Deciphering Endothelial and Mesenchymal Organ Specification in Vascularized Lung and Intestinal Organoids.

To investigate the co-development of vasculature, mesenchyme, and epithelium crucial for organogenesis and the acquisition of organ-specific characteristics, we constructed a human pluripotent stem cell-derived organoid system comprising lung or intestinal epithelium surrounded by organotypic mesenchyme and vasculature. We demonstrated the pivotal role of co-differentiating mesoderm and endoderm via precise BMP regulation in generating multilineage organoids and gut tube patterning. Single-cell RNA-seq analysis revealed organ specificity in endothelium and mesenchyme, and uncovered key ligands driving endothelial specification in the lung (e.g., WNT2B and Semaphorins) or intestine (e.g., GDF15). Upon transplantation under the kidney capsule in mice, these organoids further matured and developed perfusable human-specific sub-epithelial capillaries. Additionally, our model recapitulated the abnormal endothelial-epithelial crosstalk in patients with FOXF1 deletion or mutations. Multilineage organoids provide a unique platform to study developmental cues guiding endothelial and mesenchymal cell fate determination, and investigate intricate cell-cell communications in human organogenesis and disease. Highlights: BMP signaling fine-tunes the co-differentiation of mesoderm and endoderm.The cellular composition in multilineage organoids resembles that of human fetal organs.Mesenchyme and endothelium co-developed within the organoids adopt organ-specific characteristics.Multilineage organoids recapitulate abnormal endothelial-epithelial crosstalk in FOXF1-associated disorders.

Selection of potential targets for stratifying congenital pulmonary airway malformation patients with molecular imaging: is MUC1 the one?

Currently there is a global lack of consensus about the best treatment for asymptomatic congenital pulmonary airway malformation (CPAM) patients. The somatic KRAS mutations commonly found in adult lung cancer combined with mucinous proliferations are sometimes found in CPAM. For this risk of developing malignancy, 70% of paediatric surgeons perform a resection for asymptomatic CPAM. In order to stratify these patients into high- and low-risk groups for developing malignancy, a minimally invasive diagnostic method is needed, for example targeted molecular imaging. A prerequisite for this technique is a cell membrane bound target. The aim of this study was to review the literature to identify potential targets for molecular imaging in CPAM patients and perform a first step to validate these findings.A systematic search was conducted to identify possible targets in CPAM and adenocarcinoma in situ (AIS) patients. The most interesting targets were evaluated with immunofluorescent staining in adjacent lung tissue, KRAS+ CPAM tissue and KRAS- CPAM tissue.In 185 included studies, 143 possible targets were described, of which 20 targets were upregulated and membrane-bound. Six of them were also upregulated in lung AIS tissue (CEACAM5, E-cadherin, EGFR, ERBB2, ITGA2 and MUC1) and as such of possible interest. Validating studies showed that MUC1 is a potential interesting target.This study provides an extensive overview of all known potential targets in CPAM that might identify those patients at risk for malignancy and conducted the first step towards validation, identifying MUC1 as the most promising target.

Combating pan-coronavirus infection by indomethacin through simultaneously inhibiting viral replication and inflammatory response.

Severe infections with coronaviruses are often accompanied with hyperinflammation, requiring therapeutic strategies to simultaneously tackle the virus and inflammation. By screening a safe-in-human broad-spectrum antiviral agents library, we identified that indomethacin can inhibit pan-coronavirus infection in human cell and airway organoids models. Combining indomethacin with oral antiviral drugs authorized for treating COVID-19 results in synergistic anti-coronavirus activity. Coincidentally, screening a library of FDA-approved drugs identified indomethacin as the most potent potentiator of interferon response through increasing STAT1 phosphorylation. Combining indomethacin with interferon-alpha exerted synergistic antiviral effects against multiple coronaviruses. The anti-coronavirus activity of indomethacin is associated with activating interferon response. In a co-culture system of lung epithelial cells with macrophages, indomethacin inhibited both viral replication and inflammatory response. Collectively, indomethacin is a pan-coronavirus inhibitor that can simultaneously inhibit virus-triggered inflammatory response. The therapeutic potential of indomethacin can be further augmented by combining it with oral antiviral drugs or interferon-alpha.

Prenatal assessment of pulmonary vasculature development in fetuses with congenital diaphragmatic hernia: A literature review.

Pathophysiological studies have shown that pulmonary vascular development is impaired in fetuses with a congenital diaphragmatic hernia (CDH), leading to a simplified vascular tree and increased vascular resistance. Multiple studies have described prenatal ultrasound parameters for the assessment of the pulmonary vasculature, but none of these parameters are used in daily clinical practice. We provide a comprehensive review of the literature published between January 1990 and February 2022 describing these parameters, and aim to explain the clinical relevance of these parameters from what is known from pathophysiological studies. Prenatal detection of a smaller diameter of the contralateral (i.e. contralateral to the diaphragmatic defect) first branch of the pulmonary artery (PA), higher pulsatility indices (PI), higher peak early diastolic reverse flow values, and a lower vascularization index seem of added value for the prediction of survival and, to a lesser extent, morbidity. Integration within the routine evaluation is complicated by the lack of uniformity of the methods used. To address the main components of the pathophysiological changes, we recommend future prenatal studies in CDH with a focus on PI values, PA diameters and pulmonary vascular branching.

Clinical Relevance of Rapid FOXF1-Targeted Sequencing in Patients Suspected of Alveolar Capillary Dysplasia With Misalignment of Pulmonary Veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital lung disorder that presents shortly after birth with respiratory failure and therapy-resistant pulmonary hypertension. It is associated with heterozygous point mutations and genomic deletions that involve the FOXF1 gene or its upstream regulatory region. Patients are unresponsive to the intensive treatment regimens and suffer unnecessarily because ACDMPV is not always timely recognized and histologic diagnosis is invasive and time consuming. Here, we demonstrate the usefulness of a noninvasive, fast genetic test for FOXF1 variants that we previously developed to rapidly diagnose ACDMPV and reduce the time of hospitalization.

Longitudinal Health Status and Quality of Life in Congenital Diaphragmatic Hernia.

OBJECTIVES: To longitudinally evaluate self-reported health status (HS) and quality of life (QoL) in 8- and 12-year-old survivors of congenital diaphragmatic hernia (CDH). We hypothesized that HS would improve with age-as associated health problems tend to decline-whereas QoL would decrease, as the children start to compare themselves with peers. METHODS: Self-reported HS and QoL of 133 children born between 1999 and 2013 who had joined our standardized follow-up program were routinely assessed at the ages of 8 and 12 with generic, internationally validated, standardized instruments. Longitudinal evaluation of total and subscale scores was performed using general linear model analyses. In addition, we compared these scores to sex- and age-specific normative data. RESULTS: Between ages 8 and 12, boys born with CDH perceived a decline in HS (mean difference -7.15, P < .001). Self-reported QoL did not change over time in both boys and girls. At both ages, HS was significantly lower than that of healthy peers (effect size = 0.71, P = .001 for boys, and effect size = 0.69, P = .003 for girls), whereas differences in QoL were small. CONCLUSIONS: Children born with CDH are at risk for declining HS between 8 and 12 years, but not QoL, compared with healthy peers. Given that children born with CDH tend to grow into deficits, our findings highlight the need for continued somatic and psychological assessments in adolescent and adult CDH survivors.

Immunosuppressants exert differential effects on pan-coronavirus infection and distinct combinatory antiviral activity with molnupiravir and nirmatrelvir.

BACKGROUND: Immunocompromised populations, such as organ transplant recipients and patients with inflammatory bowel disease (IBD) receiving immunosuppressive/immunomodulatory medications, may be more susceptible to coronavirus infections. However, little is known about how immunosuppressants affect coronavirus replication and their combinational effects with antiviral drugs. OBJECTIVE: This study aims to profile the effects of immunosuppressants and the combination of immunosuppressants with oral antiviral drugs molnupiravir and nirmatrelvir on pan-coronavirus infection in cell and human airway organoids (hAOs) culture models. METHODS: Different coronaviruses (including wild type, delta and omicron variants of SARS-CoV-2, and NL63, 229E and OC43 seasonal coronaviruses) were used in lung cell lines and hAOs models. The effects of immunosuppressants were tested. RESULTS: Dexamethasone and 5-aminosalicylic acid moderately stimulated the replication of different coronaviruses. Mycophenolic acid (MPA), 6-thioguanine (6-TG), tofacitinib and filgotinib treatment dose-dependently inhibited viral replication of all tested coronaviruses in both cell lines and hAOs. The half maximum effective concentration (EC50) of tofacitinib against SARS-CoV-2 was 0.62 µM and the half maximum cytotoxic concentration (CC50) was above 30 µM, which resulted in a selective index (SI) of about 50. The anti-coronavirus effect of the JAK inhibitors tofacitinib and filgotinib is dependent on the inhibition of STAT3 phosphorylation. Combinations of MPA, 6-TG, tofacitinib, and filgotinib with the oral antiviral drugs molnupiravir or nirmatrelvir exerted an additive or synergistic antiviral activity. CONCLUSIONS: Different immunosuppressants have distinct effects on coronavirus replication, with 6-TG, MPA, tofacitinib and filgotinib possessing pan-coronavirus antiviral activity. The combinations of MPA, 6-TG, tofacitinib and filgotinib with antiviral drugs exerted an additive or synergistic antiviral activity. Thus, these findings provide an important reference for optimal management of immunocompromised patients infected with coronaviruses.

Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids.

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection.

Distinct roles for SOX2 and SOX21 in differentiation, distribution and maturation of pulmonary neuroendocrine cells.

Pulmonary neuroendocrine (NE) cells represent a small population in the airway epithelium, but despite this, hyperplasia of NE cells is associated with several lung diseases, such as congenital diaphragmatic hernia and bronchopulmonary dysplasia. The molecular mechanisms causing the development of NE cell hyperplasia remains poorly understood. Previously, we showed that the SOX21 modulates the SOX2-initiated differentiation of epithelial cells in the airways. Here, we show that precursor NE cells start to develop in the SOX2 + SOX21 + airway region and that SOX21 suppresses the differentiation of airway progenitors to precursor NE cells. During development, clusters of NE cells start to form and NE cells mature by expressing neuropeptide proteins, such as CGRP. Deficiency in SOX2 resulted in decreased clustering, while deficiency in SOX21 increased both the numbers of NE ASCL1 + precursor cells early in development, and the number of mature cell clusters at E18.5. In addition, at the end of gestation (E18.5), a number of NE cells in Sox2 heterozygous mice, did not yet express CGRP suggesting a delay in maturation. In conclusion, SOX2 and SOX21 function in the initiation, migration and maturation of NE cells.

SOX2 and SOX21 in Lung Epithelial Differentiation and Repair.

The lung originates from the ventral foregut and develops into an intricate branched structure of airways, alveoli, vessels and support tissue. As the lung develops, cells become specified and differentiate into the various cell lineages. This process is controlled by specific transcription factors, such as the SRY-related HMG-box genes SOX2 and SOX21, that are activated or repressed through intrinsic and extrinsic signals. Disturbances in any of these processes during the development of the lung may lead to various pediatric lung disorders, such as Congenital Diaphragmatic Hernia (CDH), Congenital Pulmonary Airway Malformation (CPAM) and Broncho-Pulmonary Dysplasia (BPD). Changes in the composition of the airways and the alveoli may result in reduced respiratory function and eventually lead to chronic lung disorders. In this concise review, we describe different intrinsic and extrinsic cellular processes required for proper differentiation of the epithelium during development and regeneration, and the influence of the microenvironment on this process with special focus on SOX2 and SOX21.

Lung epithelium development and airway regeneration.

The lung is composed of a highly branched airway structure, which humidifies and warms the inhaled air before entering the alveolar compartment. In the alveoli, a thin layer of epithelium is in close proximity with the capillary endothelium, allowing for an efficient exchange of oxygen and carbon dioxide. During development proliferation and differentiation of progenitor cells generates the lung architecture, and in the adult lung a proper function of progenitor cells is needed to regenerate after injury. Malfunctioning of progenitors during development results in various congenital lung disorders, such as Congenital Diaphragmatic Hernia (CDH) and Congenital Pulmonary Adenomatoid Malformation (CPAM). In addition, many premature neonates experience continuous insults on the lung caused by artificial ventilation and supplemental oxygen, which requires a highly controlled mechanism of airway repair. Malfunctioning of airway progenitors during regeneration can result in reduction of respiratory function or (chronic) airway diseases. Pathways that are active during development are frequently re-activated upon damage. Understanding the basic mechanisms of lung development and the behavior of progenitor cell in the ontogeny and regeneration of the lung may help to better understand the underlying cause of lung diseases, especially those occurring in prenatal development or in the immediate postnatal period of life. This review provides an overview of lung development and the cell types involved in repair of lung damage with a focus on the airway.

Recapitulating infection, thermal sensitivity and antiviral treatment of seasonal coronaviruses in human airway organoids.

BACKGROUND: Human seasonal coronaviruses usually cause mild upper-respiratory tract infection, but severe complications can occur in specific populations. Research into seasonal coronaviruses is limited and robust experimental models are largely lacking. This study aims to establish human airway organoids (hAOs)-based systems for seasonal coronavirus infection and to demonstrate their applications in studying virus-host interactions and therapeutic development. METHODS: The infections of seasonal coronaviruses 229E, OC43 and NL63 in 3D cultured hAOs with undifferentiated or differentiated phenotypes were tested. The kinetics of virus replication and production was profiled at 33 °C and 37 °C. Genome-wide transcriptome analysis by RNA sequencing was performed in hAOs under various conditions. The antiviral activity of molnupiravir and remdesivir, two approved medications for treating COVID19, was tested. FINDINGS: HAOs efficiently support the replication and infectious virus production of seasonal coronaviruses 229E, OC43 and NL63. Interestingly, seasonal coronaviruses replicate much more efficiently at 33 °C compared to 37 °C, resulting in over 10-fold higher levels of viral replication. Genome-wide transcriptomic analyses revealed distinct patterns of infection-triggered host responses at 33 °C compared to 37 °C temperature. Treatment of molnupiravir and remdesivir dose-dependently inhibited the replication of 229E, OC43 and NL63 in hAOs. INTERPRETATION: HAOs are capable of modeling 229E, OC43 and NL63 infections. The intriguing finding that lower temperature resembling that in the upper respiratory tract favors viral replication may help to better understand the pathogenesis and transmissibility of seasonal coronaviruses. HAOs-based innovative models shall facilitate the research and therapeutic development against seasonal coronavirus infections. FUNDING: This research is supported by funding of a VIDI grant (No. 91719300) from the Netherlands Organization for Scientific Research and the Dutch Cancer Society Young Investigator Grant (10140) to Q.P., and the ZonMw COVID project (114025011) from the Netherlands Organization for Health Research and Development to R.R.

Epigenetic reactivation of transcriptional programs orchestrating fetal lung development in human pulmonary hypertension.

Phenotypic alterations in resident vascular cells contribute to the vascular remodeling process in diseases such as pulmonary (arterial) hypertension [P(A)H]. How the molecular interplay between transcriptional coactivators, transcription factors (TFs), and chromatin state alterations facilitate the maintenance of persistently activated cellular phenotypes that consequently aggravate vascular remodeling processes in PAH remains poorly explored. RNA sequencing (RNA-seq) in pulmonary artery fibroblasts (FBs) from adult human PAH and control lungs revealed 2460 differentially transcribed genes. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed extensive differential distribution of transcriptionally accessible chromatin signatures, with 4152 active enhancers altered in PAH-FBs. Integrative analysis of RNA-seq and ChIP-seq data revealed that the transcriptional signatures for lung morphogenesis were epigenetically derepressed in PAH-FBs, including coexpression of T-box TF 4 (TBX4), TBX5, and SRY-box TF 9 (SOX9), which are involved in the early stages of lung development. These TFs were expressed in mouse fetuses and then repressed postnatally but were maintained in persistent PH of the newborn and reexpressed in adult PAH. Silencing of TBX4, TBX5, SOX9, or E1A-associated protein P300 (EP300) by RNA interference or small-molecule compounds regressed PAH phenotypes and mesenchymal signatures in arterial FBs and smooth muscle cells. Pharmacological inhibition of the P300/CREB-binding protein complex reduced the remodeling of distal pulmonary vessels, improved hemodynamics, and reversed established PAH in three rodent models in vivo, as well as reduced vascular remodeling in precision-cut tissue slices from human PAH lungs ex vivo. Epigenetic reactivation of TFs associated with lung development therefore underlies PAH pathogenesis, offering therapeutic opportunities.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Identification of SOX2 Interacting Proteins in the Developing Mouse Lung With Potential Implications for Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia is a structural birth defect of the diaphragm, with lung hypoplasia and persistent pulmonary hypertension. Aside from vascular defects, the lungs show a disturbed balance of differentiated airway epithelial cells. The Sry related HMG box protein SOX2 is an important transcription factor for proper differentiation of the lung epithelium. The transcriptional activity of SOX2 depends on interaction with other proteins and the identification of SOX2-associating factors may reveal important complexes involved in the disturbed differentiation in CDH. To identify SOX2-associating proteins, we purified SOX2 complexes from embryonic mouse lungs at 18.5 days of gestation. Mass spectrometry analysis of SOX2-associated proteins identified several potential candidates, among which were the Chromodomain Helicase DNA binding protein 4 (CHD4), Cut-Like Homeobox1 (CUX1), and the Forkhead box proteins FOXP2 and FOXP4. We analyzed the expression patterns of FOXP2, FOXP4, CHD4, and CUX1 in lung during development and showed co-localization with SOX2. Co-immunoprecipitations validated the interactions of these four transcription factors with SOX2, and large-scale chromatin immunoprecipitation (ChIP) data indicated that SOX2 and CHD4 bound to unique sites in the genome, but also co-occupied identical regions, suggesting that these complexes could be involved in co-regulation of genes involved in the respiratory system.

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

Cellular Origin(s) of Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) is a structural birth defect characterized by a diaphragmatic defect, lung hypoplasia and structural vascular defects. In spite of recent developments, the pathogenesis of CDH is still poorly understood. CDH is a complex congenital disorder with multifactorial etiology consisting of genetic, cellular and mechanical factors. This review explores the cellular origin of CDH pathogenesis in the diaphragm and lungs and describes recent developments in basic and translational CDH research.

Heritability and De Novo Mutations in Oesophageal Atresia and Tracheoesophageal Fistula Aetiology.

Tracheoesophageal Fistula (TOF) is a congenital anomaly for which the cause is unknown in the majority of patients. OA/TOF is a variable feature in many (often mono-) genetic syndromes. Research using animal models targeting genes involved in candidate pathways often result in tracheoesophageal phenotypes. However, there is limited overlap in the genes implicated by animal models and those found in OA/TOF-related syndromic anomalies. Knowledge on affected pathways in animal models is accumulating, but our understanding on these pathways in patients lags behind. If an affected pathway is associated with both animals and patients, the mechanisms linking the genetic mutation, affected cell types or cellular defect, and the phenotype are often not well understood. The locus heterogeneity and the uncertainty of the exact heritability of OA/TOF results in a relative low diagnostic yield. OA/TOF is a sporadic finding with a low familial recurrence rate. As parents are usually unaffected, de novo dominant mutations seems to be a plausible explanation. The survival rates of patients born with OA/TOF have increased substantially and these patients start families; thus, the detection and a proper interpretation of these dominant inherited pathogenic variants are of great importance for these patients and for our understanding of OA/TOF aetiology.

Disease modeling following organoid-based expansion of airway epithelial cells.

Air-liquid interface (ALI) cultures are frequently used in lung research but require substantial cell numbers that cannot readily be obtained from patients. We explored whether organoid expansion [three-dimensional (3D)] can be used to establish ALI cultures from clinical samples with low epithelial cell numbers. Airway epithelial cells were obtained from tracheal aspirates (TA) from preterm newborns and from bronchoalveolar lavage (BAL) or bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, cells were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to 1) cellular composition, 2) response to cigarette smoke exposure, and 3) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. We established well-differentiated ALI cultures from all samples. Cellular compositions (basal, ciliated, and goblet cells) were comparable. All 3D-expanded cultures showed a similar stress response following cigarette smoke exposure but differed from the 2D-expanded cultures. Higher peak levels of antioxidant genes HMOX1 and NQO1 and a more rapid return to baseline, and a lower unfolded protein response was observed after cigarette smoke exposure in 3D-derived cultures compared to 2D-derived cultures. In addition, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. Organoid-based expansion of clinical samples with low cell numbers, such as TA from preterm newborns is a valid method and tool to establish ALI cultures.