demonstrated potential for studying hitherto uncultivable human viruses and to be useful for studies of zoonotic risk. Full article

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. Currently, CFTR modulators are the most effective treatment for CF; however, they may not be suitable for all patients. A representative and convenient in vitro model is needed to screen therapeutic agents under development. This study, on the most common mutation, F508del, investigates the efficacy of human induced pluripotent stem cell-derived lung organoids (hiLOs) from NKX2.1+ lung progenitors and airway basal cells (hiBCs) as a 3D model for CFTR modulator response assessment by a forskolin-induced swelling assay. Weak swelling was observed for hiLOs from NKX2.1+ lung progenitors and hiBCs in response to modulators VX-770/VX-809 and VX-770/VX-661, whereas the VX-770/VX-661/VX-445 combination resulted in the highest swelling response, indicating superior CFTR function restoration. The ROC analysis of the FIS assay results revealed an optimal cutoff of 1.21, with 65.9% sensitivity and 71.8% specificity, and the predictive accuracy of the model was 76.4%. In addition, this study compared the response of hiLOs with the clinical response of patients to therapy and showed similar drug response dynamics. Thus, hiLOs can effectively model the CF pathology and predict patients’ specific response to modulators. Full article

Background/Objectives: Despite the introduction of innovative therapeutics, lung cancer is still the leading cause of cancer-related death. For this reason, lung cancer still requires deep characterization to identify cellular and molecular targets that can be used to develop novel therapeutic strategies. Three-dimensional cellular models, including patient-derived organoids (PDOs), represent useful tools to study lung cancer biology and may be employed in the future as predictive tools in therapeutic decisions. However, the successful establishment of lung cancer organoids cultures that faithfully represent the respective patient tissues is still challenging due to low success rate and/or overgrowth of normal airway epithelial cells. Methods: We set up a two-step protocol that allows for establishing both short-term and long-term 3D cultures, with different characteristics and success rates. Results: Cancer tissue-originated spheroids (CTOSs) show a 100% success rate and allow for the concomitant isolation of autologous tumor infiltrating leukocytes (TILs). On the contrary, PDOs can be expanded for a medium-long term and bio-banked but retain a lower success rate and the possibility of contamination with normal airway epithelial cells. To overcome these problems, we set up an optimal medium formulation and we implemented rigorous quality controls, leading to a substantial improvement in the success rate of tumoral PDO establishment. Conclusions: Overall, this protocol guarantees flexibility and reliability, also providing useful guidelines for quality control checks to support different experimental settings. The setting up of a robust protocol for lung cancer PDO culture establishment and expansion is a key requirement for their employment both in cancer research and as predictive tools in clinical practice. Full article

Swine influenza A viruses (IAVsw) are important causes of disease in pigs but also constitute a public health risk. IAVsw strains show remarkable differences in pathogenicity. We aimed to generate airway organoids from the porcine lower respiratory tract and use these to establish well-differentiated airway epithelial cell (WD-AEC) cultures grown at an air–liquid interface (ALI) for in vitro screening of IAVsw strain virulence. Epithelial cells were isolated from bronchus tissue of juvenile pigs, and airway organoids were cultured in an extracellular matrix in a culture medium containing human growth factors. Single-cell suspensions of these 3D organoids were seeded on Transwell filters and differentiated at ALI to form a pseudostratified epithelium containing ciliated cells, mucus-producing cells and tight junctions. Inoculation with a low dose of IAVsw in a low volume inoculum resulted in virus replication without requiring the addition of trypsin, and was quantified by the detection of viral genome loads in apical washes. Interestingly, inoculation of an H3N2 strain known to cause severe disease in pigs induced a greater reduction in trans-epithelial resistance and more damage to tight junctions than H1N2 or H1N1 strains associated with mild disease in pigs. We conclude that the porcine WD-AEC model is useful in assessing the virulence of IAVsw strains. Full article

Mycotoxins have the potential to increase the risk of airway or intestinal infection due to their effects on epithelial integrity and function. The bacterium Streptococcus suis (S. suis) is often carried in pigs and can cause outbreaks of invasive disease, leading to sepsis and meningitis in postweaning piglets. In this study, we tested the effect of two Fusarium mycotoxins (deoxynivalenol (DON) and T-2) on the integrity of the intestinal epithelium and their interaction with S. suis. Porcine ileal organoids were exposed to DON and T-2 individually or in combination and co-cultured with or without S. suis. Both DON and T-2 were toxic for ileal organoid monolayers at a concentration of 1 µM but not S. suis, even at a higher concentration of 4 µM. To mimic sub-clinical exposures on farms, DON was tested at a concentration of 0.1 µM and T-2 at a concentration of 0.01 µM. The mycotoxins alone did not affect cell permeability, but in combination with S. suis there was an increase in epithelial permeability. Furthermore, DON and T-2 together decreased the transepithelial electrical resistance and increased bacterial translocation. Full article

The implementation of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator drugs into clinical practice has been attaining remarkable therapeutic outcomes for CF, a life-threatening autosomal recessive genetic disease. However, there is elevated CFTR allelic heterogeneity, and various individuals carrying (ultra)rare CF genotypes remain without any approved modulator therapy. Novel translational model systems based on individuals’ own cells/tissue are now available and can be used to interrogate in vitro CFTR modulator responses and establish correlations of these assessments with clinical features, aiming to provide prediction of therapeutic effectiveness. Furthermore, because CF is a progressive disease, assessment of biomarkers in routine care is fundamental in monitoring treatment effectiveness and disease severity. In the first part of this review, we aimed to focus on the utility of individual-derived in vitro models (such as bronchial/nasal epithelial cells and airway/intestinal organoids) to identify potential responders and expand personalized CF care. Thereafter, we discussed the usage of CF inflammatory biomarkers derived from blood, bronchoalveolar lavage fluid, and sputum to routinely monitor treatment effectiveness and disease progression. Finally, we summarized the progress in investigating extracellular vesicles as a robust and reliable source of biomarkers and the identification of microRNAs related to CFTR regulation and CF inflammation as novel biomarkers, which may provide valuable information for disease prognosis. Full article

Non-polio enteroviruses (EV) belonging to species C, which are highly prevalent in Africa, mainly among children, are poorly characterized, and their pathogenesis is mostly unknown as they are difficult to culture. In this study, human airway and intestinal organotypic models were used to investigate tissue and cellular tropism of three EV-C genotypes, EV-C99, CVA-13, and CVA-20. Clinical isolates were obtained within the two passages of culture on Caco2 cells, and all three viruses were replicated in both the human airway and intestinal organotypic cultures. We did not observe differences in viral replication between fetal and adult tissue that could potentially explain the preferential infection of infants by EV-C genotypes. Infection of the airway and the intestinal cultures indicates that they both can serve as entry sites for non-polio EV-C. Ciliated airway cells and enterocytes are the target of infection for all three viruses, as well as enteroendocrine cells for EV-C99. Full article

The global burden of respiratory diseases is very high and still on the rise, prompting the need for accurate models for basic and translational research. Several model systems are currently available ranging from simple airway cell cultures to complex tissue-engineered lungs. In recent years, human lung organoids have been established as highly transferrable three-dimensional in vitro model systems for lung research. For acute infectious and chronic inflammatory diseases as well as lung cancer, human lung organoids have opened possibilities for precise in vitro research and a deeper understanding of mechanisms underlying lung injury and regeneration. Human lung organoids from induced pluripotent stem cells or from adult stem cells of patients’ samples introduce tools for understanding developmental processes and personalized medicine approaches. When further state-of-the-art technologies and protocols come into use, the full potential of human lung organoids can be harnessed. High-throughput assays in drug development, gene therapy, and organoid transplantation are current applications of organoids in translational research. In this review, we emphasize novel approaches in translational and personalized medicine in lung research focusing on the use of human lung organoids. Full article

Esophageal atresia (EA) is a rare birth defect in which respiratory tract disorders are a major cause of morbidity. It remains unclear whether respiratory tract disorders are in part caused by alterations in airway epithelial cell functions such as the activity of motile cilia. This can be studied using airway epithelial cell culture models of patients with EA. Therefore, the aim of this study was to evaluate the feasibility to culture and functionally characterize motile cilia function in the differentiated air–liquid interface cultured airway epithelial cells and 3D organoids derived from nasal brushings and bronchoalveolar lavage (BAL) fluid from children with EA. We demonstrate the feasibility of culturing differentiated airway epithelia and organoids of nasal brushings and BAL fluid of children with EA, which display normal motile cilia function. EA patient-derived airway epithelial cultures can be further used to examine whether alterations in epithelial functions contribute to respiratory disorders in EA. Full article

The respiratory epithelium, particularly the airway epithelium, is the primary infection site for respiratory pathogens. The apical surface of epithelial cells is constantly exposed to external stimuli including invading pathogens. Efforts have been made to establish organoid cultures to recapitulate the human respiratory tract. However, a robust and simple model with an easily accessible apical surface would benefit respiratory research. Here, we report the generation and characterization of apical-out airway organoids from the long-term expandable lung organoids that we previously established. The apical-out airway organoids morphologically and functionally recapitulated the human airway epithelium at a comparable level to the apical-in airway organoids. Moreover, apical-out airway organoids sustained productive and multicycle replication of SARS-CoV-2, and accurately recapitulated the higher infectivity and replicative fitness of the Omicron variants BA.5 and B.1.1.529 and an ancestral virus. In conclusion, we established a physiologically relevant and convenient apical-out airway organoid model for studying respiratory biology and diseases. Full article