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Acute respiratory distress syndrome (ARDS) is a life-threatening condition with mortality rates between 30-50%. Althoughin vitromodels replicate some aspects of ARDS, small and large animal models remain the primary research tools due to the multifactorial nature of the disease. When using these animal models, histology serves as the gold standard method to confirm lung injury and exclude other diagnoses as high-resolution chest images are often not feasible. Semi-quantitative scoring performed by independent observers is the most common form of histologic analysis in pre-clinical animal models of ARDS. Despite progress in standardizing analysis procedures, objectively comparing histological injuries remains challenging, even for highly-trained pathologists. Standardized scoring simplifies the task and allows better comparisons between research groups and across different injury models, but it is time-consuming, and interobserver variability remains a significant concern. Convolutional neural networks (CNNs), which have emerged as a key tool in image analysis, could automate this process, potentially enabling faster and more reproducible analysis. Here we explored the reproducibility of human standardized scoring for an animal model of ARDS and its suitability for training CNNs for automated scoring at the whole slide level. We found large variations between human scorers, even for pre-clinical experts and board-certified pathologies in evaluating ARDS animal models. We demonstrate that CNNs (VGG16, EfficientNetB4) are suitable for automated scoring and achieve up to 83% F1-score and 78% accuracy. Thus, CNNs for histopathological classification of acute lung injury could help reduce human variability and eliminate a time-consuming manual research task with acceptable performance.

Label-free chemical and structural imaging of complex living tissue and biological systems is the holy grail of biomedical research and clinical diagnostics. The current analysis techniques are time-consuming and/or require extensive sample preparation, often due to the presence of interfering molecules such as water, making them unsuitable for the analysis of such systems. Here, we demonstrate a proof-of-principle study using label-free optical photothermal mid-infrared microspectroscopy (O-PTIR) for fast, direct spatiotemporal chemical analysis of complex living biological systems at submicron resolution. While other analytical methods can provide only static snapshots of molecular structures, our O-PTIR approach enables time-resolved and in situ investigation of chemical and structural changes of diverse biomolecules in their native conditions. This comprises a technological breakthrough in infrared spectroscopy to analyze biomolecules under native conditions over time: in fresh unprocessed biopsies, living brain tissue, and vertebrates without compromising their viability.One-Sentence SummaryProof-of-principle application of non-destructive O-PTIR for high-resolution spatiotemporal chemical and structural analysis of unprocessed biopsies, living brain tissue, and vertebrates.

Idiopathic pulmonary fibrosis is a fatal interstitial lung disease with limited therapeutic options. Current evidence suggests that IPF may be initiated by repeated epithelial injury in the distal lung followed by abnormal wound healing responses which occur due to intrinsic and extrinsic factors. Mechanisms contributing to chronic damage of the alveolar epithelium in IPF include dysregulated cellular processes such as apoptosis, senescence, abnormal activation of developmental pathways, aging, as well as genetic mutations. Therefore, targeting the regenerative capacity of the lung epithelium is an attractive approach in the development of novel therapies for IPF. Endogenous lung regeneration is a complex process involving coordinated cross-talk between multiple cell types and re-establishment of a normal extracellular matrix environment. This review will describe the current knowledge of reparative epithelial progenitor cells in the alveolar region of the lung and discuss potential novel therapeutic approaches for IPF focusing on endogenous alveolar repair. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Repeated injury of the lung epithelium is proposed to be a main driver of idiopathic pulmonary fibrosis (IPF). However, none of the available therapies target the epithelium and there is a limited amount of human models of fibrotic epithelial damage with suitability for drug screening and discovery. We developed a model of the epithelial reprogramming seen in IPF using alveolar organoids derived from human induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. This fibrosis cocktail induced persistent epithelial reprogramming and expression of extracellular matrix. Deconvolution of RNA-seq data indicated that the fibrosis cocktail increased the proportion of cells with the KRT5-/KRT17+ aberrant basaloid phenotype, recently identified in the lungs of IPF patients. Treatment with nintedanib and pirfenidone had effects on markers of extracellular matrix, pro-fibrotic mediators and epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery.

Repeated injury of the lung epithelium is proposed to be the main driver of idiopathic pulmonary fibrosis (IPF). However, available therapies do not specifically target the epithelium and human models of fibrotic epithelial damage with suitability for drug discovery are lacking. We developed a model of the aberrant epithelial reprogramming observed in IPF using alveolar organoids derived from human-induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. Deconvolution of RNA-seq data of alveolar organoids indicated that the fibrosis cocktail rapidly increased the proportion of transitional cell types including theKRT5−/KRT17+aberrant basaloid phenotype recently identified in the lungs of IPF patients. We found that epithelial reprogramming and extracellular matrix (ECM) production persisted after removal of the fibrosis cocktail. We evaluated the effect of the two clinically approved compounds for IPF, nintedanib and pirfenidone, and found that they reduced the expression of ECM and pro-fibrotic mediators but did not completely reverse epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery.

SummaryThe respiratory epithelium consists of multiple, functionally distinct cell-types and is maintained by regionally-specific progenitor populations which repair the epithelium following injury. Several in vitro methods exist for studying lung epithelial repair using primary murine lung epithelial cells, but isolation methods are hampered by a lack of surface markers distinguishing epithelial progenitors along the respiratory epithelium. Here, we developed a 3D-printed lobe divider (3DLD) to aid in simultaneous isolation of proximal versus distal lung epithelial progenitors from individual mice which give rise to differentiated epithelia in multiple in vitro assays. In contrast to 3DLD-isolated distal progenitor cells, classic manual tracheal ligation methods followed by lobe removal resulted in co-isolation of rare proximal progenitors with distal cells which altered the transcriptional landscape of distal organoid cultures. Thus, cell isolation with the 3DLD generates reproducible distal versus proximal progenitor populations and minimizes the potential for contaminating populations to confound in vitro assays.Highlights3DLD reproducibly separates lung lobes and extrapulmonary airways (bronchi/trachea)3DLD cell isolation yields consistent isolation of distal epithelial cells (DECs)Contamination of proximal cells in classic DEC isolations may alter in vitro results3DLD allows for simultaneous isolation of proximal and DECs from single animalseTOC blurbAlsafadi et al. describes a new method for simultaneous isolation of lung epithelial proximal and distal progenitors using the aid of a 3D printed device (3DLD). Both isolated cell types differentiate in multiple in vitro assays. The 3DLD guide minimized contamination of proximal cells in distal cell isolations whose presence can alter the transcriptional landscape of distal epithelial organoids.

Acute respiratory distress syndrome (ARDS) is a common cause of death in the intensive care unit, with mortality rates of ~30–40%. To reduce invasive diagnostics such as bronchoalveolar lavage and time-consuming in-hospital transports for imaging diagnostics, we hypothesized that particle flow rate (PFR) pattern from the airways could be an early detection method and contribute to improving diagnostics and optimizing personalized therapies. Porcine models were ventilated mechanically. Lipopolysaccharide (LPS) was administered endotracheally and in the pulmonary artery to induce ARDS. PFR was measured using a customized particles in exhaled air (PExA 2.0) device. In contrast to control animals undergoing mechanical ventilation and receiving saline administration, animals who received LPS developed ARDS according to clinical guidelines and histologic assessment. Plasma levels of TNF-α and IL-6 increased significantly compared with baseline after 120 and 180 min, respectively. On the other hand, the PFR significantly increased and peaked 60 min after LPS administration, i.e., ~30 min before any ARDS stage was observed with other well-established outcome measurements such as hypoxemia, increased inspiratory pressure, and lower tidal volumes or plasma cytokine levels. The present results imply that PFR could be used to detect early biomarkers or as a clinical indicator for the onset of ARDS.

Background and purpose: Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/β-catenin signaling is reduced in emphysema and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/β-catenin signaling and asses its potential to activate lung epithelial cells and repair. Experimental approach: We screened 1216 human-approved compounds for Wnt/β-catenin signaling activation using luciferase reporter cells, and selected candidates based on their computational predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell derived lung organoids and in vivo using a murine elastase-induced emphysema model. Key results: The primary screen identified 16 compounds that significantly induced Wnt/β-catenin-dependent luciferase activity. Selected compounds activated Wnt/β-catenin signaling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/β-catenin inhibition, confirming the Wnt β-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces pro-regenerative signaling in emphysema. Conclusion and implications: Using a drug screen based on Wnt/β-catenin activity, organoid assays, and a murine emphysema model, amlexanox was identified as a novel potential therapeutic for emphysema.

Mechanical ventilation is often required in patients with pulmonary disease to maintain adequate gas exchange. Despite improved knowledge regarding the risks of over ventilating the lung, ventilator induced lung injury (VILI) remains a major clinical problem due to inhomogeneities within the diseased lung itself as well as the need to increase pressure or volume of oxygen to the lung as a life-saving measure. VILI is characterized by increased physical forces exerted within the lung, which results in cell death, inflammation and long-term fibrotic remodeling. Animal models can be used to study VILI, but it is challenging to distinguish the contributions of individual cell types in such a setup. In vitro models, which allow for controlled stretching of specific lung cell types have emerged as a potential option, but these models and the membranes used in them are unable to recapitulate some key features of the lung such as the 3D nanofibrous structure of the alveolar basement membrane while also allowing for cells to be cultured at an air liquid interface (ALI) and undergo increased mechanical stretch that mimics VILI. Here we develop a lung on a chip device with a nanofibrous synthetic membrane to provide ALI conditions and controllable stretching, including injurious stretching mimicking VILI. The lung on a chip device consists of a thin (i.e. ∼20 µm) stretchable poly(caprolactone) (PCL) nanofibrous membrane placed between two channels fabricated in polydimethylsiloxane (PDMS) using 3D printed molds. We demonstrate that this lung on a chip device can be used to induce mechanotrauma in lung epithelial cells due to cyclic pathophysiologic stretch (∼25%) that mimics clinical VILI. Pathophysiologic stretch induces cell injury and subsequently cell death, which results in loss of the epithelial monolayer, a feature mimicking the early stages of VILI. We also validate the potential of our lung on a chip device to be used to explore cellular pathways known to be altered with mechanical stretch and show that pathophysiologic stretch of lung epithelial cells causes nuclear translocation of the mechanotransducers YAP/TAZ. In conclusion, we show that a breathable lung on a chip device with a nanofibrous membrane can be easily fabricated using 3D printing of the lung on a chip molds and that this model can be used to explore pathomechanisms in mechanically induced lung injury.

In severe acute respiratory distress syndrome (ARDS), extracorporeal membrane oxygenation (ECMO) is a life‐prolonging treatment, especially among COVID‐19 patients. Evaluation of lung injury progression is challenging with current techniques. Diagnostic imaging or invasive diagnostics are risky given the difficulties of intra‐hospital transportation, contraindication of biopsies, and the potential for the spread of infections, such as in COVID‐19 patients. We have recently shown that particle flow rate (PFR) from exhaled breath could be a noninvasive, early detection method for ARDS during mechanical ventilation. We hypothesized that PFR could also measure the progress of lung injury during ECMO treatment. Lipopolysaccharide (LPS) was thus used to induce ARDS in pigs under mechanical ventilation. Eight were connected to ECMO, whereas seven animals were not. In addition, six animals received sham treatment with saline. Four human patients with ECMO and ARDS were also monitored. In the pigs, as lung injury ensued, the PFR dramatically increased and a particular spike followed the establishment of ECMO in the LPS‐treated animals. PFR remained elevated in all animals with no signs of lung recovery. In the human patients, in the two that recovered, PFR decreased. In the two whose lung function deteriorated while on ECMO, there was increased PFR with no sign of recovery in lung function. The present results indicate that real‐time monitoring of PFR may be a new, complementary approach in the clinic for measurement of the extent of lung injury and recovery over time in ECMO patients with ARDS., Particle flow rate (PFR) used in conjunction with mechanical ventilation can be used to monitor lung injury during ECMO treatment in animals with LPS induced ARDS and in humans with COVID‐19 induced ARDS. The present results indicate that real‐time monitoring of PFR may be a new, complementary approach in the clinic for measurement of the extent of lung injury and recovery over time in ECMO patients with ARDS.

The University of Vermont Larner College of Medicine, in collaboration with the National Heart, Lung, and Blood Institute (NHLBI), the Alpha-1 Foundation, the American Thoracic Society, the Cystic Fibrosis Foundation, the European Respiratory Society, the International Society for Cell & Gene Therapy, and the Pulmonary Fibrosis Foundation, convened a workshop titled “Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases” from July 24 through 27, 2017, at the University of Vermont, Burlington, Vermont. The conference objectives were to review and discuss current understanding of the following topics: 1) stem and progenitor cell biology and the role that they play in endogenous repair or as cell therapies after lung injury, 2) the emerging role of extracellular vesicles as potential therapies, 3) ex vivo bioengineering of lung and airway tissue, and 4) progress in induced pluripotent stem cell protocols for deriving lung cell types and applications in disease modeling. All of these topics are research areas in which significant and exciting progress has been made over the past few years. In addition, issues surrounding the ethics and regulation of cell therapies worldwide were discussed, with a special emphasis on combating the growing problem of unproven cell interventions being administered to patients with lung diseases. Finally, future research directions were discussed, and opportunities for both basic and translational research were identified.

Respiratory diseases, such as chronic obstructive pulmonary disease and pulmonary fibrosis, result in severely impaired quality of life and impose significant burdens on healthcare systems worldwide. Current disease management involves pharmacologic interventions, oxygen administration, reduction of infections, and lung transplantation in advanced disease stages. An increasing understanding of mechanisms of respiratory epithelial and pulmonary vascular endothelial maintenance and repair and the underlying stem/progenitor cell populations, including but not limited to airway basal cells and type II alveolar epithelial cells, has opened the possibility of cell replacement-based regenerative approaches for treatment of lung diseases. Further potential for personalized therapies, including in vitro drug screening, has been underscored by the recent derivation of various lung epithelial, endothelial, and immune cell types from human induced pluripotent stem cells. In parallel, immunomodulatory treatments using allogeneic or autologous mesenchymal stromal cells have shown a good safety profile in clinical investigations for acute inflammatory conditions, such as acute respiratory distress syndrome and septic shock. However, as yet, no cell-based therapy has been shown to be both safe and effective for any lung disease. Despite the investigational status of cell-based interventions for lung diseases, businesses that market unproven, unlicensed and potentially harmful cell-based interventions for respiratory diseases have proliferated in the United States and worldwide. The current status of various cell-based regenerative approaches for lung disease as well as the effect of the regulatory environment on clinical translation of such approaches are presented and critically discussed in this review.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo) fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored.Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF.Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies.Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-beta in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A.Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death with 65 million patients. Hallmarks include progressive and irreversible airflow limitation due to airway inflammation and alveolar tissue damage. There are limited therapeutic options for COPD which aim to decelerate disease progression but none with regenerative effects. The only option for end-stage disease is lung transplantation with limited efficacy. Thus, novel therapies are desperately needed. Intratracheal administration of alveolar type II (AT2) cells has shown regenerative effects in lung diseases, but have not yet been explored as a potential COPD therapy. The loss of structural tissue in COPD/emphysema may be a barrier for cell therapies aiming at cellular engraftment and subsequent regeneration. Here, we used a custom microfluidic device to reproducibly generate cellular microcarriers in the range of individual murine or human alveoli from alginate-reinforced decellularized extracellular matrix (rECM) as a platform to deliver alveolar type 2 (AT2) cells into COPD lungs. dECM was prepared by decellularization of porcine or human lungs followed by enzymatic digestion. This solution was mixed with alginate and used in a custom-made microfluidic device to generate monosized rECM microcarriers. The size of microcarriers was tailored by controlling flow rates and microfluidic chip geometries. In the future, we will evaluate the engraftment potential and ability of delivered cells and microcarriers to promote regeneration. This will be tested in both ex vivo and in vivo murine models of COPD.

The lung epithelium is rich in celltype diversity including several progenitors. These distinct regional progenitors have traditionally been identified using lineage tracing or single cell RNAseq. Isolation and use of such cells are hampered by the lack of specific markers that distinguish them in different airway compartments. Thus, a robust method is needed to reliably isolate progenitors from their respective location in the airway. Further, isolation of cells from the same mouse allows for direct analysis of regional disease specific effects and reduction in overall animal numbers. Here, we developed a 3D-printed lobe divider (3DLD) to simultaneously isolate proximal and distal epithelial cells from the same lung. We evaluated isolated cells by their capacity to differentiate in standard in vitro assays. Proximal and distal cells isolated with 3DLD gave rise to differentiated airway and alveolar epithelia respectively as evident by air-liquid-interface cultures and organoid assays with colony forming efficiency (CFE) matching literature. 3DLD method yielded distal organoids with increased CFE and smaller organoid diameters with median of 107µm compared to 171µm from those isolated without 3DLD. We used RNAseq in combination with computational deconvolution to evaluate differences in celltype representation among these cultures. We found that separation of proximal and distal compartments prior cell isolation altered the initial cell populations, likely due to the presence of proximal airway progenitors in single cell suspensions of distal epithelial cells. 3DLD is an inexpensive and reproducible method for isolating proximal and distal progenitors from individual mice.

Background and aim: Patients with end stage lung disease rely on mechanical ventilation (MV) to be able to breathe. However, MV often induces substantial lung injury known as ventilator-induced-lung-injury (VILI). Current in-vitro models of VILI fail to recapitulate the complex dynamic lung condition such as air-liquid interface (ALI) and 3D structures. To provide ALI conditions and customizable scaffolds for modelling VILI, we aimed to develop a lung-on-a-chip (LOC) device which allows for controllable stretch and is easily manufactured. Methods and results: We successfully fabricated a LOC using 3D printable molds and a commercially available porous membrane mimicking the lung ECM. This LOC was used to expose murine lung epithelial cells to stretch seen in MV (i.e. 25% cyclic strain). We observed higher nuclear translocation of the mechanosensors YAP/TAZ under stretched conditions (dynamic) compared to non-stretched conditions (static). Furthermore, confocal and scanning electron microscopy (SEM) images show higher amounts of cell loss in the dynamic condition compared to the static one. Cell death was measured using lactate dehydrogenase (LDH) levels in the supernatants where we confirmed higher levels of cell death under dynamic conditions compared to the static ones. Conclusion: Our study supports the potential use of 3D-printing as a fabrication technique for LOCs. Using the manufactured device, we were successfully able to accurately model lung injury on chip.

Introduction: Dysregulation of the alveolar epithelium by repeated injury is recognised as a driver of idiopathic pulmonary fibrosis (IPF). However, access to relevant pre-clinical models of alveolar epithelial injury in IPF is limited. Aims and objectives: We sought to develop a disease model of alveolar epithelial injury in IPF using alveolar epithelial organoids derived from human induced pluripotent stem cells (iPSCs) for drug discovery. Methods: Alveolar epithelial organoids were derived from iPSCs and treated with a fibrosis cocktail (FC) of the pro-fibrotic and inflammatory cytokines TGFβ, TNFα, PDGF-AB and LPA. IPF-related changes were evaluated by RNA-Seq, qRT-PCR, and Western blot. To evaluate the potential of this system for testing anti-fibrotic compounds, we performed prophylactic treatment with the marketed IPF drug nintedanib for 72 h and screened for well-known hallmarks of IPF by qRT-PCR. Results: The FC induced changes known to be observed in IPF, such as epithelial injury and reprogramming (e.g. loss of SFTPC expression and induced VIM expression), production of extracellular matrix and induction of cellular senescence- associated genes. Interestingly, FC treatment also increased expression of KRT17 and KRT8, both recently identified in single-cell RNA-Seq studies as markers of dysregulated epithelial repair in IPF. Treatment with nintedanib partially prevented transcriptional changes in extracellular matrix components, matrix-modifying enzymes and mediators of several signaling pathways associated with IPF. Conclusions: FC treatment of iPSC-derived alveolar epithelial cells successfully models several key aspects of IPF and some of these changes can be prevented with nintedanib, demonstrating that this system can be used for disease modeling of IPF and drug discovery.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with a poor prognosis and no cure. Unfortunately, over 90% of drugs fail in clinical trials for IPF due to difficulty in translating promising drugs from animal models. Human precision-cut lung slices (hPCLS) have gained increasing interest as a possible means to model IPF. An early-like stage of IPF can be induced via a fibrosis cocktail (FC) in hPCLS generated from tumor-free cancer resections to study disease and pharmacological approaches. A drawback of using PCLS from tumor-free lung cancer resections is that the tissue can be deranged (e.g. aberrant immune cells and secreted factors), which may contribute to the fibrotic response ex vivo. In the current study, we hypothesized that hPCLS generated from excess donor lung tissue could be used as a more physiologic option. hPCLS were generated from 4 excess donor lungs and treated with FC for up to 120 hours. To obtain non-biased gene expression data, we utilized RNA-seq using an RNA isolation protocol we recently developed. FC induced a fibrotic-like response after 48 hours which increased further with time. Genes associated with matrix remodeling (e.g.FN1, COL1A1, and MMP7) and Wnt-signaling (e.g.WNT2, WNT5B, WNT10A, NKD1, and AXIN2) increased, whereas genes associated with distal epithelial cell markers (e.g.SFTPC and HOPX) decreased. Genes associated with epithelial subtypes in IPF (e.g.KRT17) increased. In addition, hPCLS were treated with two novel drugs, one in pre-clinical testing and one currently in Phase II clinical trials for IPF. Each drug induced differential amelioration of fibrotic gene expression, assessed by RNA-seq. hPCLS generated from excess donor lungs and treated with FC are a promising model of IPF and to study potential therapies.

Lung transplantation is the only option for end-stage lung diseases, but organ shortage remains problematic. Bioengineering lungs ex vivo could overcome shortages and 3D bioprinting can generate relevant custom structures, but, other criteria such as the immune responses of these constructs and their integration with the host vasculature are critical for successful clinical translation. Previously, we generated a tissue-specific hybrid bioink consisting of alginate, reinforced with extracellular matrix from decellularized lung tissue (rECM) to 3D bioprint human airways comprised of regionally specified primary cells which remained patent over time.1 3D bioprinted constructs generated using batch processed rECM bioinks prevent the foreign body response when transplanted into T-cell deficient mice, mimicking clinical immunosuppression for solid organ transplantation. However, one of the major potential benefits of organ bioengineering is that the cells could be sourced from the eventual transplant recipient, thus obviating the need for long term immunosuppression. Here we sought to investigate the biocompatibility and regenerative ability of 3D printed lung rECM alginate hydrogels in immunocompetent mice. We find that the 3D printed rECM constructs do not illicit negative immune responses when syngeneic transplanted into immunocompetent mice and integrate well in the surrounding tissue without evidence of a foreign body response. Furthermore, we find that the rECM is pro-angiogenic and supports de novo vasculature upon subcutaneous implantation. rECM bioinks are thus a promising new approach for generating functional human tissue using 3D bioprinting. 1De Santis, M. M, et al., Extracellular‐Matrix‐Reinforced Bioinks for 3D Bioprinting Human Tissue. Adv. Mater. 2020, 2005476.

Background and aims: The majority of current organoids are grown within Matrigel, a protein mixture secreted by mouse sarcoma cells, due to the fact that it mimics the basement membrane and contains growth factors that support organoid formation. However, concerns have arisen as to the suitability of Matrigel for studying normal regeneration and stem cell behavior due to its cancerous origin. Hydrogels generated from pepsin extracted, decellularized extracellular matrix (dECM) solutions have recently emerged as a potential option for culturing organoids for other tissues, such as gut, but have not been evaluated for culturing primary lung stem or progenitor cells. Here, we developed protocols to generate dECM hydrogels from porcine lung and evaluated their potential for culturing organoids from human bronchial epithelial cells (HBECs) as an alternative matrix to Matrigel. Methods and results: HBECs were isolated from discarded surgical waste following resizing of airways for lung transplantation and seeded in Matrigel or dECM hydrogels. After 14 days of cell culture, we found that organoids formed in both Matrigel and in dECM hydrogels, with similar differentiation capacity, as assessed by immunofluorescence. Next, we performed bulk RNA sequencing of HBEC derived organoids from Matrigel or dECM to assess differences imposed by the surrounding matrix. We found that organoids formed in Matrigel expressed upregulation of oncogenic markers and pathways known to be aberrant in cancer as compared to organoids formed in dECM. Conclusion: We successfully developed decellularized porcine lung derived ECM gels which supported airway organoid formation. dECM hydrogels may be an alternative to Matrigel to study lung or airway regenerative capacity in a more physiologically relevant environment.

Despite improvements, lung transplantation (LTx) remains hampered by both a scarcity of donor organs and by mortality following primary graft dysfunction (PGD). Since acute respiratory distress syndrome (ARDS) limits donor lungs utilization, we investigated cytokine filtration as a means of restoring ARDS donor lungs. We induced mild to moderate ARDS using lipopolysaccharide in 12 donor pigs. Lungs were then treated with or without cytokine filtration during ex vivo lung perfusion (EVLP) and post-transplantation using extracorporeal hemoperfusion. The treatment significantly decreased cytokine levels during EVLP and decreased levels of immune cells post-transplantation. Histology demonstrated fewer signs of lung injury across both treatment periods and the incidence of PGD was significantly reduced among treated animals. Overall, cytokine filtration was able to restore lung function and reduce PGD in lung transplantation. We suggest this treatment will increase the availability of donor lungs and increase the tolerability of donor lungs in the recipient.

Engineered nanomaterials (ENMs) are increasingly produced and used today, but health risks due to their occupational airborne exposure are incompletely understood. Traditionally, nanoparticle (NP) toxicity is tested by introducing NPs to cells through suspension in the growth media, but this does not mimic respiratory exposures. Different methods to introduce aerosolized NPs to cells cultured at the air-liquid-interface (ALI) have been developed, but require specialized equipment and are associated with higher cost and time. Therefore, it is important to determine whether aerosolized setups induce different cellular responses to NPs than traditional ones, which could provide new insights into toxicological responses of NP exposure. This study evaluates the response of human alveolar epithelial cells (A549) to zinc oxide (ZnO) NPs after dry aerosol exposure in the Nano Aerosol Chamber for In Vitro Toxicity (NACIVT) system as compared to conventional, suspension-based exposure: cells at ALI or submerged. Similar to other studies using nebulization of ZnO NPs, we found that dry aerosol exposure of ZnO NPs via the NACIVT system induced different cellular responses as compared to conventional methods. ZnO NPs delivered at 1.0 µg/cm

Despite improvements, lung transplantation remains hampered by both a scarcity of donor organs and by mortality following primary graft dysfunction (PGD). Since acute respiratory distress syndrome (ARDS) limits donor lungs utilization, we investigated cytokine adsorption as a means of treating ARDS donor lungs. We induced mild to moderate ARDS using lipopolysaccharide in 16 donor pigs. Lungs were then treated with or without cytokine adsorption during ex vivo lung perfusion (EVLP) and/or post-transplantation using extracorporeal hemoperfusion. The treatment significantly decreased cytokine levels during EVLP and decreased levels of immune cells post-transplantation. Histology demonstrated fewer signs of lung injury across both treatment periods and the incidence of PGD was significantly reduced among treated animals. Overall, cytokine adsorption was able to restore lung function and reduce PGD in lung transplantation. We suggest this treatment will increase the availability of donor lungs and increase the tolerability of donor lungs in the recipient.

Fibrosis can affect any organ, resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by the expansion of connective tissue due to excessive deposition of extracellular matrix (ECM) proteins, including the fibrillar forms of collagen. A significant limitation for discovering cures for fibrosis is the availability of suitable human models and techniques to quantify mature fibrillar collagen deposition as close as possible to human physiological conditions. Here we have extensively characterised an ex vivo cultured human lung tissue-derived, precision-cut lung slices (hPCLS) model using label-free second harmonic generation (SHG) light microscopy to quantify fibrillar collagen deposition and mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint of hPCLS in ex vivo culture. We demonstrate that hPCLS are viable and metabolically active, with mesenchymal, epithelial, endothelial and immune cell types surviving for at least 2 weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed a strong induction of pulmonary fibrosis-related ECM proteins upon transforming growth factor-β1 (TGF-β1) stimulation. This upregulation of ECM proteins was not translated into an increased deposition of fibrillar collagen. In support of this observation, we revealed the presence of a pro-ECM degradation activity in our ex vivo cultures of hPCLS, inhibition of which by a metalloproteinase inhibitor resulted in increased collagen deposition in response to TGF-β1 stimulation. Together the data show that an integrated approach of measuring soluble pro-fibrotic markers alongside quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents., Multiomic and label-free imaging-based characterisation of ex vivo cultured human precision-cut lung slices (hPCLS) reveals that MMP signalling is a rate-limiting factor necessary for deposition of fibrillar collagen in ECM of hPCLS https://bit.ly/3rcUa0e

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos-exposed individuals and rapidly leads to death. MPM harbors loss-of-function mutations in BAP1, NF2, CDKN2A, and TP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here, we show that a proportion of human MPM harbor point mutations, copy number alterations, and overexpression of KRAS with or without TP53 changes. These are likely pathogenic, since ectopic expression of mutant KRAS(G12D) in the pleural mesothelium of conditional mice causes epithelioid MPM and cooperates with TP53 deletion to drive a more aggressive disease form with biphasic features and pleural effusions. Murine MPM cell lines derived from these tumors carry the initiating KRAS(G12D) lesions, secondary Bap1 alterations, and human MPM-like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate that KRAS alterations alone or in accomplice with TP53 alterations likely play an important and underestimated role in a proportion of patients with MPM, which warrants further exploration., European Union (EU); Horizon 2020; European Research Council 2010 Starting Independent Investigator; ERC Proof of Concept 2015 (ERC-PoC-2015); German Research Society; ALTERNATIVE; German Ministry for Education and Research; German Center for Lung Research Translational Research Grant; Greek State Scholarship Foundation Program; European Union (EU); European Union Social Fund; Greek National Fund; Reinforcement of Postdoctoral Researchers-1 stand 2nd cycles; General Secretariat for Research and Innovation; Hellenic Foundation for Research and Innovation; REPSIRE European Respiratory Society Fellowship; Institut National de la Sante et de la Recherche Medicale (INSERM); Centre National de la Recherche Scientifique (CNRS); Institut de Recherche en Sante Respiratoire des Pays de la Loire; National Research Agency Programme d' Investissements d' Avenir; Pays de la Loire Region Research Program; Ligue Contrele Cancer; Chancellerie des Universites de Paris; Projekt DEAL

Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology/disease and screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology/disease as compared with animal models. Several experimental readouts have been established for use with PCLS, but obtaining high-yield and -quality RNA for downstream analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for nonbiased and high-throughput analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high-quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis. We show that the RNA isolated using this method has sufficient quality for RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease.

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos-exposed individuals and rapidly leads to the development of pleural effusion and death. MPM harbours loss-of-function mutations in genes likeBAP1, NF2, CDKN2A, andTP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here we show that a significant proportion of human MPM harbour point mutations and copy number alterations in theKRASproto-oncogene. These mutations are likely pathogenic, since ectopic expression of mutantKRASG12Din the pleural mesothelium of conditional mice causes MPM. Murine MPM cell lines derived from these tumours carry the initiatingKRASG12Dlesions, secondaryBap1alterations, and human MPM-like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate thatKRASmutations likely play an important and underestimated role in MPM, which warrants further exploration.

A workshop entitled “Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases” was hosted by the University of Vermont Larner College of Medicine in collaboration with the National Heart, Lung and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, the International Society for Cell and Gene Therapy and the Pulmonary Fibrosis Foundation. The event was held from July 15 to 18, 2019 at the University of Vermont, Burlington, Vermont. The objectives of the conference were to review and discuss the current status of the following active areas of research: 1) technological advancements in the analysis and visualisation of lung stem and progenitor cells; 2) evaluation of lung stem and progenitor cells in the context of their interactions with the niche; 3) progress toward the application and delivery of stem and progenitor cells for the treatment of lung diseases such as cystic fibrosis; 4) progress in induced pluripotent stem cell models and application for disease modelling; and 5) the emerging roles of cell therapy and extracellular vesicles in immunomodulation of the lung. This selection of topics represents some of the most dynamic research areas in which incredible progress continues to be made. The workshop also included active discussion on the regulation and commercialisation of regenerative medicine products and concluded with an open discussion to set priorities and recommendations for future research directions in basic and translation lung biology., This workshop report discusses recent advances in cell therapy and bioengineering approaches for repair and regeneration of diseased lungs https://bit.ly/2DqA8eu

Chronic lung allograft dysfunction (CLAD) is a severely limiting factor to the long-term survival of lung transplant recipients (LTx). Detection of CLAD is hampered by a lack of clinically available markers. Exhaled breath particles (EBP) is proposed as a noninvasive means of identifying CLAD patients and observing their condition. This pilot study aims to capture the range of EBP expected in human samples and identify possible candidate markers for further study. EBP are collected using a PExA device. Samples were obtained from healthy subjects (a combined sample of 600 ng and a 200 ng from each) and from five patients (one grouped sample of 600 ng from three patients and two 200 ng samples from two unique patients). The samples were analyzed via tandem mass spectrometry (MS). Up to 650 proteins were identified in the EBP samples identified. The fifty proteins identified represented a breadth of biological function and displayed a variety of localizations. Ten could be traced back to immunological system processes and included a number of immunoglobulin constant regions. Sixteen of these proteins could be found in the extracellular region. EBP presents as a novel method for the non-invasive analysis of LTx with the potential to monitor for the development of CLAD. This pilot study characterizes the range of proteins that can be identified from samples across subjects and serves as the basis for the exploration of markers in LTx. The findings of this work serve as the establishing method for the ongoing effort to use EBP to identify and monitor CLAD in LTx recipients.

Precision-cut lung slices (PCLS) have gained increasing interest as a model to study lung biology and disease, as well as for screening novel therapeutics. In particular, PCLS derived from human tissue can better recapitulate some aspects of lung biology and disease as compared to PCLS derived from animals (e.g. clinical heterogeneity), but access to human tissue is limited. A number of different experimental readouts have been established for use with PCLS, but obtaining high yield and quality RNA for downstream gene expression analysis has remained challenging. This is particularly problematic for utilizing the power of next-generation sequencing techniques, such as RNA-sequencing (RNA-seq), for non-biased and high through-put analysis of PCLS human cohorts. In the current study, we present a novel approach for isolating high quality RNA from a small amount of tissue, including diseased human tissue, such as idiopathic pulmonary fibrosis (IPF). We show that the RNA isolated using this method is of sufficient quality for both RT-qPCR and RNA-seq analysis. Furthermore, the RNA-seq data from human PCLS was comparable to data generated from native tissue and could be used in several established computational pipelines, including deconvolution of bulk RNA-seq data using publicly available single-cell RNA-seq data sets. Deconvolution using Bisque revealed a diversity of cell populations in human PCLS derived from distal lung tissue, including several immune cell populations, which correlated with cell populations known to be present and aberrant in human disease, such as IPF.

Recent advances in 3D bioprinting allow for generating intricate structures with dimensions relevant for human tissue, but suitable bioinks for producing translationally relevant tissue with complex geometries remain unidentified. Here, a tissue-specific hybrid bioink is described, composed of a natural polymer, alginate, reinforced with extracellular matrix derived from decellularized tissue (rECM). rECM has rheological and gelation properties beneficial for 3D bioprinting while retaining biologically inductive properties supporting tissue maturation ex vivo and in vivo. These bioinks are shear thinning, resist cell sedimentation, improve viability of multiple cell types, and enhance mechanical stability in hydrogels derived from them. 3D printed constructs generated from rECM bioinks suppress the foreign body response, are pro-angiogenic and support recipient-derived de novo blood vessel formation across the entire graft thickness in a murine model of transplant immunosuppression. Their proof-of-principle for generating human tissue is demonstrated by 3D bioprinting human airways composed of regionally specified primary human airway epithelial progenitor and smooth muscle cells. Airway lumens remained patent with viable cells for one month in vitro with evidence of differentiation into mature epithelial cell types found in native human airways. rECM bioinks are a promising new approach for generating functional human tissue using 3D bioprinting.

ispartof: American Journal Of Physiology-Lung Cellular And Molecular Physiology vol:319 issue:3 pages:L538-L540 ispartof: location:United States status: published

Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.

Fibrotic disorders account for over one third of mortalities worldwide. Despite great efforts to study the cellular and molecular processes underlying fibrosis, there are currently few effective therapies. Dual-stage polymerization reactions are an innovative tool for recreating heterogeneous increases in extracellular matrix (ECM) modulus, a hallmark of fibrotic diseases in vivo. Here, we present a clickable decellularized ECM (dECM) crosslinker incorporated into a dynamically responsive poly(ethylene glycol)-α-methacrylate (PEGαMA) hybrid-hydrogel to recreate ECM remodeling in vitro. An off-stoichiometry thiol-ene Michael addition between PEGαMA (8-arm, 10 kg mol-1) and the clickable dECM resulted in hydrogels with an elastic modulus of E = 3.6 ± 0.24 kPa, approximating healthy lung tissue (1-5 kPa). Next, residual αMA groups were reacted via a photo-initiated homopolymerization to increase modulus values to fibrotic levels (E = 13.4 ± 0.82 kPa) in situ. Hydrogels with increased elastic moduli, mimicking fibrotic ECM, induced a significant increase in the expression of myofibroblast transgenes. The proportion of primary fibroblasts from dual-reporter mouse lungs expressing collagen 1a1 and alpha-smooth muscle actin increased by approximately 60% when cultured on stiff and dynamically stiffened hybrid-hydrogels compared to soft. Likewise, fibroblasts expressed significantly increased levels of the collagen 1a1 transgene on stiff regions of spatially patterned hybrid-hydrogels compared to the soft areas. Collectively, these results indicate that hybrid-hydrogels are a new tool that can be implemented to spatiotemporally induce a phenotypic transition in primary murine fibroblasts in vitro.