Lung Stem Cells Research Articles

Calorie restriction enhances adult mouse lung stem cells function and reverses several ageing-induced changes.

Ageing is associated with decreased lung function and an increased incidence of lung infections. Several studies have suggested that long-term calorie restriction (CR) promotes health and longevity and results in the reduced risk of several diseases. The effect of CR is thought to be through improving the function of tissue stem cells. Stem cell function is known to decline with ageing. In this study, we examined the effects of ageing on lung epithelial and stem cells and the effect of CR on young and old lungs. We found that ageing results in a decrease in tracheal basal stem cells. CR induced an increase in basal stem cells in both young and old mice. In addition, ageing induced lung inflammation, and CR tended to reduce baseline lung inflammatory cell infiltration in young mice and significantly reduced ageing-induced lung inflammation. Furthermore, ageing reduced the number and function of mitochondria in lung and increased the level of mitochondrial reactive oxygen species. CR increased the number and function of mitochondria both in young and old mice. Moreover, ageing reduced lung stem cell colony-forming efficiency (CFE), and CR increased the CFE in both young and old mice. Finally, CR improved epithelial cell survival in injured lungs of young mice. In conclusion, ageing causes several structural and functional changes/impairments in lung epithelial cells. CR induces several potentially beneficial changes in lung epithelial cells, even when it is initiated at an older age, including reversal of some ageing-induced changes.

Retraction: Kajstura J et al. Evidence for Human Lung Stem Cells. N Engl J Med 2011;364:1795-806.

We have been contacted by several authors of the article by Kajstura J et al., Evidence for Human Lung Stem Cells, N Engl J Med 2011;364:1795-1806.1 An investigation by Harvard Medical School and Brigham and Women’s Hospital has determined that some of the images in the article were manipulated. The authors tell us that “a review of the original data affirms this conclusion and has led us to conclude that we no longer have faith in the veracity of these images. As we were not aware of this manipulation of the data until long after the paper had been published, .

High fat diet activates adult mouse lung stem cells and accelerates several aging-induced effects

High fat diet (HFD) decreases the lifespan of mice, and is a risk factor for several human diseases. Here, we investigated the effects of a HFD on lung epithelial and stem cells and its interaction with aging. Young and old mice were fed with either a standard diet (SD) or a HFD then their trachea and lung were examined for histological changes, inflammation, and mitochondrial function. Their stem cell function was examined using the in vitro organoid/colony forming efficiency (CFE) assay. Aging reduced the number of tracheal basal and alveolar type-2 (AT2) cells. HFD significantly increased the number of AT2 cells. Aging also caused a significant increase in lung inflammation, and HFD caused a similar increase, in young mice. Aging reduced mitochondrial mass and function, and increased reactive oxygen species. In young mice, HFD caused mitochondrial changes similar to the aging-induced changes. Organoid culture of tracheal and lung epithelial cells collected from both young and old HFD-fed mice showed higher CFE compared to SD-fed mice. Switching the HFD to low calorie/fat diet (LCD) efficiently reversed several of the HFD-induced effects. Thus, HFD induces several histological, inflammatory, and functional changes in the lung, and exacerbates the aging-induced lung inflammation and mitochondrial deterioration. LCD can reverse many of the HFD-induced effects.

MS22.01 Lung Development and Stem Cells

MS22.01 Lung Development and Stem Cells

Characterization of distal airway stem-like cells expressing N-terminally truncated p63 and thyroid transcription factor-1 in the human lung

Distal airway stem cells (DASCs) in the mouse lung can differentiate into bronchioles and alveoli. However, it remains unclear whether the same stem cells exist in the human lung. Here, we found that human lung epithelial (HuL) cells, derived from normal, peripheral lung tissue, in monolayer, mostly express both the N-terminally truncated isoform of p63 (∆Np63), a marker for airway basal cells, and thyroid transcription factor-1 (TTF-1), a marker for alveolar epithelial cells, even though these two molecules are usually expressed in a mutually exclusive way. Three-dimensionally cultured HuL cells differentiated to form bronchiole-like and alveolus-like organoids. We also uncovered a few bronchiolar epithelial cells expressing both ∆Np63 and TTF-1 in the human lung, suggesting that these cells are the cells of origin for HuL cells. Taken together, ΔNp63+ TTF-1+ peripheral airway epithelial cells are possibly the human counterpart of mouse DASCs and may offer potential for future regenerative medicine.

Designing next generation platforms for recombinant protein production by genome engineering of Escherichia coli

The epithelial cells lining the mammalian lung are subjected to constant interaction with the external environment, necessitating robust regeneration strategies to deal with cell loss due to natural turnover or damage arising from inhaled agents or disease. Since lung epithelial function extends beyond respiratory gas exchange to include roles such as immune defense and mucociliary clearance, a diverse complement of epithelial cell types exists that are regionally distributed along the respiratory tree and extensive surface area of the alveolar interface. Although steady-state turnover of the epithelium appears to be relatively low in ideal situations, the vital role of the lung requires stem and progenitor cell populations that can promptly respond to the loss or damage of epithelial tissues. The identity and role of stem cell populations that carry out repair and replacement in the lung has begun to be clarified in recent years, led by cell lineage tracking experiments in the mouse lung, which have revealed a complex interplay of differentiation, transdifferentiation, and dedifferentiation between lung stem cells and functional respiratory cell populations. In this review article, we present the current understanding of the stem cell populations within the pulmonary epithelium and describe ongoing efforts to use these stem cell populations to generate models for exploring lung function and disease.

A basic study on self-reconstitution of alveolar epithelium-like cells by tissue stem cells in mouse lung.

In recent research on regenerative medicine, three-dimensional (3D) tissue reconstruction using the induced pluripotent stem cell (iPS cell) differentiated cells has attracted attention. In this study, mouse lungs at 1.5, 10, and 20d old were subjected to enzyme treatment, and aggregates formed in serum-free suspension culture (3D-culture) were observed. The number of aggregates formed was the highest in 1.5d. The cell aggregates in which the interior of the aggregate is filled and form small vacuoles and the organoid-like aggregates having a relatively large vacuole inside and forming the alveolar-like structure were observed. At 1.5d, the formation ratio of the organoid-like aggregates was the highest and aggregate size was small at 20d. For the cell aggregates derived from 1.5d, positive cells of SSEA-1, CD29, CD90, CD105, alveolar epithelial stem cell marker of SP-C, and Sca-1 were observed in the center. In the cell aggregates derived from 10d, the expression level of 1.5d each protein markers and OCT4 gene of transcription factor was decreased, and furthermore, markers were hardly observed in the organoid-like aggregates derived from 10d. In addition, cells surrounding the vacuole of organoid-like aggregate obtained over 10d differentiated into periodic acid-Schiff (PAS), podoplanin-positive cells. When the formed cell aggregates were dispersed, cell aggregates and organoid-like aggregates were reformed. Comparing 3D-culture and adhesion culture (2D-culture), SP-C expression of 10d of cells was maintained. Expression of markers of undifferentiated markers and alveolar tissue stem cells decreased when cell aggregates were cultured with the addition of fetal bovine serum.

Characterization, localization and comparison of c-Kit+ lung cells in never smokers and smokers with and without COPD

Backgroundc-Kit + lung stem cells have been described in the human healthy lung. Their potential relation with smoking and/or chronic obstructive pulmonary disease (COPD) is unknown.MethodsWe characterized and compared c-Kit+ cells in lung tissue of 12 never smokers (NS), 15 smokers with normal spirometry (S) and 44 COPD patients who required lung resectional surgery. Flow cytometry (FACS) was used to characterize c-Kit+ cells in fresh lung tissue disaggregates, and immunofluorescence (IF) for further characterization and to determine their location in OCT- embedded lung tissue.ResultsWe identified 4 c-Kit+ cell populations, with similar proportions in NS, S and COPD: (1) By FACS, c-Kithigh/CD45+ cells (4.03 ± 2.97% (NS), 3.96 ± 5.30% (S), and 5.20 ± 3.44% (COPD)). By IF, these cells were tryptase+ (hence, mast cells) and located around the airways; (2) By IF, c-Kitlow/CD45+/triptase- (0.07 ± 0.06 (NS), 0.03 ± 0.02 (S), and 0.06 ± 0.07 (COPD) cells/field), which likely correspond to innate lymphoid cells; (3) By FACS, c-Kitlow/CD45-/CD34+ (0.95 ± 0.84% (NS), 1.14 ± 0.94% (S) and 0.95 ± 1.38% (COPD)). By IF these cells were c-Kitlow/CD45-/CD31+, suggesting an endothelial lineage, and were predominantly located in the alveolar wall; and, (4) by FACS, an infrequent c-Kitlow/CD45-/CD34- population (0.09 ± 0.14% (NS), 0.08 ± 0.09% (S) and 0.08 ± 0.11% (COPD)) compatible with a putative lung stem cell population. Yet, IF failed to detect them and we could not isolate or grow them, thus questioning the existence of c-Kit+ lung stem-cells.ConclusionsThe adult human lung contains a mixture of c-Kit+ cells, unlikely to be lung stem cells, which are independent of smoking status and/or presence of COPD.

Tropism, replication competence, and innate immune responses of influenza virus: an analysis of human airway organoids and ex-vivo bronchus cultures

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. 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. 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. 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. US National Institute of Allergy and Infectious Diseases, Research Grants Council of the Hong Kong Special Administrative Region.

PO-293 Withaferin-A: a candidate anti-tumour natural compound for non-small cell lung cancer and stem cells

IntroductionWithaferin-A(WFA) is a natural compound extracted from Wihania somnifera plant which showed tumour growth inhibiting effects in both in vitro and in vivo models.of various cancers such as colorectal cancer, ovarian cancer, breast cancer, gastric cancers, glioma and hepatocellular carcinoma. Current commonly used therapeutic agents such as cisplatin (CDDP) are effective on cancer cells however there is a lack of efficiency on cancer stem cells (CSCs) which are considered to be responsible of recurrence and metastasis. Although anti-proliferative effect of WFA on non-small cell lunger cancer (NSCLC) cells was shown in previous studies, its effect on NSCLC CSCs remains unclear. Based on this fact, the aim of this study was to evaluate the in vitro effect of WFA on CD133 +NSCLC CSCs.Material and methodsA549 NSCLC cell line was incubated in DMEM supplemented with 10% FBS, 1% L-Glutamine, 1% Penicilline/Streptomycin at 5% CO2 and 37°C. Cells were incubated with WFA 24, 48, 72 hours of 2.5,10,20 and 50 µM WFA and 75,100 µM CDDP and their combinations. Cell viability was determined by the WST-1 assay.CD133+ A549 cells were isolated with magnetic beads. The purity of CD133 positivity was assessed by flow cytometry. A549 cells and CD133+ A549 cells were treated with 50 µM WFA, 100 µM CDDP and incombination for 24 hours. Apoptosis was evaluated by flow cytometry with annexin V and PI staining. Furthermore DNA damage and cell proliferation of CD133+ A549 cells were determined by flow cytomerty with BrdU and H2AX stainings.Results and discussionsWFA caused 18,6% early apoptosis (EA), 36,2% late apoptosis (LA), 56% anti-proliferation while CDDP caused 34,3% EA, 2,5% LA,%31,3% anti-proliferation and WFA+CDDP caused 45,6% EA, 34,5% LA 63,8% anti-proliferation on A549 cells. Also WFA 21,6% EA 38,3% LA, 6,6% DNA damage while CDDP caused 30% EA, 4,4% LA, 7,0% DNA damage and WFA+CDDP caused 21,9% EA, 42,9% LA,6,1% anti-proliferation on A549 CSCs.ConclusionWFA showed anti-proliferative and apoptosis inducing effects of both NSCLCs and their CD133 +CSCs. In addition WFA did not change anti-tumour effect of CDDP and their combination showed more anti-proliferative and apoptosis inducing effect than their administration alone. In addition, WFA caused DNA damage in NSCLC CSCs. Although CDDP has critical anti-tumour effects, it might lead to adverse effects such as ototocity, neprotoxicity, peripheral neuropathy. Therefore, systemic effects of the natural compound WFA are suggested to be evaluated in animal models in future studies.

Abstract 2003: P38α suppresses tumor initiating cell phenotypes through inhibition of NFATc4 in non-small cell lung cancer

Abstract Lung cancer development is often mediated through oxidative and inflammatory stresses caused by tobacco and environmental carcinogens, suggesting dysregulation of stress mediators might play a role during carcinogenesis. The p38 MAPK pathway is activated by multiple stress stimuli and inflammatory cytokines, and mediates a wide range of cellular processes in a context-specific manner. P38α, a member of the p38 family, is known to regulate the self-renewal and differentiation of adult lung stem cells. While p38α has been reported to mediate tumor suppressive or supportive roles in different cancers, inactivation of p38α in the lung epithelium is involved in KRASG12V induced tumorigenesis. However, whether p38α could be involved in regulating tumor initiating cells (TIC) of lung cancer remains unclear. To investigate this possibility, immunohistochemistry was first used to analyse the nuclear expression of phosphorylation-activated p38α (p-p38α) in 90 human primary lung adenocarcinomas. Results showed higher expression of p-p38α was significantly correlated with longer progression free and overall survivals, as well as a more differentiated tumor histological phenotype, indicating p38α might play a tumor suppressive role and drive tumor differentiation in lung cancer. To further address its functional role on lung TIC, p38α was suppressed by siRNA or pharmacologically inhibited by SB203580. This led to increase of the TIC subset that coexpresses ALDH and CD44 (ALDH+/CD44+-TIC), as well as up-regulated expression of the pluripotency genes SOX2, OCT4, and NANOG in lung cancer cell lines. Functionally, p38α inhibition by SB203580 promoted tumor spheres formation for two consecutive generations. NFAT is a family of transcription factors reported to regulate TIC in various cancers which are activated via de-phosphorylation leading to nuclear translocation. We hypothesize the TIC supportive function of p38α inhibition might be mediated through loss of phosphorylation and subsequent activation of NFATc4. In line with this hypothesis, we showed, using Western blot, p38α inhibition increased the expression of nuclear NFATc4. Moreover, NFATc4 knockdown led to suppressed sphere formation and pluripotency genes expression in lung cancer cell lines. Together, our results indicated p38α mediates a tumor suppressor pathway through downstream NFATc4 inactivation and TIC inhibition. The p38α -NFATc4 stress pathway might serve as a potential target for long term lung cancer treatment through TIC regulation. Citation Format: Zhi-Jie Xiao, Jing Liu, Si-Qi Wang, Xu-Yuan Gao, Vicky Pui-Chi Tin, Maria Pik Wong. P38α suppresses tumor initiating cell phenotypes through inhibition of NFATc4 in non-small cell lung cancer [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 2003.

PO-107 Notch signalling pathway is required for proliferation, repair and differentiation of primary human lung stem cells upon irradiation

IntroductionLung cancer is the leading cause of cancer death in western countries. While significant progress has been made in terms of treatment, radiotherapy is limited by dose-limiting side-effects. Reducing side-effects may improve tumour control by dose-escalation and treatment-time. The NOTCH signalling pathway plays an important role in differentiation of the airway epithelium and its deregulation is associated with lung cancer. However, the mechanism by which NOTCH inhibition integrates with airway repair is unknown. What is currently lacking are primary human lung tissue models that enable robust evaluation of the effects of treatment which can guide the selection of combination treatments for lung cancer.Material and methodsWe therefore investigated the effects of inhibiting NOTCH in human basal stem cells from normal primary bronchial epithelial cells isolated from lung cancer patients undergoing lobectomy. Basal stem cells isolated from 3 different patients were treated with the pan NOTCH inhibitor (DBZ) and irradiated with 2–4 Gy. Incucyte experiment, Edu/PI staining and clonogenic assay were used to evaluate proliferation and clonal capacity. To evaluate DNA damage response 53 bp1 staining was performed. To study Notch inhibition in basal cells differentiation the tracheal epithelium of 2–5 Gy irradiated mice was seeded in ALI system and confocal stainings were performed.Results and discussionsIncucyte experiment revealed a reduced proliferation of stem cells upon NOTCH inhibition which is further reduced upon irradiation. NOTCH inhibition induces accumulation of the cells in GO-G1 phase and a significant reduction in S phase which was exacerbated upon irradiation. The reduction in proliferation also led to a reduction in long term survival. We observed activation of the DNA damage response pathway by expression of pCHK2, pATM upon NOTCH inhibition which was enhanced by radiation. 53 BP1 foci 24 hours after RT was higher when NOTCH was inhibited demonstrating that NOTCH is required for the DNA damage response. In cultured primary stem cells from irradiated murine tracheas NOTCH inhibition induced a fate change by blocking mucous differentiation and inducing cillilary differentiation and enhancing progenitor p63 +cells. However, overall proliferation was reduced.ConclusionNOTCH pathway is needed for stem cell survival and DNA damage response upon irradiation of undifferentiated progenitors. These studies underscore the importance of studying the effect of targeted anti-cancer drugs for normal tissue effects.

Abstract PR11: Investigating lung cancer cells-of-origin using three-dimensional organoid cultures

Abstract There are many distinct epithelial cell types in the adult lung that could give rise to non-small cell lung cancer. These cell types include tracheal basal stem cells, tracheal club cells, distal lung bronchioalveolar stem cells (BASCs), and distal lung alveolar type 2 cells (AT2). My laboratory is using mouse models to test directly which lung stem and progenitor cells are most fit to serve as cells-of-origin of lung adenocarcinoma and lung squamous cell carcinoma. Previously, we demonstrated that tracheal club cells and BASCs are the likely cells-of-origin for KRASG12D; Lkb1-null adenosquamous tumors. The same strategy is now being used to test which cells can give rise to adenocarcinomas using the LSL:EGFRT90M/L858R (LSL=lox-stop-lox) mouse strain, or squamous cell carcinoma using the Lkb1flox/flox; Ptenflox/flox mouse strain. Briefly, tracheas and lungs from Cre-naïve mice are enzymatically dissociated, and fluorescence activated cell sorting (FACS) is used to enrich for four distinct populations: NGFR+/Sca1+ tracheal basal cells, NGFR-/Sca1+ tracheal club cells, Sca1+ distal lung BASCs, and Sca1- distal lung AT2 cells. Each population is then incubated with either adeno-GFP control virus or adeno-Cre virus to induce EGFR T790M/L858R expression or delete both alleles of Lkb1 and Pten. Three dimensional air-liquid-interface cultures are then used to expand the populations as organoids. For EGFR organoids, both basal and club cells give rise to larger, faster-growing bronchiolar organoids after EGFR is induced. AT2 cells similarly give rise to much larger alveolar organoids after EGFR induction, and BASCs change from giving rise to both bronchiolar and alveolar organoids to nearly exclusively alveolar. For Lkb1/Pten organoids, all four populations can tolerate deletion of the two genes, but it is only the basal cell and club cell organoids that progress to a squamous morphology in vitro. While both BASC and AT2 organoids continue to grow, they appear phenotypically normal. Transplant assays to assess malignancy and RNA-seq and ChIP-seq studies to explore gene transcription and epigenetic changes are under way. These experiments will shed light on the cells of the adult lung that give rise to genotype-specific tumors, and will aid us to better classify lung cancer heterogeneity from a cell-of-origin perspective. Citation Format: Kwok-Kin Wong, Carla Kim, Christine Fillmore Brainson. Investigating lung cancer cells-of-origin using three-dimensional organoid cultures [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr PR11.

Fluorescence Endomicroscopy Imaging of Mesenchymal Stem Cells in the Rat Lung.

Stem cell therapy has shown great promise for organ repair and regeneration. In the context of lung disease, such as radiation-induced lung damage (RILD) in cancer radiotherapy, mesenchymal stem cells (MSCs) have shown the ability to reduce damage possibly due to their immunomodulatory properties and other unknown mechanisms. However, once MSCs are transplanted into the body, little is known as to their localization or their mechanisms of action. In this work, we proposed, implemented, and validated a fluorescence endomicroscopy (FE) imaging technique that allows for the real-time detection and quantification of transplanted pre-labeled MSCs in vivo and tracking in a rat model. This protocol covers aspects related to MSCs extraction, labeling, FE imaging, and image analysis developed in a RILD rat model but applicable to other biological systems. © 2018 by John Wiley & Sons, Inc.

In silico selection approach to develop DNA aptamers for a stem-like cell subpopulation of non-small lung cancer adenocarcinoma cell line A549

BackgroundDetection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells.Materials and methodsThe human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection.ResultsThe obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences.ConclusionsThe novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.

Fibroblasts as lung stem cell niche

Stem Cells Each breath that we take provides oxygen to the bloodstream via tiny sacs in the lung called alveoli. AT1 cells line the alveoli and mediate gas exchange, whereas AT2 cells secrete lung surfactant. A subset of AT2s also serve as stem cells that slowly generate new alveolar cells throughout adult life. Nabhan et al. show that the rare AT2 stem cells have a special niche next to a fibroblast secreting Wnts. This Wnt activity is needed to select and maintain the stem cells. Injury expands the stem cell pool by transiently inducing autocrine Wnts in other surfactant-secreting alveolar cells. This simple but expandable niche sustains oxygen delivery, and it is co-opted in lung cancer. Science , this issue p. [1118][1] [1]: /lookup/doi/10.1126/science.aam6603

Claudin-18-mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis.

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells.

Alveoli, the lung's respiratory units, are tiny sacs where oxygen enters the bloodstream. They are lined by flat alveolar type 1 (AT1) cells, which mediate gas exchange, and AT2 cells, which secrete surfactant. Rare AT2s also function as alveolar stem cells. We show that AT2 lung stem cells display active Wnt signaling, and many of them are near single, Wnt-expressing fibroblasts. Blocking Wnt secretion depletes these stem cells. Daughter cells leaving the Wnt niche transdifferentiate into AT1s: Maintaining Wnt signaling prevents transdifferentiation, whereas abrogating Wnt signaling promotes it. Injury induces AT2 autocrine Wnts, recruiting "bulk" AT2s as progenitors. Thus, individual AT2 stem cells reside in single-cell fibroblast niches providing juxtacrine Wnts that maintain them, whereas injury induces autocrine Wnts that transiently expand the progenitor pool. This simple niche maintains the gas exchange surface and is coopted in cancer.

Stem cell therapies for chronic obstructive pulmonary disease: current status of pre-clinical studies and clinical trials.

Chronic obstructive pulmonary disease (COPD) is a respiratory disease that has a major impact worldwide. The currently-available drugs mainly focus on relieving the symptoms of COPD patients. Novel regenerative therapeutic approaches have been investigated with the aim of repairing or replacing the injured functional structures of the respiratory system. We summarized the progress made by regenerative therapies for COPD by analyzing results from both pre-clinical studies and completed clinical trials. These approaches include the application of exogenous stem cells or small molecules to stimulate the regeneration by endogenous lung stem/progenitor cells. Exogenous mesenchymal stem cells (MSCs) have been reported to repair the structure and improve the function of the injured respiratory system in COPD models. However, the studies that used MSCs in patients with moderate-to-severe COPD patients did not lead to clear respiratory functional improvements. Exogenous human lung stem cells applied to cryo-injured (CI) lungs of mice have been shown to organize into human-like pulmonary structures, indicating a new property of stem cells that is potentially capable of curing COPD patients. Small molecules like retinoic acid has been shown to lead to regeneration and repair of the damaged lung structures in COPD mouse models probably by activation of endogenous lung stem/progenitor cells. However, retinoic acid or agonists of retinoic acid receptor administered to moderate or severe COPD patients did not improve the density and function of the damaged lung. These novel regenerative approaches have failed in preliminary clinical trials, possibly due to the advanced severity of the disease. Further work should be done to develop the current regenerative approaches for curing patients at different stages of COPD. We suggest that some modifications of the approach in the clinical studies may lead to more successful outcomes of regenerative therapy for COPD.

Conditionally reprogrammed cells (CRC) methodology does not allow the in vitro expansion of patient-derived primary and metastatic lung cancer cells.

Availability of tumor and non-tumor patient-derived models would promote the development of more effective therapeutics for non-small cell lung cancer (NSCLC). Recently, conditionally reprogrammed cells (CRC) methodology demonstrated exceptional potential for the expansion of epithelial cells from patient tissues. However, the possibility to expand patient-derived lung cancer cells using CRC protocols is controversial. Here, we used CRC approach to expand cells from non-tumoral and tumor biopsies of patients with primary or metastatic NSCLC as well as pulmonary metastases of colorectal or breast cancers. CRC cultures were obtained from both tumor and non-malignant tissues with extraordinary high efficiency. Tumor cells were tracked in vitro through tumorigenicity assay, monitoring of tumor-specific genetic alterations and marker expression. Cultures were composed of EpCAM+ lung epithelial cells lacking tumorigenic potential. NSCLC biopsies-derived cultures rapidly lost patient-specific genetic mutations or tumor antigens. Similarly, pulmonary metastases of colon or breast cancer generated CRC cultures of lung epithelial cells. All CRC cultures examined displayed epithelial lung stem cell phenotype and function. In contrast, brain metastatic lung cancer biopsies failed to generate CRC cultures. In conclusion, patient-derived primary and metastatic lung cancer cells were negatively selected under CRC conditions, limiting the expansion to non-malignant lung epithelial stem cells from either tumor or non-tumor tissue sources. Thus, CRC approach cannot be applied for direct therapeutic testing of patient lung tumor cells, as the tumor-derived CRC cultures are composed of (non-tumoral) airway basal cells.