Congenital Pulmonary Airway malformations (CPAM) may lead to malignant degeneration, and therefore many surgeons opt to resect CPAM even in asymptomatic patients. Previously, we identified Kirsten rat sarcoma virus (KRAS) mutations in a subset of CPAM patients, possibly indicating a pre-malignant state. In order to unify treatment strategy in (asymptomatic) patients we focused on KRAS mutations as a potential risk factor for developing malignancy in CPAM. Resected lung tissue of CPAM patients was separated in affected region (“cyst”) and non-affected region (“control”) to subsequently initiate airway organoids. Cyst and control organoids from the same patients with and without KRAS mutations (KRASPOS vs. KRASNEG) (n = 3) where processed for single cell RNA sequencing (scRNA-Seq), and the cellular composition of the organoids was validated by immunofluorescent staining. The role of KRAS was identified by manipulating the expression in the organoids. ScRNA-Seq data revealed differences in cell proportions between KRASPOS and KRASNEG cyst, and control organoids. The significant differentially expressed genes in the KRASPOS cyst are comparable to those identified in lung cancer patients with KRAS mutations. Manipulation of KRAS expression showed that KRASPOS cyst organoids grew larger due to more proliferative cells and that KRAS directly affected the cell cycle. KRASPOS cyst organoids show transcriptomic similarities with KRAS mutated lung cancers, show changes in cellular composition and have increased growth and proliferation. These findings support the hypothesis that KRAS mutated CPAM cysts belong to a group of CPAM patients at higher risk of developing a malignancy.

OBJECTIVES:Children of mothers with early-onset preeclampsia (EOPE) are at increased risk of respiratory morbidity due to factors including fetal growth restriction, prematurity, and impaired pulmonary vascular development. While animal studies suggest compromised fetal pulmonary vasculature in preeclampsia, human data remain limited. This study aimed to compare fetal pulmonary vascular volume in EOPE pregnancies with healthy controls as a proof of concept. STUDY DESIGN AND MAIN OUTCOME MEASURES:Pregnant women with EOPE and healthy controls were prospectively recruited. Ultrasound assessments were performed at three gestational intervals: 19 + 0-24 + 0 weeks (US1), 24 + 1-29 + 6 weeks (US2), and 30 + 0-32 + 0 weeks (US3). Using 3D Power Doppler ultrasound, fetal right pulmonary vascular volume (R-PVV), right lung volume (R-LV), and the R-PVV/LV ratio were measured. Perinatal parameters were also evaluated. RESULTS:Seventy-two controls and 36 EOPE fetuses were included. EOPE fetuses showed significantly reduced R-PVV at US2 and US3 compared to controls. The R-PVV/LV ratio remained significantly lower at both time points, suggesting impaired vascular development independent of lung size. Maternal BMI, gestational age at birth, and neonatal outcomes differed significantly between groups. CONCLUSIONS:This is the first human study to demonstrate impaired fetal pulmonary vascular development in EOPE. These findings offer insight into the pathophysiological effects of EOPE on fetal lung health.

The pulmonary vasculature develops in close association with the airways and this network expands through the interactions between endothelial cells and the surrounding mesenchymal cells, the pericytes. Emerging evidence suggests that pericytes play a significant role in various lung diseases, such as congenital diaphragmatic hernia and chronic obstructive pulmonary disease. However, characterizing pericytes remains challenging, impeding our understanding of their exact role in lung development and disease. Therefore, we used a novel cell tracing technology based on a bacterial DNA cytosine methyltransferase (Dcm) fused to RNA polymerase II (DCM-TM) to methylate active genes. The doxycycline inducible Dcm-PolII fusion protein was activated at specific time points during gestation, while the epigenetically labeled genes were analyzed at later time points. This retrospective cell tracing was coupled to single-cell RNA sequencing to track the development of mouse pulmonary pericytes at the single cell level. This revealed the paths to differentiation of perivascular progenitors into pericytes and vascular smooth muscle cells. Temporal analysis uncovered dynamic gene expression profiles during pericyte differentiation, highlighting pathways crucial for pulmonary vascular development. Further analysis showed intricate signaling interactions between pericyte progenitors and mature pericytes, and we validated MCAM as a bona fide pulmonary pericyte marker. Our findings challenge conventional views on pericyte origin and underscore the importance of accurate pericyte identification in developmental and disease contexts. Overall, this study enhances our understanding of pulmonary pericyte ontogeny and differentiation, offering insights into their potential as therapeutic targets in pericyte-associated lung diseases.

Congenital diaphragmatic hernia (CDH) is a rare anomaly with an incidence of 1 in 3000 live births and characterized by defective closure of the diaphragm, lung hypoplasia, and pulmonary vascular remodeling. Although several genes associated with the occurrence of CDH, no clear genetic component has been identified. Previously, we showed that CDH patients have vascular abnormalities already early in development, and that perivascular cells are affected in CDH. In the current study, we focused on early abnormalities in the nitrofen-induced CDH mouse model. Transcriptome analysis of FACS-sorted perivascular and endothelial cell populations from lungs of control and experimental mouse CDH revealed a high similarity in GO terms of differentially expressed genes (DEGs) between these populations, suggesting a disturbed interaction between endothelial cells and pericytes. The disturbed interaction is the basis for the aberrant development of the pulmonary vasculature in CDH. Furthermore, the RNA sequence data revealed reduced expression of the Kruppel like factor 4 (Klf4) gene in endothelial cells of CDH, which was confirmed with protein expression analysis. Furthermore, we show that KLF4 is an important up stream regulator of genes associated with vascular development and confirm that members of the NOTCH signaling pathway are differentially expressed, indicating that NOTCH signaling is disturbed in CDH. Collectively, our data support the importance of KLF4 in pulmonary angiogenesis and contribute to our previous data that pulmonary vessels in CDH patients are already affected before birth, which in turn may impact therapeutic strategies to reduce pulmonary hypertension associated with CDH.

Human lung models replicate various aspects to address diverse research questions. The complexity of human lung models, such as co-cultures and lung-on-chip devices, is increasing, but details on culture methodologies are often lacking. Here, we describe steps for the isolation, maintenance, and co-culturing of primary epithelial, endothelial, and mesenchymal cells derived from human lung resection material. We then detail procedures for 3D printing the simple-flow device, setting it up with co-cultures of human primary epithelial and endothelial cells under fluidic conditions.

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

Current animal and in vitro cell culture models do not fully recapitulate the physiological and pathophysiological characteristics of the human lung. As a result, the translation of these models to clinical practice is very limited, and clinical trials initiated on the extrapolation of such data fail. Although current models are beneficial in fundamental research, there is a need to constantly improve models to more accurately predict outcomes in clinical trials and personalized medicine. Here, we report important strategies to develop a 3D lung model with human primary lung cells. Starting from the well-established air-liquid interface (ALI) culture system, we describe a gradual increase in the complexity of the system by co-culturing different primary cell types, by testing different coatings, and by adding a three-dimensional matrix. As a result, we have established a reproducible 3D in vitro model of the airway consisting of human primary cells representing a differentiated mucociliary airway epithelium, an underlying submucosa with fibroblasts, and an endothelial interface.

Airborne respiratory viruses, such as coronaviruses and influenza, pose major threats to public health and the economy, as highlighted by the COVID-19 pandemic. Preclinical research is hindered by models that poorly mimic human tissue structure and function, often relying on immortalized cell lines and low-throughput animal studies. This limits accurate prediction of disease mechanisms, drug effects, and target suitability. Here, we report a custom-engineered, passive-flow, high-containment chip for culturing human primary bronchial epithelial cells (hPBECs) at air-liquid interface (ALI) on a large-area membrane. The dual-chamber microfluidic chip, separated by a horizontal support membrane, is enclosed in a 35 mm sealed Petri dish, enabling safe use in standard incubators without leakage or biosafety concerns. The platform supports high-resolution in-situ imaging, apical viral infection, and retrieval of cells and secretions (e.g., mucus, viral lysate) for molecular analysis. We demonstrate robust infection and replication of human coronavirus NL63 (HCoV-NL63) in differentiated hPBECs cultured up to 4 weeks at ALI. Epithelial differentiation was confirmed by immunofluorescence (e.g., ciliated cells), and infection kinetics were monitored by RT-qPCR over 7 days. The interferon-based immune response showed increased activity, with upregulation of viral response pathways (e.g., replication, inflammation, immunoregulation), and consistent activation across donors (e.g., ISG15, IFIT1). Collectively, we present a reproducible, small-scale chip model that enables high-containment in vitro studies of respiratory viruses and their effects on human airway epithelia.

Bronchopulmonary dysplasia (BPD) is the most common adverse outcome in preterm neonates and a high risk for early-onset emphysema and asthma. BPD is characterized by disrupted alveolar and microvascular development due to a variety of pathogenic factors, such as hyperoxia, inflammation, and dysbiosis. The resulting clinical manifestations are challenging, and current treatment options are limited. To improve therapeutic options, it is imperative to understand underlying causes. Resident lung mesenchymal stromal cells (L-MSCs) are important for alveolar microvascularization, repair, and regeneration. Here, we report the immediate effects of hyperoxia- and antibiotics-induced reduced bacterial load on L-MSCs and alveolar development using the hyperoxia-induced BPD mouse model. Newborn mice were exposed to hyperoxia from postnatal day 4 (P4) to P14, with room air recovery from P14 to P21. Dams received antibiotics-supplemented water (ampicillin, gentamycin, and vancomycin) from embryonic day 15 (E15) to P21. Hyperoxia significantly impaired alveolar development between P14 and P21, whereas both hyperoxia and antibiotic exposure impaired lung microvascular development. Moreover, hyperoxia reduced the number of pericytes, proliferative mesenchymal progenitors, Col13a1POS matrix fibroblasts, and P2RY14POS alveolar myofibroblasts. RNA sequencing (RNA-seq) of LY6A-sorted L-MSCs revealed differential expression of 103 genes in hyperoxia, 10 of which are related to mast cell biology. Antibiotic exposure also altered mesenchymal cell distribution, suggesting an additional impact on lung development. The transcriptomic landscape and distribution of important L-MSC subtypes and microvascular development are affected by hyperoxia and antibiotic exposure in a BPD mouse model. In conclusion, we show that hyperoxia- and antibiotics-induced reduced bacterial load affect the mesenchymal cell population, which may contribute to the development of BPD.NEW & NOTEWORTHY Bronchopulmonary dysplasia (BPD) is associated with preterm-born children, and antibiotic treatment increases the incidence. Lung repair is affected in BPD, and here we focused on the LY6APOS lung mesenchymal cells (L-MSCs), which modulate repair. We show that hyperoxia, which induces BPD in rodents, and antibiotics affect the transcriptome of these cells, resulting in altered signaling to mast cells. Antibiotics also affected the hyperoxia-induced changes in the cellular composition of L-MSCs at early alveologenesis.

Background: Gastroesophageal reflux disease (GERD) is a common comorbidity associated with congenital diaphragmatic hernia (CDH), with reported cases of Barrett's esophagus (BE) and esophageal adenocarcinoma before the age of 25. The prevalence and natural course of GERD in CDH survivors remain uncertain due to variations in diagnostic methods. We aimed to analyse the GERD prevalence from infancy through young adulthood. Methods: We retrospectively analyzed pH-impedance measurements and endoscopic findings in 96 CDH survivors evaluated as routine care using well established clinical protocols. GERD was defined as an abnormal acid exposure time for pH-MII measurements and as presence of reflux esophagitis or BE at upper endoscopy. Clinical data including symptoms at time of follow-up and use of antireflux medication were collected. Results: GERD prevalence remained consistently low (<10%) across all age groups, yet many patients experienced GER symptoms. Histological abnormalities were observed in 80% of adolescents and young adults, including microscopic esophagitis in 50%. BE was diagnosed in 7% before the age of 18, all had GER symptoms. CDH severity, anatomy at the time of CDH correction, alcohol usage, and smoking did not emerge as significant risk factors for GERD. Conclusions: Given the low GERD prevalence in CDH survivors, a symptom-driven approach to diagnosis and follow-up is warranted. We advise long-term follow-up for all adult patients due to the early onset of BE and the limited evidence available. The longitudinal course and impact of GERD on other long-term CDH-related comorbidities should be explored in larger cohorts. Level of Evidence: Not applicable (c) 2024 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).

Immortalized epithelial cell lines and animal models have been used in fundamental and preclinical research to study pulmonary diseases. However valuable, though, these models incompletely recapitulate the in vivo human lung, which leads to low predictive outcomes in potential respiratory treatments. Advanced technology and cell culture techniques stimulate the development of improved models that more closely mimic the physiology of the human lung. Nonetheless, most of these models are technically demanding and have a low throughput and reproducibility. Here, we describe a robust fluidic device consisting of a biocompatible and customizable 3D-printed cell culture plate, the Simple-Flow, which has medium throughput, is simple to manufacture, and is easy to set up. As a proof of principle, human primary bronchial epithelial cells (hPBECs) and human pulmonary microvascular endothelial cells (hMVECs) were cocultured on the apical and basolateral sides of the inset membranes, respectively. While hPBECs were cultured at the air-liquid interface to induce mucociliary differentiation, hMVECs were exposed to flow medium for up to 2 weeks. We show the versatility of 3D-printing technology in designing in vitro models for cell culturing applications, such as pediatric lung diseases or other pulmonary disorders.

Emerging evidence suggests pericytes play a significant role in various lung diseases. However, characterizing pericytes remains challenging, impeding our understanding of their role in lung development and disease. Using single-cell RNA sequencing and DCM-time machine technology, we tracked the development of mouse pulmonary pericytes. Our study revealed the differentiation of perivascular progenitors into pericytes and vascular smooth muscle cells. Temporal analysis uncovered dynamic gene expression profiles during pericyte differentiation, highlighting pathways crucial for pulmonary vascular development. Further analysis showed intricate signaling interactions between pericyte progenitors and mature pericytes, and we validated Mcam as a bona fide pulmonary pericyte marker. These findings challenge conventional views on pericyte origin and underscore the importance of accurate pericyte identification in developmental and disease contexts. Overall, this study enhances our understanding of pulmonary pericyte ontogeny and differentiation, offering insights into their potential as therapeutic targets in pericyte-associated lung diseases.

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.

Climate and justice are interconnected. However, simply raising ethical issues associated with the links between climate change, technology, and health is insufficient. Rather, policies and practices need to consider ethics ahead of time. If it is only added “after the fact,” policy will be less efficient and opportunities for carbon minimization will be lost. This will require the cooperation of people at many levels and can be guided by two essential ethical principles: distributive justice and environmental sustainability.

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.

AbstractBackgroundImmunocompromised 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.ObjectiveThis 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.MethodsDifferent 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.ResultsDexamethasone 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.ConclusionsDifferent 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.

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.

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.

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.