Bronchioalveolar Stem Cells Research Articles

Tracing the origin of alveolar stem cells in lung repair and regeneration

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

NLRP3 inflammasome mediates abnormal epithelial regeneration and distal lung remodeling in silica‑induced lung fibrosis.

NOD-like receptor protein 3 (NLRP3) inflammasome is closely related to silica particle‑induced chronic lung inflammation but its role in epithelial remodeling, repair and regeneration in the distal lung during development of silicosis remains to be elucidated. The present study aimed to determine the effects of the NLRP3 inflammasome on epithelial remodeling and cellular regeneration and potential mechanisms in the distal lung of silica‑treated mice at three time points. Pulmonary function assessment, inflammatory cell counting, enzyme‑linked immunosorbent assay, histological and immunological analyses, hydroxyproline assay and western blotting were used in the study. Single intratracheal instillation of a silica suspension caused sustained NLRP3 inflammasome activation in the distal lung. Moreover, a time‑dependent increase in airway resistance and a decrease in lung compliance accompanied progression of pulmonary fibrosis. In the terminal bronchiole, lung remodeling including pyroptosis (membrane‑distributed GSDMD+), excessive proliferation (Ki67+), mucus overproduction (mucin 5 subtype AC and B) and epithelial‑mesenchymal transition (decreased E‑Cadherin+ and increased Vimentin+), was observed by immunofluorescence analysis. Notably, aberrant spatiotemporal expression of the embryonic lung stem/progenitor cell markers SOX2 and SOX9 and ectopic distribution of bronchioalveolar stem cells were observed in the distal lung only on the 7th day after silica instillation (the early inflammatory phase of silicosis). Western blotting revealed that the Sonic hedgehog/Glioma‑associated oncogene (Shh/Gli) and Wnt/β‑catenin pathways were involved in NLRP3 inflammasome activation‑mediated epithelial remodeling and dysregulated regeneration during the inflammatory and fibrotic phases. Overall, sustained NLRP3 inflammasome activation led to epithelial remodeling in the distal lung of mice. Moreover, understanding the spatiotemporal profile of dysregulated epithelial repair and regeneration may provide a novel therapeutic strategy for inhalable particle‑related chronic inflammatory and fibrotic lung disease.

Age-associated H3K9me2 loss alters the regenerative equilibrium between murine lung alveolar and bronchiolar progenitors

The lung contains multiple progenitor cell types, but how their responses are choreographed during injury repair and whether this changes with age is poorly understood. We report that histone H3 lysine 9 di-methylation (H3K9me2), mediated by the methyltransferase G9a, regulates the dynamics of distal lung epithelial progenitor cells and that this regulation deteriorates with age. In aged mouse lungs, H3K9me2 loss coincided with fewer alveolar type 2 (AT2) cell progenitors and reduced alveolar regeneration but increased the frequency and activity of multipotent bronchioalveolar stem cells (BASCs) and bronchiolar progenitor club cells. H3K9me2 depletion in young mice decreased AT2 progenitor activity and impaired alveolar injury repair. Conversely, H3K9me2 depletion increased chromatin accessibility of bronchiolar cell genes, increased BASC frequency, and accelerated bronchiolar cell injury repair. These findings indicate that during aging, the epigenetic regulation that coordinates lung progenitor cells' regenerative responses becomes dysregulated, aiding our understanding of age-related susceptibility to lung disease.

Angiotensin 1-7 Stimulates Proliferation of Lung Bronchoalveolar Progenitors-Implications for SARS-CoV-2 Infection.

SARS-CoV-2 infection leads to severe lung damage due to pneumonia and, in more severe cases, leads to acute respiratory distress syndrome, or ARDS. This affects the viability of bronchoalveolar cells. An important role in the pathogenesis of these complications is the hyperactivation of the renin-angiotensin-aldosterone (RAA) pathway and induction of cytokine storm that occurs in an Nlrp3 inflammasome-dependent manner. To shed more light on the susceptibility of lung tissue to SARS-CoV-2 infection, we evaluated murine bronchioalveolar stem cells (BASC), alveolar type II cells (AT2), and 3D-derived organoids expression of mRNA encoding genes involved in virus entry into cells, components of RAA, and genes that comprise elements of the Nlrp3 inflammasome pathway. We noticed that all these genes are expressed by lung alveolar stem cells and organoids-derived from these cells. Interestingly, all these cells express a high level of ACE2 that, on the one hand, serves as an entry receptor for SARS-CoV-2 and, on the other, converts angiotensin II into its physiological antagonist, angiotensin 1–7 (Ang 1–7), which has been reported to have a protective role in lung damage. To shed more light on the role of Ang 1–7 on lung tissue, we exposed lung-derived BASC and AT2 cells to this mediator of RAA and noticed that it increases the proliferation of these cells. Based on this, Ang 1–7 could be employed to alleviate the damage to lung alveolar stem/progenitor cells during SARS-CoV-2 infection.

Identification of Active Bronchioalveolar Stem Cells as the Cell of Origin in Lung Adenocarcinoma.

Identification of active bronchioalveolar stem cells as lung adenocarcinoma cells of origin provides insights into mechanisms of lung tumorigenesis and could facilitate development of effective strategies for cancer prevention and therapy. See related commentary by Osborne and Minna, p. 972.

Cellular Origins of EGFR-Driven Lung Cancer Cells Determine Sensitivity to Therapy.

Targeting the epidermal growth factor receptor (EGFR) with tyrosine kinase inhibitors (TKIs) is one of the major precision medicine treatment options for lung adenocarcinoma. Due to common development of drug resistance to first‐ and second‐generation TKIs, third‐generation inhibitors, including osimertinib and rociletinib, have been developed. A model of EGFR‐driven lung cancer and a method to develop tumors of distinct epigenetic states through 3D organotypic cultures are described here. It is discovered that activation of the EGFR T790M/L858R mutation in lung epithelial cells can drive lung cancers with alveolar or bronchiolar features, which can originate from alveolar type 2 (AT2) cells or bronchioalveolar stem cells, but not basal cells or club cells of the trachea. It is also demonstrated that these clones are able to retain their epigenetic differences through passaging orthotopically in mice and crucially that they have distinct drug vulnerabilities. This work serves as a blueprint for exploring how epigenetics can be used to stratify patients for precision medicine decisions.

Effect of High Fat Diet on the Severity and Repair of Lung Fibrosis in Mice.

Lung fibrosis is a progressive fatal disease, and the underlying mechanisms remain unclear. These involve a combination of altered fibroblasts, excessive accumulation of extracellular matrix, inflammation, and aberrant activation of epithelial cells. Previously, we showed that high-fat diet (HFD) induces lung inflammation, aberrant activation of stem cells, and lung mitochondria impairment. Therefore, we hypothesized that HFD-induced changes would influence lung fibrosis. Mice were fed standard diet (SD) or HFD, administered bleomycin, then examined for fibrosis severity and the start of repair 3 weeks after injury, and for fibrosis repair/resolution 6-9 weeks after injury. At 3 weeks, no significant differences in inflammation and fibrosis severity were observed between SD- and HFD-fed mice. However, infiltration of alveolar type (AT)-2 cells and bronchioalveolar stem cells (BASCs) into the fibrotic areas (the start of repair) was impaired in HFD-fed mice. At 6 weeks, SD-fed mice showed near-complete resolution/repair of fibrosis and inflammation, while HFD-fed mice still showed residual fibrosis and inflammation. Infiltration of the fibrotic areas with AT2 cells was observed, but very few BASCs were detectable. At 9 weeks, mice from both groups showed complete resolution/repair of fibrosis and inflammation, indicating that HFD induced delayed, rather than failed, resolution of fibrosis and alveolar repair. To further confirm the direct role of enhanced fatty-acid oxidation (FAO) in delayed resolution/repair, we administered etomoxir, a FAO inhibitor, to HFD-fed mice for 3-6 weeks after bleomycin injury. Inhibition of FAO abolished the HFD-induced delay in alveolar repair and fibrosis resolution at both time points. In conclusion, after a fibrosis-inducing injury, HFD slows resolution of fibrosis/inflammation and delays alveolar repair by slowing the contribution of AT2 stem cells and abolishing the contribution of BASCs in the repair process. FAO activation appears to be involved in this delay mechanism; thus, inhibiting FAO may be useful in the treatment of lung injury and fibrosis.

Bone Marrow-Derived VSELs Engraft as Lung Epithelial Progenitor Cells after Bleomycin-Induced Lung Injury.

Background: Alveolar type 2 (AT2) cells and bronchioalveolar stem cells (BASC) perform critical regenerative functions in response to lung damage. Published data show that nonhematopoietic, bone marrow-derived “very small embryonic-like stem cells” (VSELs) can differentiate in vivo into surfactant protein C (SPC)-producing AT2 cells in the lung. Here, we test directly whether VSEL-derived BASC and AT2 cells function to produce differentiated progeny. Methods: using a reporter mouse in which the H2B-GFP fusion protein is driven from the murine SPC promoter, we tested whether bone marrow-derived VSELs or non-VSEL/nonhematopoietic stem cells (non-VSEL/non-HSCs) can differentiate into AT2 and BASC cells that function as progenitor cells. Immediately following bleomycin administration, WT recipient mice underwent intravenous administration of VSELs or non-VSEL/non-HSCs from SPC H2B-GFP mice. GFP+ AT2 and BASC were isolated and tested for progenitor activity using in vitro organoid assays. Results: after 21 days in vivo, we observed differentiation of VSELs but not non-VSEL/non-HSCs into phenotypic AT2 and BASC consistent with previous data in irradiated recipients. Subsequent in vitro organoid assays revealed that VSEL-derived AT2 and BASC maintained physiological potential for differentiation and self-renewal. Conclusion: these findings prove that VSELs produce functional BASC and AT2 cells, and this may open new avenues using VSELs to develop effective cell therapy approaches for patients with lung injury.

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway.

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.

Prematurity negatively affects regenerative properties of human amniotic epithelial cells in the context of lung repair.

There is a growing appreciation of the role of lung stem/progenitor cells in the development and perpetuation of chronic lung disease including idiopathic pulmonary fibrosis. Human amniotic epithelial cells (hAECs) were previously shown to improve lung architecture in bleomycin-induced lung injury, with the further suggestion that hAECs obtained from term pregnancies possessed superior anti-fibrotic properties compared with their preterm counterparts. In the present study, we aimed to elucidate the differential effects of hAECs from term and preterm pregnancies on lung stem/progenitor cells involved in the repair. Here we showed that term hAECs were better able to activate bronchioalveolar stem cells (BASCs) and type 2 alveolar epithelial cells (AT2s) compared with preterm hAECs following bleomycin challenge. Further, we observed that term hAECs restored TGIF1 and TGFβ2 expression levels, while increasing c-MYC expression despite an absence of significant changes to Wnt/β-catenin signaling. In vitro, term hAECs increased the average size and numbers of BASC and AT2 colonies. The gene expression levels of Wnt ligands were higher in term hAECs, and the expression levels of BMP4, CCND1 and CDC42 were only increased in the BASC and AT2 organoids co-cultured with hAECs from term pregnancies but not preterm pregnancies. In conclusion, term hAECs were more efficient at activating the BASC niche compared with preterm hAECs. The impact of gestational age and/or complications leading to preterm delivery should be considered when applying hAECs and other gestational tissue-derived stem and stem-like cells therapeutically.

Bronchioalveolar stem cells derived from mouse-induced pluripotent stem cells promote airway epithelium regeneration

BackgroundBronchioalveolar stem cells (BASCs) located at the bronchioalveolar-duct junction (BADJ) are stem cells residing in alveoli and terminal bronchioles that can self-renew and differentiate into alveolar type (AT)-1 cells, AT-2 cells, club cells, and ciliated cells. Following terminal-bronchiole injury, BASCs increase in number and promote repair. However, whether BASCs can be differentiated from mouse-induced pluripotent stem cells (iPSCs) remains unreported, and the therapeutic potential of such cells is unclear. We therefore sought to differentiate BASCs from iPSCs and examine their potential for use in the treatment of epithelial injury in terminal bronchioles.MethodsBASCs were induced using a modified protocol for differentiating mouse iPSCs into AT-2 cells. Differentiated iPSCs were intratracheally transplanted into naphthalene-treated mice. The engraftment of BASCs into the BADJ and their subsequent ability to promote repair of injury to the airway epithelium were evaluated.ResultsFlow cytometric analysis revealed that BASCs represented ~ 7% of the cells obtained. Additionally, ultrastructural analysis of these iPSC-derived BASCs via transmission electron microscopy showed that the cells containing secretory granules harboured microvilli, as well as small and immature lamellar body-like structures. When the differentiated iPSCs were intratracheally transplanted in naphthalene-induced airway epithelium injury, transplanted BASCs were found to be engrafted in the BADJ epithelium and alveolar spaces for 14 days after transplantation and to maintain the BASC phenotype. Notably, repair of the terminal-bronchiole epithelium was markedly promoted after transplantation of the differentiated iPSCs.ConclusionsMouse iPSCs could be differentiated in vitro into cells that display a similar phenotype to BASCs. Given that the differentiated iPSCs promoted epithelial repair in the mouse model of naphthalene-induced airway epithelium injury, this method may serve as a basis for the development of treatments for terminal-bronchiole/alveolar-region disorders.

Bronchioalveolar stem cells in lung repair, regeneration and disease.

Bronchioalveolar stem cells (BASCs) are a lung resident stem cell population located at bronchioalveolar duct junctions that contribute to the maintenance of bronchiolar club cells and alveolar epithelial cells of the distal lung. Their transformed counterparts are considered to be likely progenitors of lung adenocarcinomas, which has been a major area of research in relation to BASCs. A critical limitation in addressing the function of BASCs in vivo has been the lack of a unique BASC marker, which has prevented specific targeting of BASCs in animal models of respiratory conditions. Recently, there have been several studies describing genetically modified mice that allow in vivo quantification, tracing, and functional analysis of BASCs to address this long-standing issue. These cutting-edge experimental tools will likely have significant implications for future experimental studies involving BASCs and the elucidation of their role in various lung diseases. To date, this has been largely explored in models of lung injury including naphthalene-induced airway injury, bleomycin-induced alveolar injury, hyperoxia-induced models of bronchopulmonary dysplasia, and influenza virus infection. These novel experimental mouse tools will facilitate the assessment of the impact of BASC loss on additional respiratory conditions including infection-induced severe asthma and chronic obstructive pulmonary disease, as well as respiratory bacterial infections, both in early life and adulthood. These future studies may shed light on the potential broad applicability of targeting BASCs for a diverse range of respiratory conditions during lung development and in promoting effective regeneration and repair of the lung in respiratory diseases. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Multilineage murine stem cells generate complex organoids to model distal lung development and disease

Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three‐dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self‐organization of FACS‐sorted bronchioalveolar stem cells (BASCs) upon co‐culture with lung‐resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single‐cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue‐resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial–mesenchymal‐myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro.

Abstract 5185: Squamous cell cancers and chronic obstructive pulmonary disease share polycomb repressive cComplex 2 dysregulation

Abstract Non-small cell lung cancers often share the aberrant differentiation patterns observed in the bronchiolar epithelium of chronic obstructive pulmonary disease (COPD), including squamous and mucinous phenotypes. Additionally, smokers with COPD are 3.5 times more likely to develop squamous lung cancer than smokers with pathologically normal lung epithelium. The Polycomb Repressive Complex 2 (PRC2), which contains the methyltransferase EZH2, mediates proper cell differentiation via tri-methylation of histone H3 at lysine 27 (H3K27me3), leading to epigenetic silencing of genomic regions. Our previous data demonstrate that squamous lung cancers often have low H3K27me3. Therefore, we hypothesized that loss of proper PRC2-mediated gene repression changes lung epithelial cell fate and drives squamous and mucinous phenotypes in COPD as well. To examine this hypothesis, we stained lung tissue from healthy patients and patients with COPD for H3K27me3 and observed that areas of squamous and mucinous metaplasia had a significantly decreased abundance of nuclear H3K27me3 stain. To demonstrate that this effect was due to loss of EZH2 activity, EZH2 transcripts were knocked down with short hairpins in human bronchiolar epithelial cells. When grown in air-liquid-interface cultures, shEZH2 cells had significantly more goblet cells and significantly fewer club cells than control cultures. We also tested this hypothesis in genetically engineered mice in which we could conditionally delete Ezh2. To examine growth, differentiation and self-renewal potentials of different lung epithelial cells, we FACS-isolated distal lung bronchioalveolar stem cells (BASCs) and cultured them as organoids. We observed a 2-fold decrease in organoid number of Ezh2 null vs wild type bronchiolar cells and a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. Organoids with squamous morphology were only seen in the Ezh2 null genotype. Lastly, we performed Gene Set Enrichment Analysis and found that genes up-regulated in human COPD patients relative to healthy smokers were also up-regulated in murine EZH2 knock-out adenocarcinoma cells. Together, these data suggest that loss of proper PRC2-mediated gene silencing is a shared epigenetic state in COPD and squamous lung cancers. Our laboratory is actively investigating the mechanisms through which EZH2 activity is lost in bronchiolar epithelium and plan to use our knowledge in attempt to ‘renormalize’ dysregulated epithelium to a more normal epigenetic state. Funded by American Cancer Society IRG-85-001-25, K22 CA20103 and Molecular Mechanisms of Toxicity Training Grant T32ES07266 Citation Format: Aria L. Byrd, Christine F. Brainson. Squamous cell cancers and chronic obstructive pulmonary disease share polycomb repressive cComplex 2 dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5185.

BASC-ing in the glow: bronchioalveolar stem cells get their place in the lung.

Lung epithelial progenitor cell populations fulfill the needs of this complex facultative regenerative organ when exposed to insult and contribute to repair in either the airway or alveolar compartments. The presence of a cell that can populate both epithelia had been proposed previously but has remained elusive. In this issue, Salwig et al (2019) provide compelling, genetic evidence that supports a growing narrative—a rare population of bronchioalveolar stem cells (BASCs) that can contribute to both airway and alveolar epithelium in the distal murine lung.

Bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia in vivo.

Bronchioalveolar stem cells (BASCs) are a potential source for lung regeneration, but direct invivo evidence for a multipotential lineage contribution during homeostasis and disease is critically missing, since specific genetic labeling of BASCs has not been possible. We developed a novel cell tracing approach based on intein-mediated assembly of newly engineered split-effectors, allowing selective targeting of dual-marker expressing BASCs in the mouse lung. RNA sequencing of isolated BASCs demonstrates that BASCs show a distinct transcriptional profile, characterized by co-expression of bronchiolar and alveolar epithelial genes. We found that BASCs generate the majority of distal lung airway cells after bronchiolar damage but only moderately contribute to cellular turnover under homeostatic conditions. Importantly, DTA-mediated ablation of BASCs compromised proper regeneration of distal airways. The study defines BASCs as crucial components of the lung repair machinery and provides a paradigmatic example for the detection and manipulation of stem cells that cannot be recognized by a single marker alone.

Loss of Polycomb Repressive Complex 2 impairs lung bronchiolar cell growth and underlies chronic obstructive pulmonary disease

EZH2, the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), is a histone methyl transferase that tri-methylates histone H3 at lysine 27 (H3K27me3). This protein plays a significant role in maintaining tissue specificity by silencing gene programs that control lineage fate. We hypothesized that PRC2 controls cell fate of the lung stem cells. In order to test this hypothesis, we generated mice in which we can conditionally delete Ezh2 upon ingestion of doxycycline. To examine growth, differentiation and self-renewal potentials of different lung epithelial cells, we FACS-isolated distal lung bronchioalveolar stem cells (BASCs) and cultured them in air-liquid-interface Matrigel cultures. We observed a 2fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells and a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. To test in vivo potential of EZH2 depleted epithelium to repair, we performed naphthalene injury, which specifically injures the bronchiolar club cells. We observed that 7 days post naphthalene injury, Ezh2 knock-out lungs had significantly less repair, as measured by repopulation of bronchiolar lung cells. Given that we observed that lung stem cells were impaired when PRC2-mediated gene repression was perturbed, we sought to learn if PRC2 is also dysregulated in Chronic Obstructive Pulmonary Disease (COPD). We first performed Gene Set Enrichment Analysis and found that genes up-regulated in COPD patients relative to healthy smokers were also up-regulated in Ezh2 knock-out adenocarcinoma cells. We next obtained lung tissue from healthy patients and patients with COPD, and performed immunohistochemistry for the PRC2 mark H3K27me3 and immunofluorescence for basal and club cells. There was a significant decrease in H3K27me3, coupled with an increase in basal cells and goblet cells that contained high expressions of club cell markers in COPD lung compared to healthy controls. Finally, to determine if loss of PRC2 function drives basal cell fate and goblet cell differentiation, EZH2 transcripts were knocked down with short hairpins in human bronchiolar epithelial cells, and basal cell differentiation capacity was analyzed. We observed an increased expression of a goblet cell marker and decreased expression of a club cell marker in EZH2 knockdown cultures. Together our results indicate that PRC2 plays an important role in lung stem cell function as well as club cell differentiation fate. These mechanisms may also be perturbed in COPD patients. ChIP-seq and RNA-seq studies to understand the transcriptional and epigenetic changes leading to these phenotypes are underway Support or Funding Information NIH grant K22 CA 20103, Molecular Mechanisms of Toxicity Training Grant T32ES07266 There was a 2-fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells. There was a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. Immunohistochemistry staining of EZH2 shows distinct differences in EZH2 expression between wild type, heterozygous and null organoids. Naphthalene exposure to mouse lung has been shown to kill club cells in the trachea and bronchioles. This injury typically results in club cell regeneration and subsequent lung cell repopulation. Club cells without EZH2 were unable to regenerate and repopulate the airway after naphthalene injury. Club cells that did regenerate in the Ezh2-null lungs lacked the GFP tag, indicating that these cells were likely not responding to doxycycline to delete Ezh2. Gene Set Enrichment Analysis was used to compare gene expression between murine Ezh2-deleted adenocarcinoma cells and COPD patients. It revealed that a significant number of the same genes are highly expressed in both states when compared to healthy lungs or the Ezh2 WT setting. Immunohistochemistry shows significantly lower abundance of H3K27me3 in the nuclei of COPD bronchiolar cells compared to normal. Immunofluorescence staining of club (CCSP and SOX2) and basal cells (KRT5 and SOX2) in normal vs COPD lung airway shows a mixture of metaplasia and hyperplasia along with collapsed airways in COPD airway compared to normal. There is an increase in basal cells and goblet cells that also contain high expressions of CCSP (a club cell marker). EZH2 transcripts were knocked down in immortalized, Human Bronchiolar Epithelial Cells (HBEC) which were, then, prompted to differentiate in air-liquid-interface cultures. Acetylated-Mucin5 was highly expressed while club cell secretory protein (CCSP) was lowly expressed in EZH2 knockdown cells compared to wild type. This suggest that EZH2 plays a role in club and goblet cell differentiation. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bronchioalveolar stem cells vindicated!

Fourteen years ago, Kim et al . (1) reported the existence of bronchioalveolar stem cells (BASCs) in the lung. These cells expressed both secretoglobin family 1A member 1 (Scgb1a1) and surfactant protein c (Sftpc), the differentiation marker of club cells and alveolar epithelial type 2 cells, respectively.

Lung regeneration by multipotent stem cells residing at the bronchioalveolar-duct junction.

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.

Abstract 1983: Enriching EpCAM+/Sca-1+ mouse bronchioalveolar stem cells to generate lung adenocarcinoma

Abstract Background: Clarifying the cells of origin in human lung adenocarcinoma will contribute to a better understanding of oncogene-induced lung adenocarcinomas and to the development of new treatments. Alveolar type II cells and club cells (Clara cells) have been shown to be the cells of origin by lineage-tracing approaches and conditional transgenic mice. Additionally, bronchioalveolar stem cells (BASCs), which are adult murine distal lung epithelial stem cells, are considered to be the cells of origin in lung adenocarcinomas because BASCs have both self-renewal abilities and differentiation properties. We attempted to enrich BASCs from mouse lungs and determine whether or not BASCs have the potential to form tumors driven by the oncogene. Methods: The epithelial cell adhesion molecule (EpCAM)+/CD45-/CD31- lung cells were isolated from the Ink4a/Arf-/- C57BL/6 mice by using a fluorescence activated cell sorting (FACS). The cells were then subjected to 3D culture on Matrigel with B27, EGF, KGF, ROCK inhibitor in serum-free DMEM/F-12. For detection of BASCs, we investigated the expressions of EpCAM and Sca-1 by flow cytometric analyses. We also investigated the expressions of SP-C and CC-10 by immunofluorescence analyses. The cells were then infected with bicistronic retroviruses having green fluorescent protein (GFP) and either KRASG12V or EML4-ALK genes. The GFP-positive oncogene-expressing cells were sorted by FACS and expanded under 3D culture conditions. Cells were then transplanted into the recipient C57BL/6 mice so that we could investigate their tumorigenic activities. Results: Flow cytometric analyses revealed that the EpCAM+/Sca-1+ cells, which are putative BASCs, were enriched in the 3D culture of the isolated EpCAM+/CD45-/CD31- lung cells from Ink4a/Arf-/- mice. The percentage of EpCAM+/Sca-1+ cell fraction was 36.97%, 92.63%, 99.98%, at primary EpCAM+/CD45-/CD31- lung cells and after the first and second passage, respectively. We also detected a small population of SP-C+/CC-10+ cells in colonies formed under our 3D culture conditions. The oncogene-expressing cells maintained the population of EpCAM+/Sca-1+ cells; 88.64-99.89% and 72.72-99.12%, KRASG12V and EML4-ALK, respectively. These cells formed differentiated tumors which were similar to human lung adenocarcinomas, having papillary, acinar, and solid components. Conclusions: The EpCAM+/Sca-1+ lung cells, putative BASCs, from Ink4a/Arf-/- mice were enriched in 3D culture. These cells formed histologically different types of lung adenocarcinomas after the transduction of KRASG12V or EML4-ALK, suggesting that we had established the source of the lung adenocarcinoma cells. Citation Format: Takashi Semba, Ryo Sato, Akiyoshi Kasuga, Makoto Suzuki, Hideyuki Saya, Yoshimi Arima. Enriching EpCAM+/Sca-1+ mouse bronchioalveolar stem cells to generate lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1983.