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Small cell lung cancer (SCLC) remains a lethal disease with a dismal overall survival rate of 6% despite promising responses to upfront combination chemotherapy. The key drivers of such rapid mortality include early metastatic dissemination in the natural course of the disease and the near guaranteed emergence of chemoresistant disease. Here, we found that we could model the regression and relapse seen in clinical SCLC in vitro. We utilized time-course resolved RNA-sequencing to globally profile transcriptome changes as SCLC cells responded to a combination of cisplatin and etoposide—the standard-of-care in SCLC. Comparisons across time points demonstrated a distinct transient transcriptional state resembling embryonic diapause. Differential gene expression analysis revealed that expression of the PEA3 transcription factors ETV4 and ETV5 were transiently upregulated in the surviving fraction of cells which we determined to be necessary for efficient clonogenic expansion following chemotherapy. The FGFR-PEA3 signaling axis guided the identification of a pan-FGFR inhibitor demonstrating in vitro and in vivo efficacy in delaying progression following combination chemotherapy, observed inhibition of phosphorylation of the FGFR adaptor FRS2 and corresponding downstream MAPK and PI3K-Akt signaling pathways. Taken together, these data nominate PEA3 transcription factors as key mediators of relapse progression in SCLC and identify a clinically actionable small molecule candidate for delaying relapse of SCLC.

XLSX file - 48KB, IPA biological functions analysis.

This perspective on Varella-Garcia et al. (beginning on p. 447 in this issue of the journal) discusses the role of sputum-based biomarkers in the risk assessment and early detection of lung cancer. The importance of the detection of sputum epithelial chromosomal aneusomy by fluorescence in situ hybridization (FISH) as a potential risk or early-detection biomarker is discussed in the context of other biomarkers and models in lung carcinogenesis. The presently reported findings on FISH in sputum cells are an important contribution worthy of further investigation in defined clinical settings. Cancer Prev Res; 3(4); 420–3. ©2010 AACR.

XLSX file - 343KB, Genes associated with SCC carcinogenesis.

XLS file - 32KB, MYC activity prediction and its target genes.

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PDF file - 690KB, Supplementary Figure S1. Laser capture microdissection. Supplementary Figure S2. Experimental validation of PTBP3 expression. Supplementary Table S1. Patient demographics. List of SCC patients with their age, gender, grade of premalignant lesion, and assays performed. Supplementary Table S2. Alignment statistics. The number and percentage of reads aligning uniquely to the nuclear (autosomal and sex) or mitochondrial chromosomes, or not uniquely aligned, are shown for each sample.

XLS file - 35KB, TP53 activity prediction and its target genes.

Related Perspective and Related Article from Lung Cancer Biomarkers: FISHing in the Sputum for Risk Assessment and Early Detection

Figures S5-S6 provide additional evidence that Snail malignantly transforms epithelial cells in vitro. Information on the mutational background and stem phenotype of the cells transformed by Snail is provided.

Detailed supplemental methods and key references are provided. Tables S1-S2-S3-S4 provide antibody information, mouse study design information, PCR primer information, and an abbreviations list, respectively.

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated, including in non–small cell lung cancer (NSCLC). Here, we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo. Snail-mediated transformation relied upon silencing of the tumor-suppressive RNA splicing regulatory protein ESRP1. In clinical specimens of NSCLC, ESRP1 loss was documented in Snail-expressing premalignant pulmonary lesions. Mechanistic investigations showed that Snail drives malignant progression in an ALDH+CD44+CD24− pulmonary stem cell subset in which ESRP1 and stemness-repressing microRNAs are inhibited. Collectively, our results show how ESRP1 loss is a critical event in lung carcinogenesis, and they identify new candidate directions for targeted therapy of NSCLC.Significance: This study defines a Snail-ESRP1 cancer axis that is crucial for human lung carcinogenesis, with implications for new intervention strategies and translational opportunities. Cancer Res; 78(8); 1986–99. ©2018 AACR.

Figures S2-S3-S4 provide additional evidence that Snail is a potent driver of EMT, carcinogenic signaling programs, and stem cell expansion in vitro.

Figure S8 provides evidence that ESRP1 loss alone (without Snail expression) is not sufficient to transform epithelial cells in vitro.

Smoking is the most important known risk factor for the development of lung cancer. Tobacco exposure results in chronic inflammation, tissue injury, and repair. A recent hypothesis argues for a stem/progenitor cell involved in airway epithelial repair that may be a tumor-initiating cell in lung cancer and which may be associated with recurrence and metastasis. We used immunostaining, quantitative real-time PCR, Western blots, and lung cancer tissue microarrays to identify subpopulations of airway epithelial stem/progenitor cells under steady-state conditions, normal repair, aberrant repair with premalignant lesions and lung cancer, and their correlation with injury and prognosis. We identified a population of keratin 14 (K14)–expressing progenitor epithelial cells that was involved in repair after injury. Dysregulated repair resulted in the persistence of K14+ cells in the airway epithelium in potentially premalignant lesions. The presence of K14+ progenitor airway epithelial cells in NSCLC predicted a poor prognosis, and this predictive value was strongest in smokers, in which it also correlated with metastasis. This suggests that reparative K14+ progenitor cells may be tumor-initiating cells in this subgroup of smokers with NSCLC. Cancer Res; 70(16); 6639–48. ©2010 AACR.

Supplementary Methods, Tables 1-4, Figures 1-6 from Presence of a Putative Tumor-Initiating Progenitor Cell Population Predicts Poor Prognosis in Smokers with Non–Small Cell Lung Cancer

Small-cell-lung-cancer (SCLC) has the worst prognosis of all lung cancers because of a high incidence of relapse after therapy. We developed a bioengineered 3-dimensional (3D) SCLC co-culture organoid as a phenotypic tool to study SCLC tumor kinetics and SCLC-fibroblast interactions during relapse. We used functionalized alginate microbeads as a scaffold to mimic lung alveolar architecture and co-cultured SCLC cell lines with primary adult lung fibroblasts (ALF). We found that SCLCs in the model proliferated extensively, invaded the microbead scaffold and formed tumors within just 7 days. We compared the bioengineered tumors with patient tumors and found them to recapitulate the pathology and immunophenotyping of the patient tumors better than the PDX model developed from the same SCLC cell line. When treated with standard chemotherapy drugs, etoposide and cisplatin, the organoid recapitulated relapse after chemotherapy. Co-culture of the SCLC cells with ALFs revealed that the fibroblasts play a key role in inducing faster and more robust SCLC cell regrowth in the model. This was a paracrine effect as conditioned medium from the same fibroblasts was responsible for this accelerated cell regrowth. This model is also amenable to high throughput phenotypic or targeted drug screening to find new therapeutics for SCLC.

Cystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts more than 70,000 people. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most important determinant of morbidity and mortality. Here we report results from a multi-institute consortium in which single-cell transcriptomics were applied to define disease-related changes by comparing the proximal airway of CF donors (n = 19) undergoing transplantation for end-stage lung disease with that of previously healthy lung donors (n = 19). Disease-dependent differences observed include an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subsets coupled with an unexpected decrease in cycling basal cells. Our study yields a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease. Single-cell RNA profiling of human cystic fibrosis proximal airway tissue reveals an overabundance of epithelial cells transitioning to specialized ciliated and secretory cells coupled with a decrease in cycling basal cells.

SummaryTo date, there is no effective oral antiviral against SARS-CoV-2 that is also anti-inflammatory. Herein, we show that the mitochondrial antioxidant mitoquinone/mitoquinol mesylate (Mito-MES), a dietary supplement, has potent antiviral activity against SARS-CoV-2 and its variants of concern in vitro and in vivo. Mito-MES had nanomolar in vitro antiviral potency against the Beta and Delta SARS-CoV-2 variants as well as the murine hepatitis virus (MHV-A59). Mito-MES given in SARS-CoV-2 infected K18-hACE2 mice through oral gavage reduced viral titer by nearly 4 log units relative to the vehicle group. We found in vitro that the antiviral effect of Mito-MES is attributable to its hydrophobic dTPP+ moiety and its combined effects scavenging reactive oxygen species (ROS), activating Nrf2 and increasing the host defense proteins TOM70 and MX1. Mito-MES was efficacious reducing increase in cleaved caspase-3 and inflammation induced by SARS-CoV2 infection both in lung epithelial cells and a transgenic mouse model of COVID-19. Mito-MES reduced production of IL-6 by SARS-CoV-2 infected epithelial cells through its antioxidant properties (Nrf2 agonist, coenzyme Q10 moiety) and the dTPP moiety. Given established safety of Mito-MES in humans, our results suggest that Mito-MES may represent a rapidly applicable therapeutic strategy that can be added in the therapeutic arsenal against COVID-19. Its potential long-term use by humans as diet supplement could help control the SARS-CoV-2 pandemic, especially in the setting of rapidly emerging SARS-CoV-2 variants that may compromise vaccine efficacy.One-Sentence SummaryMitoquinone/mitoquinol mesylate has potent antiviral and anti-inflammatory activity in preclinical models of SARS-CoV-2 infection.

Small cell lung cancer (SCLC) remains a lethal disease with a dismal overall survival rate of 6% despite promising responses to upfront combination chemotherapy. The key drivers of such rapid mortality include early metastatic dissemination in the natural course of the disease and the near guaranteed emergence of chemoresistant disease. Here, we found that we could model the regression and relapse seen in clinical SCLC in vitro. We utilized time-course resolved RNA-sequencing to globally profile transcriptome changes as SCLC cells responded to a combination of cisplatin and etoposide-the standard-of-care in SCLC. Comparisons across time points demonstrated a distinct transient transcriptional state resembling embryonic diapause. Differential gene expression analysis revealed that expression of the PEA3 transcription factors ETV4 and ETV5 were transiently upregulated in the surviving fraction of cells which we determined to be necessary for efficient clonogenic expansion following chemotherapy. The FGFR-PEA3 signaling axis guided the identification of a pan-FGFR inhibitor demonstrating in vitro and in vivo efficacy in delaying progression following combination chemotherapy, observed inhibition of phosphorylation of the FGFR adaptor FRS2 and corresponding downstream MAPK and PI3K-Akt signaling pathways. Taken together, these data nominate PEA3 transcription factors as key mediators of relapse progression in SCLC and identify a clinically actionable small molecule candidate for delaying relapse of SCLC.

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Although infection initiates in the proximal airways, severe and sometimes fatal symptoms of the disease are caused by infection of the alveolar type 2 (AT2) cells of the distal lung and associated inflammation. In this study, we develop primary human lung epithelial infection models to understand initial responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells are readily infected by SARS-CoV-2, leading to an epithelial cell-autonomous proinflammatory response with increased expression of interferon signaling genes. Studies to validate the efficacy of selected candidate COVID-19 drugs confirm that remdesivir strongly suppresses viral infection/replication. We provide a relevant platform for study of COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and emergent respiratory pathogens., Graphical abstract, In vitro models of human lung epithelium, including diverse cell types of the proximo-distal axis, are critical for modeling infection. Mulay et al. show that alveospheres, with epithelial type 2- and type 1-like cells, are infected by SARS-CoV-2, initiating an interferon response, and serve as a platform for screening antiviral drugs.

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients., The pandemic of COVID-19, caused by SARS-CoV-2 infection, warrants immediate investigation for therapy options. Here the authors show, using epithelial and air-liquid interface cultures, that SARS-CoV-2 hijacks host cell metabolism to facilitate viral replication, and that inhibition of mTORC1, a master metabolic regulator, suppresses viral replication.

Emergence of a highly contagious novel coronavirus, SARS-CoV-2 that causes COVID-19, has precipitated the current global health crisis with over 479,000 deaths and more than 9.3 million confirmed cases. Currently, our knowledge of the mechanisms of COVID-19 disease pathogenesis is very limited which has hampered attempts to develop targeted antiviral strategies. Therefore, we urgently need an effective therapy for this unmet medical need. Viruses hijack and dysregulate cellular machineries in order for them to replicate and infect more cells. Thus, identifying and targeting dysregulated signaling pathways that have been taken over by viruses is one strategy for developing an effective antiviral therapy. We have developed a high-throughput drug screening system to identify potential antiviral drugs targeting SARS-CoV-2. We utilized a small molecule library of 430 protein kinase inhibitors, which are in various stages of clinical trials. Most of the tested kinase antagonists are ATP competitive inhibitors, a class of nucleoside analogs, which have been shown to have potent antiviral activity. From the primary screen, we have identified 34 compounds capable of inhibiting viral cytopathic effect in epithelial cells. Network of drug and protein relations showed that these compounds specifically targeted a limited number of cellular kinases. More importantly, we have identified mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-Damage Response (DDR) pathways as key cellular signaling pathways critical for SARS-CoV-2 infection. Subsequently, a secondary screen confirmed compounds such as Berzosertib (VE-822), Vistusertib (AZD2014), and Nilotinib with anti SARS-CoV-2 activity. Finally, we found that Berzosertib, an ATR kinase inhibitor in the DDR pathway, demonstrated potent antiviral activity in a human epithelial cell line and human induced pluripotent stem cell (hIPSC)-derived cardiomyocytes. These inhibitors are already in clinical trials of phase 2 or 3 for cancer treatment, and can be repurposed as promising drug candidates for a host-directed therapy of SARS-CoV-2 infection. In conclusion, we have identified small molecule inhibitors exhibiting anti SARS-CoV-2 activity by blocking key cellular kinases, which gives insight on important mechanism of host-pathogen interaction. These compounds can be further evaluated for the treatment of COVID-19 patients following additional in vivo safety and efficacy studies.DisclosuresNone declared.

Introduction/AbstractCystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts in excess of 70,000 people globally. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most significant determinant of morbidity and mortality. In this study we report results from a multi-institute consortium in which single cell transcriptomics were applied to define disease-related changes to the proximal airway of CF donors (n=19) undergoing transplantation for end-stage lung disease compared to the proximal airway of previously healthy lung donors (n=19). We found that all major airway epithelial cell types were conserved between control and CF donors. Disease-dependent differences were observed, including an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subtypes coupled with an unexpected decrease in cycling basal cells. This study developed a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

The respiratory tract is a vital, intricate system for several important biological processes including mucociliary clearance, airway conductance, and gas exchange. The Wnt signaling pathway plays several crucial and indispensable roles across lung biology in multiple contexts. This review highlights the progress made in characterizing the role of Wnt signaling across several disciplines in lung biology, including development, homeostasis, regeneration following injury, in vitro directed differentiation efforts, and disease progression. We further note uncharted directions in the field that may illuminate important biology. The discoveries made collectively advance our understanding of Wnt signaling in lung biology and have the potential to inform therapeutic advancements for lung diseases., Cody Aros, Carla Pantoja, and Brigitte Gomperts review the key role of Wnt signaling in all aspects of lung development, repair, and disease progression. They provide an overview of recent research findings and highlight where research is needed to further elucidate mechanisms of action, with the aim of improving disease treatments.

Background Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. Methods We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Results Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. Conclusions This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0652-9) contains supplementary material, which is available to authorized users.

Summary The mission of the California Institute of Regenerative Medicine (CIRM) is to accelerate treatments to patients with unmet medical needs. In September 2016, CIRM sponsored a workshop held at the University of California, Los Angeles, to discuss regenerative medicine approaches for treatment of lung diseases and to identify the challenges remaining for advancing such treatments to the clinic and market approval. Workshop participants discussed current preclinical and clinical approaches to regenerative medicine in the lung, as well as the biology of lung stem cells and the role of stem cells in the etiology of various lung diseases. The outcome of this effort was the recognition that whereas transient cell delivery approaches are leading the way in the clinic, recent advances in the understanding of lung stem cell biology, in vitro and in vivo disease modeling, gene editing and replacement methods, and cell engraftment approaches raise the prospect of developing cures for some lung diseases in the foreseeable future. In addition, advances in in vitro modeling using lung organoids and “lung on a chip” technology are setting the stage for high quality small molecule drug screening to develop treatments for lung diseases with complex biology.

Bronchopulmonary dysplasia (BPD) is a common complication of premature birth. The histopathology of BPD is characterized by an arrest of alveolarization with fibroblast activation. The Wnt/β-catenin signaling pathway is important in early lung development. When Wnt signaling is active, phosphorylation of β-catenin by tyrosine kinases at activating sites, specifically at tyrosine 489 (Y489), correlates with nuclear localization of β-catenin. We examined fetal lung tissue, lung tissue from term newborns, and lung tissue from infants who died with BPD; we found nuclear β-catenin phosphorylation at Y489 in epithelial and mesenchymal cells in fetal tissue and BPD tissue, but not in the lungs of term infants. Using a 3D human organoid model, we found increased nuclear localization of β-catenin phosphorylated at Y489 (p-β-cateninY489) after exposure to alternating hypoxia and hyperoxia compared with organoids cultured in normoxia. Exogenous stimulation of the canonical Wnt pathway in organoids was sufficient to cause nuclear localization of p-β-cateninY489 in normoxia and mimicked the pattern of α-smooth muscle actin (α-SMA) expression seen with fibroblastic activation from oxidative stress. Treatment of organoids with a tyrosine kinase inhibitor prior to cyclic hypoxia-hyperoxia inhibited nuclear localization of p-β-cateninY489 and prevented α-SMA expression by fibroblasts. Posttranslational phosphorylation of β-catenin is a transient feature of normal lung development. Moreover, the persistence of p-β-cateninY489 is a durable marker of fibroblast activation in BPD and may play an important role in BPD disease pathobiology.

Small cell neuroendocrine (SCN) cancers (SCNCs) are an aggressive cancer subtype. Transdifferentiation towards a SCN phenotype has been reported as a resistance route in response to targeted therapies. Here, we identified a convergence to a SCN state that is widespread across epithelial cancers and is associated with poor prognosis. More broadly, non-SCN metastases have higher expression of SCN-associated transcription factors than non-SCN primary tumors. Drug sensitivity and gene dependency screens demonstrate that these convergent SCNCs have shared vulnerabilities. These common vulnerabilities are found across unannotated SCN-like epithelial cases, small round blue cell tumors, and unexpectedly in hematological malignancies. The SCN convergent phenotype and common sensitivity profiles with hematological cancers can guide treatment options beyond tissue-specific targeted therapies.

Summary Our understanding of dynamic interactions between airway basal stem cells (ABSCs) and their signaling niches in homeostasis, injury, and aging remains elusive. Using transgenic mice and pharmacologic studies, we found that Wnt/β-catenin within ABSCs was essential for proliferation post-injury in vivo. ABSC-derived Wnt ligand production was dispensable for epithelial proliferation. Instead, the PDGFRα+ lineage in the intercartilaginous zone (ICZ) niche transiently secreted Wnt ligand necessary for ABSC proliferation. Strikingly, ABSC-derived Wnt ligand later drove early progenitor differentiation to ciliated cells. We discovered additional changes in aging, as glandular-like epithelial invaginations (GLEIs) derived from ABSCs emerged exclusively in the ICZ of aged mice and contributed to airway homeostasis and repair. Further, ABSC Wnt ligand secretion was necessary for GLEI formation, and constitutive activation of β-catenin in young mice induced their formation in vivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging.

Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation.We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions.Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function.This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.

This unit describes a protocol for generation of lung organoids. A lung organoid is a 3D cell/hydrogel composite that resembles the morphology and cellular composition of the human distal lung. These tissue-engineered constructs provide an in vitro model of human lung and are best suited for disease modeling applications. The organoid generation methodology is flexible, allowing for easy scalability in the number of organoids produced and in the ability to accommodate a wide range of cell types. © 2018 by John WileySons, Inc.

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated, including in non–small cell lung cancer (NSCLC). Here, we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo. Snail-mediated transformation relied upon silencing of the tumor-suppressive RNA splicing regulatory protein ESRP1. In clinical specimens of NSCLC, ESRP1 loss was documented in Snail-expressing premalignant pulmonary lesions. Mechanistic investigations showed that Snail drives malignant progression in an ALDH+CD44+CD24− pulmonary stem cell subset in which ESRP1 and stemness-repressing microRNAs are inhibited. Collectively, our results show how ESRP1 loss is a critical event in lung carcinogenesis, and they identify new candidate directions for targeted therapy of NSCLC. Significance: This study defines a Snail-ESRP1 cancer axis that is crucial for human lung carcinogenesis, with implications for new intervention strategies and translational opportunities. Cancer Res; 78(8); 1986–99. ©2018 AACR.

Lung tissue can robustly regenerate functional alveolar units after injury, but the mechanisms are unknown. Lechner et al. (2017) in this issue of Cell Stem Cell demonstrate that lung regeneration is facilitated by bone-marrow-derived myeloid cells that are recruited to the lung through a CCL2-CCR2 chemokine axis and by IL-13 expressing innate lymphoid cells.

Stem cell technologies, especially patient-specific, induced stem cell pluripotency and directed differentiation, hold great promise for changing the landscape of medical therapies. Proper exploitation of these methods may lead to personalized organ transplants, but to regenerate organs, it is necessary to develop methods for assembling differentiated cells into functional, organ-level tissues. The generation of three-dimensional human tissue models also holds potential for medical advances in disease modeling, as full organ functionality may not be necessary to recapitulate disease pathophysiology. This is specifically true of lung diseases where animal models often do not recapitulate human disease. Here, we present a method for the generation of self-assembled human lung tissue and its potential for disease modeling and drug discovery for lung diseases characterized by progressive and irreversible scarring such as idiopathic pulmonary fibrosis (IPF). Tissue formation occurs because of the overlapping processes of cellular adhesion to multiple alveolar sac templates, bioreactor rotation, and cellular contraction. Addition of transforming growth factor-β1 to single cell-type mesenchymal organoids resulted in morphologic scarring typical of that seen in IPF but not in two-dimensional IPF fibroblast cultures. Furthermore, this lung organoid may be modified to contain multiple lung cell types assembled into the correct anatomical location, thereby allowing cell-cell contact and recapitulating the lung microenvironment. Our bottom-up approach for synthesizing patient-specific lung tissue in a scalable system allows for the development of relevant human lung disease models with the potential for high throughput drug screening to identify targeted therapies.

Both basal and submucosal gland (SMG) duct stem cells of the airway epithelium are capable of sphere formation in the in vitro sphere assay, although the efficiency at which this occurs is very low. We sought to improve this efficiency of sphere formation by identifying subpopulations of airway basal stem cells (ABSC) and SMG duct cells based on their aldehyde dehydrogenase (ALDH) activity. ALDH(hi) ABSCs and SMG duct cells were highly enriched for the population of cells that could make spheres, while the co-culture of ALDH(hi) differentiated cells with the ALDH(hi) ABSCs increased their sphere-forming efficiency. Specific ALDH agonists and antagonists were used to show that airway specific ALDH isozymes are important for ABSC proliferation. Pathway analysis of gene expression profiling of ALDH(hi) and ALDH(lo) ABSCs revealed a significant upregulation of the arachidonic acid (AA) metabolism pathway in ALDH(hi) ABSCs. We confirmed the importance of this pathway in the metabolism of proliferating ALDH(hi) ABSCs using bioenergetics studies as well as agonists and antagonists of the AA pathway. These studies could lead to the development of novel strategies for altering ABSC proliferation in the airway epithelium.

Background and objective: The heterotopic syngeneic tracheal transplant mouse model is an acute hypoxic-ischemic injury model that undergoes complete repair and regeneration. We hypothesized that the repair and regeneration process of the surface epithelium and submucosal glands would occur in a reproducible pattern that could be followed by the expression of specific markers of epithelial cell types. Methods: We used the syngeneic heterotopic tracheal transplant model to develop a temporal and spatial map of cellular repair and regeneration by examining the tracheal grafts at post-transplant days 1, 3, 5, 7, 10 and 14. We used pulsed BrdU and immunofluorescent staining to identify and follow proliferating and repairing cell populations. Results: We confirmed the reproducibility of the injury and repair in the model and we found a distinct sequence of reappearance of the various stem/progenitor and differentiated cell populations of the tracheal surface epithelium and submucosal glands. In the initial phase, the basal and duct cells that survived the injury proliferated to re-epithelialize the basement membrane with K5 and K14 expressing cells. Then these cells proliferated further and differentiated to restore the function of the epithelium. During this repair process, TROP-2 marked all repairing submucosal gland tubules and ducts. Non-CCSP-expressing serous cells were found to differentiate 4–5 days before Clara, mucus and ciliated cells. Conclusions: Improving our understanding of the reparative process of the airway epithelium will allow us to identify cell-specific mechanisms of repair that could be used as novel therapeutic approaches for abnormal repair leading to airway diseases.

Bronchopulmonary dysplasia (BPD) is a leading complication of premature birth and occurs primarily in infants delivered during the saccular stage of lung development. Histopathology shows decreased alveolarization and a pattern of fibroblast proliferation and differentiation to the myofibroblast phenotype. Little is known about the molecular pathways and cellular mechanisms that define BPD pathophysiology and progression. We have developed a novel three-dimensional human model of the fibroblast activation associated with BPD, and using this model we have identified the Notch pathway as a key driver of fibroblast activation and proliferation in response to changes in oxygen. Fetal lung fibroblasts were cultured on sodium alginate beads to generate lung organoids. After exposure to alternating hypoxia and hyperoxia, the organoids developed a phenotypic response characterized by increased α-smooth muscle actin (α-SMA) expression and other genes known to be upregulated in BPD and also demonstrated increased expression of downstream effectors of the Notch pathway. Inhibition of Notch with a γ-secretase inhibitor prevented the development of the pattern of cellular proliferation and α-SMA expression in our model. Analysis of human autopsy tissue from the lungs of infants who expired with BPD demonstrated evidence of Notch activation within fibrotic areas of the alveolar septae, suggesting that Notch may be a key driver of BPD pathophysiology.

The airway epithelium is in direct contact with the environment and therefore constantly at risk for injury. Basal cells (BCs) have been found to repair the surface epithelium (SE), but the contribution of other stem cell populations to airway epithelial repair has not been identified. We demonstrated that airway submucosal gland (SMG) duct cells, in addition to BCs, survived severe hypoxic-ischemic injury. We developed a method to isolate duct cells from the airway. In vitro and in vivo models were used to compare the self-renewal and differentiation potential of duct cells and BCs. We found that only duct cells were capable of regenerating SMG tubules and ducts, as well as the SE overlying the SMGs. SMG duct cells are therefore a multipotent stem cell for airway epithelial repair This is of importance to the field of lung regeneration as determining the repairing cell populations could lead to the identification of novel therapeutic targets and cell-based therapies for patients with airway diseases.