Regulation of Human Airway Epithelial Tissue Stem Cell Differentiation by β-Catenin, P300, and CBP. Malleske Daniel T,Hayes Don,Lallier Scott W,Hill Cynthia L,Reynolds Susan D Stem cells (Dayton, Ohio) The wingless/integrase-1 (WNT)/β-catenin signaling pathway is active in several chronic lung diseases including idiopathic pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease. Although this WNT/β-catenin pathway activity is associated with an increase in mucus cell frequency and a decrease in ciliated cell frequency, a cause and consequence relationship between signaling and cell frequency has not been established. We previously demonstrated that genetic stabilization of β-catenin inhibited differentiation of mouse bronchiolar tissue stem cells (TSC). This study determined the effect of β-catenin and its co-factors P300 (E1A-binding protein, 300 kDa) and cAMP response element binding (CREB)-binding protein (CBP) on human bronchial epithelial TSC differentiation to mucus and ciliated cells. We developed a modified air-liquid interface (ALI) culture system in which mucus and ciliated cell frequency is similar. These cultures were treated with the β-catenin agonist CHIR99021 (CHIR) and antagonists to β-catenin (XAV939), P300 (IQ1), and CBP (ICG001). We report that human TSC differentiation to mucus and ciliated cells can be divided into two stages, specification and commitment. CHIR treatment inhibited mucus and ciliated cell commitment while XAV939 treatment demonstrated that β-catenin was necessary for mucus and ciliated cell specification. Additional studies demonstrate that a β-catenin/P300 complex promotes mucus cell specification and that β-catenin interacts with either P300 or CBP to inhibit ciliated cell commitment. These data indicate that activation of β-catenin-dependent signaling in chronic lung disease leads to changes in mucus and ciliated cell frequency and that P300 and CBP tune the β-catenin signal to favor mucus cell differentiation. Stem Cells 2018;36:1905-12. 10.1002/stem.2906
    EGF-Amphiregulin Interplay in Airway Stem/Progenitor Cells Links the Pathogenesis of Smoking-Induced Lesions in the Human Airway Epithelium. Zuo Wu-Lin,Yang Jing,Gomi Kazunori,Chao IonWa,Crystal Ronald G,Shaykhiev Renat Stem cells (Dayton, Ohio) The airway epithelium of cigarette smokers undergoes dramatic remodeling with hyperplasia of basal cells (BC) and mucus-producing cells, squamous metaplasia, altered ciliated cell differentiation and decreased junctional barrier integrity, relevant to chronic obstructive pulmonary disease and lung cancer. In this study, we show that epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) is induced by smoking in human airway epithelium as a result of epidermal growth factor (EGF)-driven squamous differentiation of airway BC stem/progenitor cells. In turn, AREG induced a unique EGFR activation pattern in human airway BC, distinct from that evoked by EGF, leading to BC- and mucous hyperplasia, altered ciliated cell differentiation and impaired barrier integrity. Further, AREG promoted its own expression and suppressed expression of EGF, establishing an autonomous self-amplifying signaling loop in airway BC relevant for promotion of EGF-independent hyperplastic phenotypes. Thus, EGF-AREG interplay in airway BC stem/progenitor cells is one of the mechanisms that mediates the interconnected pathogenesis of all major smoking-induced lesions in the human airway epithelium. Stem Cells 2017;35:824-837. 10.1002/stem.2512
    TGF-β activation impairs fibroblast ability to support adult lung epithelial progenitor cell organoid formation. Ng-Blichfeldt John-Poul,de Jong Tristan,Kortekaas Rosa K,Wu Xinhui,Lindner Michael,Guryev Victor,Hiemstra Pieter S,Stolk Jan,Königshoff Melanie,Gosens Reinoud American journal of physiology. Lung cellular and molecular physiology Transforming growth factor-β (TGF-β)-induced fibroblast-to-myofibroblast differentiation contributes to remodeling in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis, but whether this impacts the ability of fibroblasts to support lung epithelial repair remains little explored. We pretreated human lung fibroblasts [primary (phFB) or MRC5 cells] with recombinant human TGF-β to induce myofibroblast differentiation, then cocultured them with adult mouse lung epithelial cell adhesion molecule-positive cells (EpCAM) to investigate their capacity to support epithelial organoid formation in vitro. While control phFB and MRC5 lung fibroblasts supported organoid formation of mouse EpCAM cells, TGF-β pretreatment of both phFB and MRC5 impaired organoid-supporting ability. We performed RNA sequencing of TGF-β-treated phFB, which revealed altered expression of key Wnt signaling pathway components and Wnt/β-catenin target genes, and modulated expression of secreted factors involved in mesenchymal-epithelial signaling. TGF-β profoundly skewed the transcriptional program induced by the Wnt/β-catenin activator CHIR99021. Supplementing organoid culture media recombinant hepatocyte growth factor or fibroblast growth factor 7 promoted organoid formation when using TGF-β pretreated fibroblasts. In conclusion, TGF-β-induced myofibroblast differentiation results in Wnt/β-catenin pathway skewing and impairs fibroblast ability to support epithelial repair likely through multiple mechanisms, including modulation of secreted growth factors. 10.1152/ajplung.00400.2018
    Retinoic acid signaling balances adult distal lung epithelial progenitor cell growth and differentiation. Ng-Blichfeldt John-Poul,Schrik Anneke,Kortekaas Rosa K,Noordhoek Jacobien A,Heijink Irene H,Hiemstra Pieter S,Stolk Jan,Königshoff Melanie,Gosens Reinoud EBioMedicine BACKGROUND:Despite compelling data describing pro-regenerative effects of all-trans retinoic acid (ATRA) in pre-clinical models of chronic obstructive pulmonary disease (COPD), clinical trials using retinoids for emphysema patients have failed. Crucial information about the specific role of RA signaling in adult rodent and human lung epithelial progenitor cells is largely missing. METHODS:Adult lung organoid cultures were generated from isolated primary mouse and human lung epithelial cells, and incubated with pharmacological pathway modulators and recombinant proteins. Organoid number and size were measured, and differentiation was assessed with quantitative immunofluorescence and gene expression analyses. FINDINGS:We unexpectedly found that ATRA decreased lung organoid size, whereas RA pathway inhibition increased mouse and human lung organoid size. RA pathway inhibition stimulated mouse lung epithelial proliferation via YAP pathway activation and epithelial-mesenchymal FGF signaling, while concomitantly suppressing alveolar and airway differentiation. HDAC inhibition rescued differentiation in growth-augmented lung organoids. INTERPRETATION:In contrast to prevailing notions, our study suggests that regenerative pharmacology using transient RA pathway inhibition followed by HDAC inhibition might hold promise to promote lung epithelial regeneration in diseased adult lung tissue. FUND: This project is funded by the Lung Foundation Netherlands (Longfonds) grant (RG, MK, JS, PSH) and W2/W3 Professorship Award by the Helmholtz Association, Berlin, Germany (MK). 10.1016/j.ebiom.2018.09.002
    Very Small Embryonic-like Stem Cells Are Mobilized in Human Peripheral Blood during Hypoxemic COPD Exacerbations and Pulmonary Hypertension. Guerin Coralie L,Blandinières Adeline,Planquette Benjamin,Silvestre Jean-Sébastien,Israel-Biet Dominique,Sanchez Olivier,Smadja David M Stem cell reviews and reports Very small embryonic-like stem cells (VSELs) are major pluripotent stem cells involved in vascular and tissue regeneration and constitute a recruitable pool of stem/progenitor cells with putative instrumental role in organ repair. Here, we hypothesized that VSELs might be mobilized from the bone marrow (BM) to peripheral blood (PB) in patients with hypoxic lung disease or pulmonary hypertension (PH). The objective of the present study was then to investigate the changes in VSELs number in peripheral blood of patients with hypoxic lung disease and PH. We enrolled 26 patients with Chronic Obstructive Pulmonary Disease (COPD) with or without hypoxemia, 13 patients with PH and 20 controls without any respiratory or cardiovascular diseases. In PH patients, VSELs levels have been determined during right heart catheterization in pulmonary blood and PB. For this purpose, mononuclear cells were separated by density gradient and VSELs have been quantified by using a multiparametric flow cytometry approach. The number of PB-VSELs in hypoxic COPD patients was significantly increased compared with non-hypoxic COPD patients or controls (p = 0.0055). In patients with PH, we did not find any difference in VSELs numbers between arterial pulmonary blood and venous PB (p = 0.93). However, we found an increase in VSELs in the peripheral blood of patients with PH (p = 0.03). In conclusion, we unraveled that circulating VSELs were increased in peripheral blood of patients with hypoxic COPD or with PH. Thus, VSELs may serve as a reservoir of pluripotent stem cells that can be recruited into PB and may play an important role in promoting lung repair. 10.1007/s12015-017-9732-6
    Shared epithelial pathways to lung repair and disease. Spella Magda,Lilis Ioannis,Stathopoulos Georgios T European respiratory review : an official journal of the European Respiratory Society Chronic lung diseases present tremendous health burdens and share a common pathobiology of dysfunctional epithelial repair. Lung adenocarcinoma, the leading cancer killer worldwide, is caused mainly by chemical carcinogens of tobacco smoke that induce mutations in pulmonary epithelial cells leading to uncontrolled epithelial proliferation. Lung epithelial cells that possess the capacity for self-renewal and regeneration of other lung cell types are believed to underlie the pathobiology of chronic obstructive, fibrotic and neoplastic lung disorders. However, the understanding of lung epithelial progenitor cell hierarchy and turnover is incomplete and a comprehensive model of the cellular and transcriptional events that underlie lung regeneration and carcinogenesis is missing. The mapping of these processes is extremely important, since their modulation would potentially allow effective cure and/or prevention of chronic lung diseases. In this review we describe current knowledge on cellular and molecular pathways at play during lung repair and carcinogenesis and summarise the critical lung cell populations with regenerative and cancerous potential. 10.1183/16000617.0048-2017
    Wnt/β-catenin signaling is critical for regenerative potential of distal lung epithelial progenitor cells in homeostasis and emphysema. Hu Yan,Ng-Blichfeldt John-Poul,Ota Chiharu,Ciminieri Chiara,Ren Wenhua,Hiemstra Pieter S,Stolk Jan,Gosens Reinoud,Königshoff Melanie Stem cells (Dayton, Ohio) Wnt/β-catenin signaling regulates progenitor cell fate decisions during lung development and in various adult tissues. Ectopic activation of Wnt/β-catenin signaling promotes tissue repair in emphysema, a devastating lung disease with progressive loss of parenchymal lung tissue. The identity of Wnt/β-catenin responsive progenitor cells and the potential impact of Wnt/β-catenin signaling on adult distal lung epithelial progenitor cell function in emphysema are poorly understood. Here, we used TCF/Lef:H2B/GFP reporter mice to investigate the role of Wnt/β-catenin signaling in lung organoid formation. We identified an organoid-forming adult distal lung epithelial progenitor cell population characterized by a low Wnt/β-catenin activity, which was enriched in club and alveolar epithelial type (AT)II cells. Endogenous Wnt/β-catenin activity was required for the initiation of multiple subtypes of distal lung organoids derived from the Wnt epithelial progenitors. Further ectopic Wnt/β-catenin activation specifically led to an increase in alveolar organoid number; however, the subsequent proliferation of alveolar epithelial cells in the organoids did not require constitutive Wnt/β-catenin signaling. Distal lung epithelial progenitor cells derived from the mouse model of elastase-induced emphysema exhibited reduced organoid forming capacity. This was rescued by Wnt/β-catenin signal activation, which largely increased the number of alveolar organoids. Together, our study reveals a novel mechanism of lung epithelial progenitor cell activation in homeostasis and emphysema. 10.1002/stem.3241
    Rho-Kinase 1/2 Inhibition Prevents Transforming Growth Factor-β-Induced Effects on Pulmonary Remodeling and Repair. Wu Xinhui,Verschut Vicky,Woest Manon E,Ng-Blichfeldt John-Poul,Matias Ana,Villetti Gino,Accetta Alessandro,Facchinetti Fabrizio,Gosens Reinoud,Kistemaker Loes E M Frontiers in pharmacology Transforming growth factor (TGF)-β-induced myofibroblast transformation and alterations in mesenchymal-epithelial interactions contribute to chronic lung diseases such as chronic obstructive pulmonary disease (COPD), asthma and pulmonary fibrosis. Rho-associated coiled-coil-forming protein kinase (ROCK) consists as two isoforms, ROCK1 and ROCK2, and both are playing critical roles in many cellular responses to injury. In this study, we aimed to elucidate the differential role of ROCK isoforms on TGF-β signaling in lung fibrosis and repair. For this purpose, we tested the effect of a non-selective ROCK 1 and 2 inhibitor (compound 31) and a selective ROCK2 inhibitor (compound A11) in inhibiting TGF-β-induced remodeling in lung fibroblasts and slices; and dysfunctional epithelial-progenitor interactions in lung organoids. Here, we demonstrated that the inhibition of ROCK1/2 with compound 31 represses TGF-β-driven actin remodeling as well as extracellular matrix deposition in lung fibroblasts and PCLS, whereas selective ROCK2 inhibition with compound A11 did not. Furthermore, the TGF-β induced inhibition of organoid formation was functionally restored in a concentration-dependent manner by both dual ROCK 1 and 2 inhibition and selective ROCK2 inhibition. We conclude that dual pharmacological inhibition of ROCK 1 and 2 counteracts TGF-β induced effects on remodeling and alveolar epithelial progenitor function, suggesting this to be a promising therapeutic approach for respiratory diseases associated with fibrosis and defective lung repair. 10.3389/fphar.2020.609509
    Increased tissue endothelial progenitor cells in end-stage lung diseases with pulmonary hypertension. Schiavon Marco,Fadini Gian Paolo,Lunardi Francesca,Agostini Carlo,Boscaro Elisa,Calabrese Fiorella,Marulli Giuseppe,Rea Federico The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation BACKGROUND:Diffuse lung diseases promote the development of vascular changes and pulmonary hypertension (PH). Endothelial progenitor cells (EPCs) seem to be involved in pulmonary vascular remodeling. We evaluated circulating and intra-pulmonary EPCs in end-stage lung diseases in relation to pulmonary arterial pressure (PAP). METHODS:The study included 19 patients affected by different end-stage lung diseases, with or without PH. Six lung donors were considered as control group. EPCs were measured in blood samples taken at the time of transplant from pulmonary arteries and veins (by flow cytometry) as well as in lung specimen sections (by confocal microscopy) and expressed as percentage of total number of cells. RESULTS:The amount of EPC in lung specimens was significantly different according to type of disease (p = 0.001). Specifically, a higher number of EPCs was detected in idiopathic pulmonary hypertension and idiopathic pulmonary fibrosis with high (> 25 mm Hg) mean PAP (p = 0.03 for both) compared with chronic obstructive pulmonary disease and control group. There was a direct correlation between intrapulmonary EPCs and PAP. According to receiver operating characteristic curve analysis, the presence of > 3% EPCs had a 91% sensitivity and 93% specificity in identifying high mean PAP. There were no differences in circulating arterial or venous EPCs among groups. CONCLUSIONS:Intra-pulmonary EPCs are increased in lung diseases with high PAP, suggesting that EPCs may contribute to vascular remodeling in end-stage pulmonary disease. 10.1016/j.healun.2012.06.005
    Circulating endothelial progenitor cells and chronic pulmonary diseases. Huertas A,Palange P The European respiratory journal Circulating endothelial progenitor cells (EPCs) are bone marrow-derived cells that contribute to vascular healing and remodelling under physiological and pathological conditions. Although controversies exist regarding the definition and origin of EPCs, it has been widely demonstrated that they are involved in several diseases and that they have therapeutic implications. Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation that is not fully reversible, associated with abnormalities of airways (bronchitis) and parenchyma (emphysema), reduced exercise tolerance and systemic inflammation. Growing evidence has also suggested that endothelial dysfunction may play a role in COPD. Although it is not clear whether endothelial dysfunction represents a cause or a consequence of COPD, several studies have highlighted the importance of EPCs in this disease, suggesting that the bone marrow could be a novel target of COPD. The present review summarises the role of EPCs in pulmonary diseases, with particular emphasis on COPD. The aim is to improve understanding as to the possible role of EPCs in COPD pathophysiology. This may help in the identification of novel diagnostic and therapeutic tools in COPD. 10.1183/09031936.00034810
    Airway basal cells. The "smoking gun" of chronic obstructive pulmonary disease. Crystal Ronald G American journal of respiratory and critical care medicine The earliest abnormality in the lung associated with smoking is hyperplasia of airway basal cells, the stem/progenitor cells of the ciliated and secretory cells that are central to pulmonary host defense. Using cell biology and 'omics technologies to assess basal cells isolated from bronchoscopic brushings of nonsmokers, smokers, and smokers with chronic obstructive pulmonary disease (COPD), compelling evidence has been provided in support of the concept that airway basal cells are central to the pathogenesis of smoking-associated lung diseases. When confronted by the chronic stress of smoking, airway basal cells become disorderly, regress to a more primitive state, behave as dictated by their inheritance, are susceptible to acquired changes in their genome, lose the capacity to regenerate the epithelium, are responsible for the major changes in the airway that characterize COPD, and, with persistent stress, can undergo malignant transformation. Together, these observations led to the conclusion that accelerated loss of lung function in susceptible individuals begins with disordered airway basal cell biology (i.e., that airway basal cells are the "smoking gun" of COPD, a potential target for the development of therapies to prevent smoking-related lung disorders). 10.1164/rccm.201408-1492PP
    Dysfunction of endothelial progenitor cells from smokers and chronic obstructive pulmonary disease patients due to increased DNA damage and senescence. Paschalaki Koralia E,Starke Richard D,Hu Yanhua,Mercado Nicolas,Margariti Andriana,Gorgoulis Vassilis G,Randi Anna M,Barnes Peter J Stem cells (Dayton, Ohio) Cardiovascular disease (CVD) is a major cause of death in smokers, particularly in those with chronic obstructive pulmonary disease (COPD). Circulating endothelial progenitor cells (EPC) are required for endothelial homeostasis, and their dysfunction contributes to CVD. To investigate EPC dysfunction in smokers, we isolated and expanded blood outgrowth endothelial cells (BOEC) from peripheral blood samples from healthy nonsmokers, healthy smokers, and COPD patients. BOEC from smokers and COPD patients showed increased DNA double-strand breaks and senescence compared to nonsmokers. Senescence negatively correlated with the expression and activity of sirtuin-1 (SIRT1), a protein deacetylase that protects against DNA damage and cellular senescence. Inhibition of DNA damage response by silencing of ataxia telangiectasia mutated (ATM) kinase resulted in upregulation of SIRT1 expression and decreased senescence. Treatment of BOEC from COPD patients with the SIRT1 activator resveratrol or an ATM inhibitor (KU-55933) also rescued the senescent phenotype. Using an in vivo mouse model of angiogenesis, we demonstrated that senescent BOEC from COPD patients are dysfunctional, displaying impaired angiogenic ability and increased apoptosis compared to cells from healthy nonsmokers. Therefore, this study identifies epigenetic regulation of DNA damage and senescence as pathogenetic mechanisms linked to endothelial progenitors' dysfunction in smokers and COPD patients. These defects may contribute to vascular disease and cardiovascular events in smokers and could therefore constitute therapeutic targets for intervention. 10.1002/stem.1488
    Early events in the pathogenesis of chronic obstructive pulmonary disease. Smoking-induced reprogramming of airway epithelial basal progenitor cells. Shaykhiev Renat,Crystal Ronald G Annals of the American Thoracic Society The airway epithelium is the primary site of the earliest pathologic changes induced by smoking, contributing to the development of chronic obstructive pulmonary disease (COPD). The normal human airway epithelium is composed of several major cell types, including differentiated ciliated and secretory cells, intermediate undifferentiated cells, and basal cells (BC). BC contain the stem/progenitor cell population responsible for maintenance of the normally differentiated airway epithelium. Although inflammatory and immune processes play a significant role in the pathogenesis of COPD, the earliest lesions include hyperplasia of the BC population, suggesting that the disease may start with this cell type. Apart from BC hyperplasia, smoking induces a number of COPD-relevant airway epithelial remodeling phenotypes that are likely initiated in the BC population, including mucous cell hyperplasia, squamous cell metaplasia, epithelial-mesenchymal transition, altered ciliated and nonmucous secretory cell differentiation, and suppression of junctional barrier integrity. Significant progress has been recently made in understanding the biology of human airway BC, including gene expression features, stem/progenitor, and other functions, including interaction with other airway cell types. Accumulating evidence suggests that human airway BC function as both sensors and cellular sources of various cytokines and growth factors relevant to smoking-associated airway injury, as well as the origin of various molecular and histological phenotypes relevant to the pathogenesis of COPD. In the context of these considerations, we suggest that early BC-specific smoking-induced molecular changes are critical to the pathogenesis of COPD, and these represent a candidate target for novel therapeutic approaches to prevent COPD progression in susceptible individuals. 10.1513/AnnalsATS.201402-049AW
    Exaggerated BMP4 signalling alters human airway basal progenitor cell differentiation to cigarette smoking-related phenotypes. Zuo Wu-Lin,Yang Jing,Strulovici-Barel Yael,Salit Jacqueline,Rostami Mahboubeh,Mezey Jason G,O'Beirne Sarah L,Kaner Robert J,Crystal Ronald G The European respiratory journal Airway remodelling in chronic obstructive pulmonary disease (COPD) originates, in part, from smoking-induced changes in airway basal stem/progenitor cells (BCs). Based on the knowledge that bone morphogenetic protein 4 (BMP4) influences epithelial progenitor function in the developing and adult mouse lung, we hypothesised that BMP4 signalling may regulate the biology of adult human airway BCs relevant to COPD.BMP4 signalling components in human airway epithelium were analysed at the mRNA and protein levels, and the differentiation of BCs was assessed using the BC expansion and air-liquid interface models in the absence/presence of BMP4, BMP receptor inhibitor and/or small interfering RNAs against BMP receptors and downstream signalling.The data demonstrate that in cigarette smokers, BMP4 is upregulated in ciliated and intermediate undifferentiated cells, and expression of the BMP4 receptor BMPR1A is enriched in BCs. BMP4 induced BCs to acquire a smoking-related abnormal phenotype mediated by BMPR1A/Smad signalling, characterised by decreased capacity to differentiate into normal mucociliary epithelium, while generating squamous metaplasia.Exaggerated BMP4 signalling promotes cigarette smoking-relevant airway epithelial remodelling by inducing abnormal phenotypes in human airway BCs. Targeting of BMP4 signalling in airway BCs may represent a novel target to prevent/treat COPD-associated airway disease. 10.1183/13993003.02553-2017
    Exhaustion of Airway Basal Progenitor Cells in Early and Established Chronic Obstructive Pulmonary Disease. Ghosh Moumita,Miller York E,Nakachi Ichiro,Kwon Jennifer B,Barón Anna E,Brantley Alexandra E,Merrick Daniel T,Franklin Wilbur A,Keith Robert L,Vandivier R William American journal of respiratory and critical care medicine RATIONALE:Up to 40% of smokers develop chronic obstructive pulmonary disease (COPD) over a period that spans decades. Despite the importance of COPD, much remains to be learned about susceptibility and pathogenesis, especially during early, prediagnostic stages of disease. Airway basal progenitor cells are crucial for lung health and resilience because of their ability to repair injured airways. In COPD, the normal airway epithelium is replaced with increased basal and secretory (mucous) cells and decreased ciliated cells, suggesting that progenitors are impaired. OBJECTIVES:To examine airway basal progenitor cells and lung function in smokers with and without COPD. METHODS:Bronchial biopsies taken from smokers at risk for COPD and lung cancer were used to acquire airway basal progenitor cells. They were evaluated for count, self-renewal, and multipotentiality (ability to differentiate to basal, mucous, and ciliated cells), and progenitor count was examined for its relationship with lung function. MEASUREMENTS AND MAIN RESULTS:Basal progenitor count, self-renewal, and multipotentiality were all reduced in COPD versus non-COPD. COPD progenitors produced an epithelium with increased basal and mucous cells and decreased ciliated cells, replicating the COPD phenotype. Progenitor depletion correlated with lung function and identified a subset of subjects without COPD with lung function that was midway between non-COPD with high progenitor counts and those with COPD. CONCLUSIONS:Basal progenitor dysfunction relates to the histologic and physiologic manifestations of COPD and identifies a subset that may represent an early, prediagnostic stage of COPD, indicating that progenitor exhaustion is involved in COPD pathogenesis. 10.1164/rccm.201704-0667OC
    Single-cell RNA sequencing profiling of the effects of aging on alveolar stem cells. Lv Tingting,Jiang Kewu,Wang Jiawen,Tang Nan,Dai Huaping,Wang Chen Science China. Life sciences The aging of alveolar stem cells has been linked to many chronic lung diseases, including pulmonary fibrosis. However, the effects of aging on alveolar stem cells during homeostasis and post-injury alveolar repair have not been well characterized. Here we conducted a single-cell RNA sequencing (scRNA-seq) analysis of alveolar stem cells of 3-month-old and 12-month-old mice to characterize the aging effect on alveolar stem cells. Our results have shown that the transcriptomes of alveolar stem cells of 3-month-old and 12-month-old mice are not significantly different under the steady condition. However, after a bleomycin-induced lung injury, the alveolar stem cells of 12-month-old mice show enhanced inflammatory responses and decreased lipid metabolism. Our study suggests a close relationship among aging, lipid metabolism, inflammatory responses and chronic lung diseases. 10.1007/s11427-019-9583-9
    Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung. McQualter Jonathan L,Yuen Karen,Williams Brenda,Bertoncello Ivan Proceedings of the National Academy of Sciences of the United States of America The role of lung epithelial stem cells in maintenance and repair of the adult lung is ill-defined, and their identity remains contentious because of the lack of definitive markers for their prospective isolation and the absence of clonogenic assays able to measure their stem/progenitor cell potential. In this study, we show that replication of epithelial-mesenchymal interactions in a previously undescribed matrigel-based clonogenic assay enables the identification of lung epithelial stem/progenitor cells by their colony-forming potential in vitro. We describe a population of EpCAM(hi) CD49f(pos) CD104(pos) CD24(low) epithelial cfus that generate colonies comprising airway, alveolar, or mixed lung epithelial cell lineages when cocultured with EpCAM(neg) Sca-1(pos) lung mesenchymal cells. We show that soluble fibroblast growth factor-10 and hepatocyte growth factor partially replace the requirement for mesenchymal support of epithelial colony formation, allowing clonal passaging and demonstration of their capacity for self-renewal. These data support a model in which the adult mouse lung contains a minor population of multipotent epithelial stem/progenitor cells with the capacity for self-renewal and whose descendants give rise to airway and alveolar epithelial cell lineages in vitro. 10.1073/pnas.0909207107
    Identification of a Repair-Supportive Mesenchymal Cell Population during Airway Epithelial Regeneration. Moiseenko Alena,Vazquez-Armendariz Ana Ivonne,Kheirollahi Vahid,Chu Xuran,Tata Aleksandra,Rivetti Stefano,Günther Stefan,Lebrigand Kevin,Herold Susanne,Braun Thomas,Mari Bernard,De Langhe Stijn,Kwapiszewska Grazyna,Günther Andreas,Chen Chengshui,Seeger Werner,Tata Purushothama Rao,Zhang Jin-San,Bellusci Saverio,El Agha Elie Cell reports Tissue regeneration requires coordinated and dynamic remodeling of stem and progenitor cells and the surrounding niche. Although the plasticity of epithelial cells has been well explored in many tissues, the dynamic changes occurring in niche cells remain elusive. Here, we show that, during lung repair after naphthalene injury, a population of PDGFRα cells emerges in the non-cartilaginous conducting airway niche, which is normally populated by airway smooth muscle cells (ASMCs). This cell population, which we term "repair-supportive mesenchymal cells" (RSMCs), is distinct from conventional ASMCs, which have previously been shown to contribute to epithelial repair. Gene expression analysis on sorted lineage-labeled cells shows that RSMCs express low levels of ASMC markers, but high levels of the pro-regenerative marker Fgf10. Organoid co-cultures demonstrate an enhanced ability for RSMCs in supporting club-cell growth. Our study highlights the dynamics of mesenchymal cells in the airway niche and has implications for chronic airway-injury-associated diseases. 10.1016/j.celrep.2020.108549
    The circadian clock protein REVERBα inhibits pulmonary fibrosis development. Cunningham Peter S,Meijer Peter,Nazgiewicz Alicja,Anderson Simon G,Borthwick Lee A,Bagnall James,Kitchen Gareth B,Lodyga Monika,Begley Nicola,Venkateswaran Rajamiyer V,Shah Rajesh,Mercer Paul F,Durrington Hannah J,Henderson Neil C,Piper-Hanley Karen,Fisher Andrew J,Chambers Rachel C,Bechtold David A,Gibbs Julie E,Loudon Andrew S,Rutter Martin K,Hinz Boris,Ray David W,Blaikley John F Proceedings of the National Academy of Sciences of the United States of America Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinβ1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach. 10.1073/pnas.1912109117
    Long-term expanding human airway organoids for disease modeling. Sachs Norman,Papaspyropoulos Angelos,Zomer-van Ommen Domenique D,Heo Inha,Böttinger Lena,Klay Dymph,Weeber Fleur,Huelsz-Prince Guizela,Iakobachvili Nino,Amatngalim Gimano D,de Ligt Joep,van Hoeck Arne,Proost Natalie,Viveen Marco C,Lyubimova Anna,Teeven Luc,Derakhshan Sepideh,Korving Jeroen,Begthel Harry,Dekkers Johanna F,Kumawat Kuldeep,Ramos Emilio,van Oosterhout Matthijs Fm,Offerhaus G Johan,Wiener Dominique J,Olimpio Eduardo P,Dijkstra Krijn K,Smit Egbert F,van der Linden Maarten,Jaksani Sridevi,van de Ven Marieke,Jonkers Jos,Rios Anne C,Voest Emile E,van Moorsel Coline Hm,van der Ent Cornelis K,Cuppen Edwin,van Oudenaarden Alexander,Coenjaerts Frank E,Meyaard Linde,Bont Louis J,Peters Peter J,Tans Sander J,van Zon Jeroen S,Boj Sylvia F,Vries Robert G,Beekman Jeffrey M,Clevers Hans The EMBO journal Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the study of hereditary, malignant, and infectious pulmonary disease. 10.15252/embj.2018100300
    Syndecan 4 Mediates Nrf2-dependent Expansion of Bronchiolar Progenitors That Protect Against Lung Inflammation. Santoso Arif,Kikuchi Toshiaki,Tode Naoki,Hirano Taizou,Komatsu Riyo,Damayanti Triya,Motohashi Hozumi,Yamamoto Masayuki,Kojima Tetsuhito,Uede Toshimitsu,Nukiwa Toshihiro,Ichinose Masakazu Molecular therapy : the journal of the American Society of Gene Therapy The use of lung progenitors for regenerative medicine appears promising, but their biology is not fully understood. Here, we found anti-inflammatory attributes in bronchiolar progenitors that were sorted as a multipotent subset of mouse club cells and found to express secretory leukocyte protease inhibitor (SLPI). Notably, the impaired expression of SLPI in mice increased the number of bronchiolar progenitors and decreased the lung inflammation. We determined a transcriptional profile for the bronchiolar progenitors of Slpi-deficient mice and identified syndecan 4, whose expression was markedly elevated as compared to that of wild-type mice. Systemic administration of recombinant syndecan 4 protein caused a substantial increase in the number of bronchiolar progenitors with concomitant attenuation of both airway and alveolar inflammation. The syndecan 4 administration also resulted in activation of the Keap1-Nrf2 antioxidant pathway in lung cells, which is critically involved in the therapeutic responses to the syndecan 4 treatment. Moreover, in 3D culture, the presence of syndecan 4 induced differentiated club cells to undergo Nrf2-dependent transition into bronchiolar progenitors. Our observations reveal that differentiative switches between bronchiolar progenitors and club cells are under the Nrf2-mediated control of SLPI and syndecan 4, suggesting the possibility of new therapeutic approaches in inflammatory lung diseases. 10.1038/mt.2015.153
    p53 Regulates Progenitor Cell Quiescence and Differentiation in the Airway. McConnell Alicia M,Yao Changfu,Yeckes Alyson R,Wang Yizhou,Selvaggio Anna S,Tang Jie,Kirsch David G,Stripp Barry R Cell reports Mechanisms that regulate progenitor cell quiescence and differentiation in slowly replacing tissues are not fully understood. Here, we demonstrate that the tumor suppressor p53 regulates both proliferation and differentiation of progenitors in the airway epithelium. p53 loss decreased ciliated cell differentiation and increased the self-renewal and proliferative capacity of club progenitors, increasing epithelial cell density. p53-deficient progenitors generated a pseudostratified epithelium containing basal-like cells in vitro and putative bronchioalveolar stem cells in vivo. Conversely, an additional copy of p53 increased quiescence and ciliated cell differentiation, highlighting the importance of tight regulation of p53 levels. Using single-cell RNA sequencing, we found that loss of p53 altered the molecular phenotype of progenitors and differentially modulated cell-cycle regulatory genes. Together, these findings reveal that p53 is an essential regulator of progenitor cell behavior, which shapes our understanding of stem cell quiescence during homeostasis and in cancer development. 10.1016/j.celrep.2016.11.007
    Tropism, replication competence, and innate immune responses of influenza virus: an analysis of human airway organoids and ex-vivo bronchus cultures. Hui Kenrie P Y,Ching Rachel H H,Chan Stan K H,Nicholls John M,Sachs Norman,Clevers Hans,Peiris J S Malik,Chan Michael C W The Lancet. Respiratory medicine BACKGROUND:Human airway organoids are three-dimensional cultures derived from stem cells, which self-organise in ex-vivo conditions to form so-called mini-airways. The cellular morphology of these cultures is physiologically similar to the human airway, with cilia, goblet cells, and club cells facing the inner lumen and basal cells situated at the outer layer. The aim of this study was to compare replication competence, tissue tropism, and host responses elicited by human and avian strains of influenza A virus in ex-vivo human bronchus and human airway organoids. METHODS:Between Sept 29, 2016, and Jan 4, 2017, we obtained ex-vivo cultures of the human bronchus and cultured human airway organoids from lung stem cells obtained from human lung tissues removed as part of the routine clinical care of patients undergoing surgical resection at the Department of Cardiothoracic Surgery, University of Hong Kong, Queen Mary Hospital, Hong Kong. We compared viral replication competence, tissue tropism, and cytokine and chemokine induction of avian influenza A viruses isolated from humans (Sh2/H7N9, H5N1/483, H5N6/39715), and human H1N1pdm/415742 in airway organoids and ex-vivo bronchus explant cultures. FINDINGS:Virus tropism and replication kinetics of human and avian influenza A viruses in human airway organoids mimicked those found in ex-vivo cultures of human bronchus explants. In both airway organoids and bronchus explants, influenza A H1N1 subtype (H1N1) and avian influenza A H7N9 viruses replicated to significantly higher titres than did the highly pathogenic avian influenza (HPAI) H5N1, whereas HPAI H5N6 replication was moderate. H1N1, H7N9, and H5N6 viruses infected ciliated cells and goblet cells, but not basal cells in both airway organoids and bronchus explants. The expression of cytokines, interleukin 6, and interferon β, and the chemokine regulated-on-activation, normal T-cell expressed and secreted, was significantly higher in human airway organoids infected with HPAI H5N1 virus than H1N1pdm/415742, Sh2/H7N9, and H5N6/39715 viruses, and the expression of monocyte chemoattractant protein-1 was significantly higher in human organoids infected with HPAI H5N1 virus than H1N1pdm/415742 and Sh2/H7N9 viruses. INTERPRETATION:Human airway organoid cultures provided results that were comparable to those observed in human ex-vivo bronchus cultures, and thus provide an alternative physiologically relevant experimental model for investigating virus tropism and replication competence that could be used to assess the pandemic threat of animal influenza viruses. FUNDING:US National Institute of Allergy and Infectious Diseases, Research Grants Council of the Hong Kong Special Administrative Region. 10.1016/S2213-2600(18)30236-4
    Distinct Airway Epithelial Stem Cells Hide among Club Cells but Mobilize to Promote Alveolar Regeneration. Kathiriya Jaymin J,Brumwell Alexis N,Jackson Julia R,Tang Xiaodan,Chapman Harold A Cell stem cell Lung injury activates specialized adult epithelial progenitors to regenerate the epithelium. Depending on the extent of injury, both remaining alveolar type II cells (AEC2s) and distal airway stem/progenitors mobilize to cover denuded alveoli and restore normal barriers. The major source of airway stem/progenitors other than basal-like cells remains uncertain. Here, we define a distinct subpopulation (∼5%) of club-like lineage-negative epithelial progenitors (LNEPs) marked by high H2-K1 expression critical for alveolar repair. Quiescent H2-K1 cells account for virtually all in vitro regenerative activity of airway lineages. After bleomycin injury, H2-K1 cells expand and differentiate in vivo to alveolar lineages. However, injured H2-K1 cells eventually develop impaired self-renewal with features of senescence, limiting complete repair. Normal H2-K1 cells transplanted into injured lungs differentiate into alveolar cells and rescue lung function. These findings indicate that small subpopulations of specialized stem/progenitors are required for effective lung regeneration and are a potential therapeutic adjunct after major lung injury. 10.1016/j.stem.2019.12.014
    Lung regeneration by multipotent stem cells residing at the bronchioalveolar-duct junction. Liu Qiaozhen,Liu Kuo,Cui Guizhong,Huang Xiuzhen,Yao Shun,Guo Wenke,Qin Zhen,Li Yan,Yang Rui,Pu Wenjuan,Zhang Libo,He Lingjuan,Zhao Huan,Yu Wei,Tang Muxue,Tian Xueying,Cai Dongqing,Nie Yu,Hu Shengshou,Ren Tao,Qiao Zengyong,Huang Hefeng,Zeng Yi Arial,Jing Naihe,Peng Guangdun,Ji Hongbin,Zhou Bin Nature genetics Characterizing the stem cells responsible for lung repair and regeneration is important for the treatment of pulmonary diseases. Recently, a unique cell population located at the bronchioalveolar-duct junctions has been proposed to comprise endogenous stem cells for lung regeneration. However, the role of bronchioalveolar stem cells (BASCs) in vivo remains debated, and the contribution of such cells to lung regeneration is not known. Here we generated a genetic lineage-tracing system that uses dual recombinases (Cre and Dre) to specifically track BASCs in vivo. Fate-mapping and clonal analysis showed that BASCs became activated and responded distinctly to different lung injuries, and differentiated into multiple cell lineages including club cells, ciliated cells, and alveolar type 1 and type 2 cells for lung regeneration. This study provides in vivo genetic evidence that BASCs are bona fide lung epithelial stem cells with deployment of multipotency and self-renewal during lung repair and regeneration. 10.1038/s41588-019-0346-6
    Targeting signalling pathways and the immune microenvironment of cancer stem cells - a clinical update. Clara Joseph A,Monge Cecilia,Yang Yingzi,Takebe Naoko Nature reviews. Clinical oncology Cancer stem cells (CSCs) have important roles in tumour development, relapse and metastasis; the intrinsic self-renewal characteristics and tumorigenic properties of these cells provide them with unique capabilities to resist diverse forms of anticancer therapy, seed recurrent tumours, and disseminate to and colonize distant tissues. The findings of several studies indicate that CSCs originate from non-malignant stem or progenitor cells. Accordingly, inhibition of developmental signalling pathways that are crucial for stem and progenitor cell homeostasis and function, such as the Notch, WNT, Hedgehog and Hippo signalling cascades, continues to be pursued across multiple cancer types as a strategy for targeting the CSCs hypothesized to drive cancer progression - with some success in certain malignancies. In addition, with the renaissance of anticancer immunotherapy, a better understanding of the interplay between CSCs and the tumour immune microenvironment might be the key to unlocking a new era of oncological treatments associated with a reduced propensity for the development of resistance and with enhanced antimetastatic activity, thus ultimately resulting in improved patient outcomes. Herein, we provide an update on the progress to date in the clinical development of therapeutics targeting the Notch, WNT, Hedgehog and Hippo pathways. We also discuss the interactions between CSCs and the immune system, including the potential immunological effects of agents targeting CSC-associated developmental signalling pathways, and provide an overview of the emerging approaches to CSC-targeted immunotherapy. 10.1038/s41571-019-0293-2
    Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Huang Sarah X L,Islam Mohammad Naimul,O'Neill John,Hu Zheng,Yang Yong-Guang,Chen Ya-Wen,Mumau Melanie,Green Michael D,Vunjak-Novakovic Gordana,Bhattacharya Jahar,Snoeck Hans-Willem Nature biotechnology The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP-agonists to signaling pathways critical for early lung development in the mouse-recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells. 10.1038/nbt.2754
    Parabronchial smooth muscle constitutes an airway epithelial stem cell niche in the mouse lung after injury. Volckaert Thomas,Dill Erik,Campbell Alice,Tiozzo Caterina,Majka Susan,Bellusci Saverio,De Langhe Stijn P The Journal of clinical investigation During lung development, parabronchial SMC (PSMC) progenitors in the distal mesenchyme secrete fibroblast growth factor 10 (Fgf10), which acts on distal epithelial progenitors to promote their proliferation. β-catenin signaling within PSMC progenitors is essential for their maintenance, proliferation, and expression of Fgf10. Here, we report that this Wnt/Fgf10 embryonic signaling cascade is reactivated in mature PSMCs after naphthalene-induced injury to airway epithelium. Furthermore, we found that this paracrine Fgf10 action was essential for activating surviving variant Clara cells (the cells in the airway epithelium from which replacement epithelial cells originate) located at the bronchoalveolar duct junctions and adjacent to neuroendocrine bodies. After naphthalene injury, PSMCs secreted Fgf10 to activate Notch signaling and induce Snai1 expression in surviving variant Clara cells, which subsequently underwent a transient epithelial to mesenchymal transition to initiate the repair process. Epithelial Snai1 expression was important for regeneration after injury. We have therefore identified PSMCs as a stem cell niche for the variant Clara cells in the lung and established that paracrine Fgf10 signaling from the niche is critical for epithelial repair after naphthalene injury. These findings also have implications for understanding the misregulation of lung repair in asthma and cancer. 10.1172/JCI58097
    Identification of bronchioalveolar stem cells in normal lung and lung cancer. Kim Carla F Bender,Jackson Erica L,Woolfenden Amber E,Lawrence Sharon,Babar Imran,Vogel Sinae,Crowley Denise,Bronson Roderick T,Jacks Tyler Cell Injury models have suggested that the lung contains anatomically and functionally distinct epithelial stem cell populations. We have isolated such a regional pulmonary stem cell population, termed bronchioalveolar stem cells (BASCs). Identified at the bronchioalveolar duct junction, BASCs were resistant to bronchiolar and alveolar damage and proliferated during epithelial cell renewal in vivo. BASCs exhibited self-renewal and were multipotent in clonal assays, highlighting their stem cell properties. Furthermore, BASCs expanded in response to oncogenic K-ras in culture and in precursors of lung tumors in vivo. These data support the hypothesis that BASCs are a stem cell population that maintains the bronchiolar Clara cells and alveolar cells of the distal lung and that their transformed counterparts give rise to adenocarcinoma. Although bronchiolar cells and alveolar cells are proposed to be the precursor cells of adenocarcinoma, this work points to BASCs as the putative cells of origin for this subtype of lung cancer. 10.1016/j.cell.2005.03.032
    The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium. Rawlins Emma L,Okubo Tadashi,Xue Yan,Brass David M,Auten Richard L,Hasegawa Hiroshi,Wang Fan,Hogan Brigid L M Cell stem cell To directly test the contribution of Scgb1a1(+) Clara cells to postnatal growth, homeostasis, and repair of lung epithelium, we generated a Scgb1a1-CreER "knockin" mouse for lineage-tracing these cells. Under all conditions tested, the majority of Clara cells in the bronchioles both self-renews and generates ciliated cells. In the trachea, Clara cells give rise to ciliated cells but do not self-renew extensively. Nevertheless, they can contribute to tracheal repair. In the postnatal mouse lung, it has been proposed that bronchioalveolar stem cells (BASCs) which coexpress Scgb1a1 (Secretoglobin1a1) and SftpC (Surfactant Protein C), contribute descendants to both bronchioles and alveoli. The putative BASCs were lineage labeled in our studies. However, we find no evidence for the function of a special BASC population during postnatal growth, adult homeostasis, or repair. Rather, our results support a model in which the trachea, bronchioles, and alveoli are maintained by distinct populations of epithelial progenitor cells. 10.1016/j.stem.2009.04.002
    Conditional depletion of airway progenitor cells induces peribronchiolar fibrosis. Perl Anne-Karina T,Riethmacher Dieter,Whitsett Jeffrey A American journal of respiratory and critical care medicine RATIONALE:The respiratory epithelium has a remarkable capacity to respond to acute injury. In contrast, repeated epithelial injury is often associated with abnormal repair, inflammation, and fibrosis. There is increasing evidence that nonciliated epithelial cells play important roles in the repair of the bronchiolar epithelium after acute injury. Cellular processes underlying the repair and remodeling of the lung after chronic epithelial injury are poorly understood. OBJECTIVES:To identify cell processes mediating epithelial regeneration and remodeling after acute and chronic Clara cell depletion. METHODS:A transgenic mouse model was generated to conditionally express diphtheria toxin A to ablate Clara cells in the adult lung. Epithelial regeneration and peribronchiolar fibrosis were assessed after acute and chronic Clara cell depletion. MEASUREMENTS AND MAIN RESULTS:Acute Clara cell ablation caused squamous metaplasia of ciliated cells and induced proliferation of residual progenitor cells. Ciliated cells in the bronchioles and pro-surfactant protein C-expressing cells in the bronchiolar alveolar duct junctions did not proliferate. Epithelial cell proliferation occurred at multiple sites along the airways and was not selectively associated with regions around neuroepithelial bodies. Chronic Clara cell depletion resulted in ineffective repair and caused peribronchiolar fibrosis. CONCLUSIONS:Colocalization of proliferation and cell type-specific markers demonstrate that Clara cells are critical airway progenitor cells. Continuous depletion of Clara cells resulted in persistent squamous metaplasia, lack of normal reepithelialization, and peribronchiolar fibrosis. Induction of proliferation in subepithelial fibroblasts supports the concept that chronic epithelial depletion caused peribronchiolar fibrosis. 10.1164/rccm.201005-0744OC
    Integrin α6β4 identifies an adult distal lung epithelial population with regenerative potential in mice. Chapman Harold A,Li Xiaopeng,Alexander Jonathan P,Brumwell Alexis,Lorizio Walter,Tan Kevin,Sonnenberg Arnoud,Wei Ying,Vu Thiennu H The Journal of clinical investigation Laminins and their integrin receptors are implicated in epithelial cell differentiation and progenitor cell maintenance. We report here that a previously unrecognized subpopulation of mouse alveolar epithelial cells (AECs) expressing the laminin receptor α6β4, but little or no pro-surfactant C (pro-SPC), is endowed with regenerative potential. Ex vivo, this subpopulation expanded clonally as progenitors but also differentiated toward mature cell types. Integrin β4 itself was not required for AEC proliferation or differentiation. An in vivo embryonic lung organoid assay, which we believe to be novel, was used to show that purified β4+ adult AECs admixed with E14.5 lung single-cell suspensions and implanted under kidney capsules self-organized into distinct Clara cell 10-kDa secretory protein (CC10+) airway-like and SPC+ saccular structures within 6 days. Using a bleomycin model of lung injury and an SPC-driven inducible cre to fate-map AECs, we found the majority of type II AECs in fibrotic areas were not derived from preexisting type II AECs, demonstrating that SPC- progenitor cells replenished type II AECs during repair. Our findings support the idea that there is a stable AEC progenitor population in the adult lung, provide in vivo evidence of AEC progenitor cell differentiation after parenchymal injury, and identify a strong candidate progenitor cell for maintenance of type II AECs during lung repair. 10.1172/JCI57673
    Stem cells are dispensable for lung homeostasis but restore airways after injury. Giangreco Adam,Arwert Esther N,Rosewell Ian R,Snyder Joshua,Watt Fiona M,Stripp Barry R Proceedings of the National Academy of Sciences of the United States of America Local tissue stem cells have been described in airways of the lung but their contribution to normal epithelial maintenance is currently unknown. We therefore developed aggregation chimera mice and a whole-lung imaging method to determine the relative contributions of progenitor (Clara) and bronchiolar stem cells to epithelial maintenance and repair. In normal and moderately injured airways chimeric patches were small in size and not associated with previously described stem cell niches. This finding suggested that single, randomly distributed progenitor cells maintain normal epithelial homeostasis. In contrast we found that repair following severe lung injury resulted in the generation of rare, large clonal cell patches that were associated with stem cell niches. This study provides evidence that epithelial stem cells are dispensable for normal airway homeostasis. We also demonstrate that stem cell activation and robust clonal cellular expansion occur only during repair from severe lung injury. 10.1073/pnas.0900668106
    Identification of pulmonary Oct-4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus (SARS-CoV) infection in vitro. Ling Thai-Yen,Kuo Ming-Der,Li Chung-Leung,Yu Alice L,Huang Yen-Hua,Wu Tsai-Jung,Lin You-Chin,Chen Shu-Hwa,Yu John Proceedings of the National Academy of Sciences of the United States of America In this study, we report a serum-free culture system for primary neonatal pulmonary cells that can support the growth of octamer-binding transcription factor 4+ (Oct-4+) epithelial colonies with a surrounding mesenchymal stroma. In addition to Oct-4, these cells also express other stem cell markers such as stage-specific embryonic antigen 1 (SSEA-1), stem cell antigen 1 (Sca-1), and Clara cell secretion protein (CCSP) but not c-Kit, CD34, and p63, indicating that they represent a subpopulation of Clara cells that have been implicated as lung stem/progenitor cells in lung injury models. These colony cells can be kept for weeks in primary cultures and undergo terminal differentiation to alveolar type-2- and type-1-like pneumocytes sequentially when removed from the stroma. In addition, we have demonstrated the presence of Oct-4+ long-term BrdU label-retaining cells at the bronchoalveolar junction of neonatal lung, providing a link between the Oct-4+ cells in vivo and in vitro and strengthening their identity as putative neonatal lung stem/progenitor cells. Lastly, these Oct-4+ epithelial colony cells, which also express angiotensin-converting enzyme 2, are the target cells for severe acute respiratory syndrome coronavirus infection in primary cultures and support active virus replication leading to their own destruction. These observations imply the possible involvement of lung stem/progenitor cells, in addition to pneumocytes, in severe acute respiratory syndrome coronavirus infection, accounting for the continued deterioration of lung tissues and apparent loss of capacity for lung repair. 10.1073/pnas.0510232103
    Neuroepithelial body microenvironment is a niche for a distinct subset of Clara-like precursors in the developing airways. Guha Arjun,Vasconcelos Michelle,Cai Yan,Yoneda Mitsuhiro,Hinds Anne,Qian Jun,Li Guihua,Dickel Lauren,Johnson Jane E,Kimura Shioko,Guo Jinjin,McMahon Jill,McMahon Andrew P,Cardoso Wellington V Proceedings of the National Academy of Sciences of the United States of America Clara cells of mammalian airways have multiple functions and are morphologically heterogeneous. Although Notch signaling is essential for the development of these cells, it is unclear how Notch influences Clara cell specification and if diversity is established among Clara cell precursors. Here we identify expression of the secretoglobin Scgb3a2 and Notch activation as early events in a program of secretory cell fate determination in developing murine airways. We show that Scgb3a2 expression in vivo is Notch-dependent at early stages and ectopically induced by constitutive Notch1 activation, and also that in vitro Notch signaling together with the pan-airway transcription factor Ttf1 (Nkx2.1) synergistically regulate secretoglobin gene transcription. Furthermore, we identified a subpopulation of secretory precursors juxtaposed to presumptive neuroepithelial bodies (NEBs), distinguished by their strong Scgb3a2 and uroplakin 3a (Upk3a) signals and reduced Ccsp (Scgb1a1) expression. Genetic ablation of Ascl1 prevented NEB formation and selectively interfered with the formation of this subpopulation of cells. Lineage labeling of Upk3a-expressing cells during development showed that these cells remain largely uncommitted during embryonic development and contribute to Clara and ciliated cells in the adult lung. Together, our findings suggest a role for Notch in the induction of a Clara cell-specific program of gene expression, and reveals that the NEB microenvironment in the developing airways is a niche for a distinct subset of Clara-like precursors. 10.1073/pnas.1204710109
    Identification of cancer initiating cells in K-Ras driven lung adenocarcinoma. Mainardi Sara,Mijimolle Nieves,Francoz Sarah,Vicente-Dueñas Carolina,Sánchez-García Isidro,Barbacid Mariano Proceedings of the National Academy of Sciences of the United States of America Ubiquitous expression of a resident K-Ras(G12V) oncogene in adult mice revealed that most tissues are resistant to K-Ras oncogenic signals. Indeed, K-Ras(G12V) expression only induced overt tumors in lungs. To identify these transformation-permissive cells, we induced K-Ras(G12V) expression in a very limited number of adult lung cells (0.2%) and monitored their fate by X-Gal staining, a surrogate marker coexpressed with the K-Ras(G12V) oncoprotein. Four weeks later, 30% of these cells had proliferated to form small clusters. However, only SPC(+) alveolar type II (ATII) cells were able to form hyperplastic lesions, some of which progressed to adenomas and adenocarcinomas. In contrast, induction of K-Ras(G12V) expression in lung cells by intratracheal infection with adenoviral-Cre particles generated hyperplasias in all regions except the proximal airways. Bronchiolar and bronchioalveolar duct junction hyperplasias were primarily made of CC10(+) Clara cells. Some of them progressed to form benign adenomas. However, only alveolar hyperplasias, exclusively made up of SPC(+) ATII cells, progressed to yield malignant adenocarcinomas. Adenoviral infection induced inflammatory infiltrates primarily made of T and B cells. This inflammatory response was essential for the development of K-Ras(G12V)-driven bronchiolar hyperplasias and adenomas, but not for the generation of SPC(+) ATII lesions. Finally, activation of K-Ras(G12V) during embryonic development under the control of a Sca1 promoter yielded CC10(+), but not SPC(+), hyperplasias, and adenomas. These results, taken together, illustrate that different types of lung cells can generate benign lesions in response to K-Ras oncogenic signals. However, in adult mice, only SPC(+) ATII cells were able to yield malignant adenocarcinomas. 10.1073/pnas.1320383110
    Human airway secretory cells during development and in mature airway epithelium. Jeffery P K,Gaillard D,Moret S The European respiratory journal The combined secretions of distinct secretory cells of the airway lining mucosa serve to keep the inspired air moist and free of potentially harmful dust particles, organisms and adsorbed gases. Apart from their role in protecting the respiratory zone of the lung, mucus-secreting cells act as pluripotential stem cells during foetal development and, in the adult, following mucosal injury. The variety of secretory cells include the mucous and serous cells of the surface and glandular epithelium, the non-ciliated bronchiolar (Clara) cell and the less frequent dense-core granulated (neuroendocrine) cell. The last-mentioned is the first type to differentiate at about 10 weeks of gestation; mucus-secreting cells are present from the 13th week of gestation, when mature ciliated cells are already present, and Clara cells begin to mature during the 19th week of human development. The alteration of secretory cell number and chemical composition of their secretions during the second trimester of foetal life is similar to that which occurs in chronic bronchitis in the adult. However, in hypersecretory disease the extent and site of the major change appear to be inappropriate to the defence of the lung.
    Number and proliferation of clara cells in normal human airway epithelium. Boers J E,Ambergen A W,Thunnissen F B American journal of respiratory and critical care medicine Experimental pathologic studies suggest that Clara cells are one of the types of airway stem cells but the proliferation of Clara cells in human lungs has not yet been examined. The purpose of this study was to assess in conducting airways of normal human lungs: (1) the number of Clara cells; and (2) the contribution of Clara cells to the proliferation compartment. Samples of histologic normal tissue were taken from seven lungs obtained by autopsy. A triple sequential (immuno)histochemical staining was performed, using MIB-1 as a proliferation marker and anti-CC10 for the identification of Clara cells; subsequently, a PAS stain was carried out as a marker for goblet cells, as these cells were reported to be CC10-immunoreactive in an unknown proportion. Clara cells were virtually absent in the proximal airway epithelium. The number of Clara cells in the terminal bronchioles was 11 +/- 3% (mean +/- SD) and in respiratory bronchioles 22 +/- 5%. The overall proliferation compartment of the conducting airway epithelium was 0.83 +/- 0.47%; the contribution of Clara cells was 9%. In the terminal bronchioles 15% of proliferating airway epithelial cells were Clara cells, and in the respiratory bronchioles this number increased to 44%. The contribution of Clara cells to the proliferation compartment of normal human tracheobronchial epithelium is substantial, demonstrating a role of the Clara cell in the maintenance of the normal epithelium of the distal conducting airways in humans. 10.1164/ajrccm.159.5.9806044
    Breathing life into the lung stem cell field. Fine Alan Cell stem cell In this issue of Cell Stem Cell, Rawlins et al. (2009) use an elegant lineage-tracing system to circumvent technical obstacles that have long limited advances in lung stem cell research and, as a result, definitively clarify the role of Clara cells in lung growth, homeostasis, and repair. 10.1016/j.stem.2009.05.012