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Organoids have been an exciting advancement in stem cell research. Here we describe a strategy for directed differentiation of human pluripotent stem cells into distal lung organoids. This protocol recapitulates lung development by sequentially specifying human pluripotent stem cells to definitive endoderm, anterior foregut endoderm, ventral anterior foregut endoderm, lung bud organoids and finally lung organoids. The organoids take ~40 d to generate and can be maintained more than 180 d, while progressively maturing up to a stage consistent with the second trimester of human gestation. They are unique because of their branching morphology, the near absence of non-lung endodermal lineages, presence of mesenchyme and capacity to recapitulate interstitial lung diseases. This protocol can be performed by anyone familiar with cell culture techniques, is conducted in serum-free conditions and does not require lineage-specific reporters or enrichment steps. We also provide a protocol for the generation of single-cell suspensions for single-cell RNA sequencing.
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Enfermedades Pulmonares Intersticiales , Células Madre Pluripotentes , Virosis , Humanos , Pulmón , Organoides , Diferenciación CelularRESUMEN
Human lungs contain unique cell populations in distal respiratory airways (RAs). These populations accumulate in patients with lung injury, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Their lineage potentials and roles are unknown, however. As they are absent in rodents, deeper understanding of these cells requires a human in vitro model. Here we report the generation from human pluripotent stem cells (hPSCs) of expandable spheres (induced respiratory airway progenitors (iRAPs)) consisting of all RA-associated cell types. iRAPs could differentiate into type 1 (AT1) and type 2 alveolar (AT2) epithelial cells in defined conditions, showing that alveolar cells can be derived from RAs. iRAPs with deletion of HPS1, which causes pulmonary fibrosis in humans, display defects that are hallmarks of IPF, indicating involvement of intrinsic dysfunction of RA-associated cells in IPF. iRAPs thus provide a model to gain insight into human lung regeneration and into pathogenesis of IPF.
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There is mounting evidence that shows the association between chronic exposure to air pollutants (particulate matter and gaseous) and onset of various respiratory impairments. However, the corresponding toxicological mechanisms of mixed exposure are poorly understood. Therefore, in this study, we aimed to establish a repeated exposure setting for evaluating the pulmonary toxicological effects of diesel exhaust particles (DEP), nitrogen dioxide (NO2), and sulfur dioxide (SO2) as representative criterial air pollutants. Single, combined (DEP with NO2 and SO2), and repeated exposures were performed using physiologically relevant human bronchial mucosa models developed at the air−liquid interface (bro-ALI). The bro-ALI models were generated using human primary bronchial epithelial cells (3−4 donors; 2 replicates per donor). The exposure regime included the following: 1. DEP (12.5 µg/cm2; 3 min/day, 3 days); 2. low gaseous (NO2: 0.1 ppm + SO2: 0.2 ppm); (30 min/day, 3 days); 3. high gaseous (NO2: 0.2 ppm + SO2: 0.4 ppm) (30 min/day, 3 days); and 4. single combined (DEP + low gaseous for 1 day). The markers for pro-inflammatory (IL8, IL6, NFKB, TNF), oxidative stress (HMOX1, GSTA1, SOD3,) and tissue injury/repair (MMP9, TIMP1) responses were assessed at transcriptional and/ or secreted protein levels following exposure. The corresponding sham-exposed samples under identical conditions served as the control. A non-parametric statistical analysis was performed and p < 0.05 was considered as significant. Repeated exposure to DEP and single combined (DEP + low gaseous) exposure showed significant alteration in the pro-inflammatory, oxidative stress and tissue injury responses compared to repeated exposures to gaseous air pollutants. The study demonstrates that it is feasible to predict the long-term effects of air pollutants using the above explained exposure system.
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Secretoglobin family 1A member 1 (SCGB1A1) alternatively known as club cell protein 16 is a protective pneumo-protein. Decreased serum levels of SCGB1A1 have been associated with tobacco smoke induced chronic obstructive pulmonary disease (TS-COPD). Exposure to biomass smoke (BMS) is an important COPD risk factor among women in low and lower-middle income countries. Therefore, in a cross-sectional study (n = 50/group; total 200 subjects) we assessed serum SCGB1A1 levels in BMS-COPD subjects (11 male, 39 female) compared to TS-COPD (all male) along with TS-CONTROL (asymptomatic smokers, all male) and healthy controls (29 male, 21 female) in an Indian population. Normal and chronic bronchitis like bronchial mucosa models developed at the air-liquid interface using human primary bronchial epithelial cells (3 donors, and three replicates per donor) were exposed to cigarette smoke condensate (CSC; 0.25, 0.5, and 1%) to assess SCGB1A1 transcript expression and protein secretion. Significantly (p < 0.0001) decreased serum SCGB1A1 concentrations (median, interquartile range, ng/mL) were detected in both BMS-COPD (1.6; 1.3-2.4) and TS-COPD (1.8; 1.4-2.5) subjects compared to TS-CONTROL (3.3; 2.9-3.5) and healthy controls (5.1; 4.5-7.2). The levels of SCGB1A1 were positively correlated (r = 0.7-0.8; p < 0.0001) with forced expiratory volume in 1 s, forced vital capacity, their ratios, and exercise capacity. The findings are also consistent within the BMS-COPD sub-group as well. Significantly (p < 0.03) decreased SCGB1A1 concentrations were detected with severity of COPD, dyspnea, quality of life, and mortality indicators. In vitro studies demonstrated significantly (p < 0.05) decreased SCGB1A1 transcript and/or protein levels following CSC exposure. Circulating SCGB1A1 levels may therefore also be considered as a potent marker of BMS-COPD and warrant studies in larger independent cohorts.
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Limited toxicity data on electronic cigarette (ECIG) impede evidence-based policy recommendations. We compared two popular mixed fruit flavored ECIG-liquids with and without nicotine aerosolized at 40 W (E-smoke) with respect to particle number concentrations, chemical composition, and response on physiologically relevant human bronchial and alveolar lung mucosa models cultured at air-liquid interface. E-smoke was characterized by significantly increased particle number concentrations with increased wattage (25, 40, and 55 W) and nicotine presence. The chemical composition of E-smoke differed across the two tested flavors in terms of cytotoxic compounds including p-benzoquinone, nicotyrine, and flavoring agents (for example vanillin, ethyl vanillin). Significant differences in the expression of markers for pro-inflammation, oxidative stress, tissue injury/repair, alarm anti-protease, anti-microbial defense, epithelial barrier function, and epigenetic modification were observed between the flavors, nicotine content, and/ or lung models (bronchial or alveolar). Our findings indicate that ECIG toxicity is influenced by combination of multiple factors including flavor, nicotine content, vaping regime, and the region of respiratory tree (bronchial or alveolar). Toxic chemicals and flavoring agents detected in high concentrations in the E-smoke of each flavor warrant independent evaluation for their specific role in imparting toxicity. Therefore, multi-disciplinary approaches are warranted for comprehensive safety profiling of ECIG.
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Bronquios/citología , Marcadores Genéticos/efectos de los fármacos , Nicotina/efectos adversos , Alveolos Pulmonares/citología , Vapeo/efectos adversos , Bronquios/química , Bronquios/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Sistemas Electrónicos de Liberación de Nicotina , Aromatizantes/efectos adversos , Aromatizantes/química , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Biológicos , Tamaño de la Partícula , Alveolos Pulmonares/química , Alveolos Pulmonares/efectos de los fármacosRESUMEN
Diacetyl is an artificial flavouring agent, known to cause bronchiolitis obliterans. Diacetyl-induced pulmonary effects were assessed in human primary bronchial epithelial cells (PBEC) cultured at air-liquid interface (ALI). The PBEC-ALI models were exposed to clean air (sham) and diacetyl vapour (1, 3, 10 and 30â¯ppm) for 30â¯min. At 6 and 24â¯h post-exposure, cell medium was sampled for assessment of cytotoxicity measurement, and CXCL8, MMP9 secretion by ELISA. Pro-inflammatory, oxidative stress, tissue injury/repair, anti-protease and beta-defensin markers were assessed using qRT-PCR. Additionally, epidermal growth factor receptor ligands (amphiregulin) and anti-protease (SLPI) were analysed at 6â¯h, 8â¯h and 24â¯h post exposure to 1 and 10â¯ppm diacetyl. No significant cytotoxicity was observed at any exposure level. MMP9 was significantly increased in both apical and basal media at 24â¯h. Both SLPI and amphiregulin secretion were significantly increased following exposure to 10â¯ppm diacetyl. Exposure of PBEC-ALI model to diacetyl vapour resulted in significantly altered transcript expression of pro-inflammatory, oxidative stress, anti-protease, tissue injury/repair markers. Changes in transcript expression of significantly altered markers were more prominent 24â¯h post-exposure compared to 6â¯h. This study warrants further mechanistic investigations to elucidate the pulmonary effects of inhaled diacetyl vapour using physiologically relevant in vitro models.
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Bronquios/citología , Diacetil/toxicidad , Células Epiteliales/efectos de los fármacos , Aromatizantes/toxicidad , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Humanos , Ligandos , Metaloproteinasa 9 de la Matriz/genética , Modelos Biológicos , FN-kappa B/metabolismoRESUMEN
BACKGROUND: Failure to attain peak lung function by early adulthood is a risk factor for chronic lung diseases. Previously, we reported that C3H/HeJ mice have about twice total lung capacity (TLC) compared to JF1/MsJ mice. We identified seven lung function quantitative trait loci (QTL: Lfnq1-Lfnq7) in backcross/intercross mice derived from these inbred strains. We further demonstrated, superoxide dismutase 3, extracellular (Sod3), Kit oncogene (Kit) and secreted phosphoprotein 1 (Spp1) located on these Lfnqs as lung function determinants. Emanating from the concept of early origin of lung disease, we sought to identify novel candidate genes for pulmonary function by investigating lung transcriptome in C3H/HeJ and JF1/MsJ mice at the completion of embryonic development, bulk alveolar formation and maturity. METHODS: Design-based stereological analysis was performed to study lung structure in C3H/HeJ and JF1/MsJ mice. Microarray was used for lung transcriptomic analysis [embryonic day 18, postnatal days 28, 70]. Quantitative real time polymerase chain reaction (qRT-PCR), western blot and immunohistochemical analysis were used to confirm selected differences. RESULTS: Stereological analysis revealed decreased alveolar number density, elastin to collagen ratio and increased mean alveolar volume in C3H/HeJ mice compared to JF1/MsJ. Gene ontology term "extracellular region" was enriched among the decreased JF1/MsJ transcripts. Candidate genes identified using the expression-QTL strategy include: ATP-binding cassette, sub-family G (WHITE), member 1 (Abcg1), formyl peptide receptor 1 (Fpr1), gamma-aminobutyric acid (GABA) B receptor, 1 (Gabbr1); histocompatibility 2 genes: class II antigen E beta (H2-Eb1), D region locus 1 (H2-D1), and Q region locus 4 (H2-Q4); leucine rich repeat containing 6 (testis) (Lrrc6), radial spoke head 1 homolog (Rsph1), and surfactant associated 2 (Sfta2). Noteworthy genes selected as candidates for their consistent expression include: Wnt inhibitor factor 1 (Wif1), follistatin (Fst), chitinase-like 1 (Chil1), and Chil3. CONCLUSIONS: Comparison of late embryonic, adolescent and adult lung transcript profiles between mouse strains with extreme TLCs lead to the identification of candidate genes for pulmonary function that has not been reported earlier. Further mechanistic investigations are warranted to elucidate their mode of action in determining lung function.
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Perfilación de la Expresión Génica/métodos , Estudios de Asociación Genética/métodos , Pulmón/fisiología , Capacidad Pulmonar Total/genética , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C3H , Pruebas de Función Respiratoria/métodos , Especificidad de la EspecieRESUMEN
Superoxide dismutase 3, extracellular (SOD3) polymorphisms have been implicated in reduced pulmonary function development and altered risk for chronic obstructive pulmonary disease. We previously reported that gene-targeted Sod3-/- mice have impaired lung function and human SOD3 variants are associated with reduced pulmonary function in children. Reduced lung SOD3 levels were reported in mice with lower lung function with the greatest difference occurring during alveogenesis phase [postnatal (P) days 14-28]. Interactions between homeobox (HOX), wingless-type MMTV integration site member (WNT), and fibroblast growth factor (FGF) signaling govern complex developmental processes in several organs. A subset of HOX family members, HOXA5 and HOXB5, is expressed in the developing lung. Therefore, in this study we assessed the transcript expression of these family members and their downstream targets in Sod3-/- mice during alveogenesis (P14). In the lung of Sod3-/- mice, Hoxa5 and Hoxb5 increased. These transcription factors regulate WNT gene expression and were accompanied by increases in their downstream targets Wnt2 and Wnt5A, canonical and noncanonical WNT members, respectively. The WNT signaling target, lymphoid enhancer binding factor 1 (Lef1), also increased along with its downstream targets Fgf2, Fgf7, and Fgf10 in the lungs of Sod3-/- mice. Due to limited knowledge on the role of FGF2 in lung development, we further examined FGF2 protein and found increased levels in the bronchial and alveolar type II epithelial cells of Sod3-/- mice compared to age-matched controls. Thus, our findings suggest that deficient management of extracellular superoxide can lead to altered lung developmental signaling during alveogenesis in mice.
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Factor 2 de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/genética , Proteínas de Homeodominio/genética , Alveolos Pulmonares/crecimiento & desarrollo , Superóxido Dismutasa/genética , Vía de Señalización Wnt/genética , Animales , Proteína Axina/genética , Proteínas Dishevelled/genética , Células Epiteliales/metabolismo , Femenino , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Receptores Frizzled/genética , Regulación de la Expresión Génica , Factor de Unión 1 al Potenciador Linfoide/genética , Ratones , Ratones Noqueados , Fosfoproteínas/genética , Superóxido Dismutasa/deficiencia , Superóxido Dismutasa/metabolismo , Factores de Transcripción , Transcripción Genética , Proteína Wnt-5a/genética , Proteína wnt2/genéticaRESUMEN
Secreted phosphoprotein 1 (Spp1) is located within quantitative trait loci associated with lung function that was previously identified by contrasting C3H/HeJ and JF1/Msf mouse strains that have extremely divergent lung function. JF1/Msf mice with diminished lung function had reduced lung SPP1 transcript and protein during the peak stage of alveologenesis (postnatal day [P]14-P28) as compared with C3H/HeJ mice. In addition to a previously identified genetic variant that altered runt-related transcription factor 2 (RUNX2) binding in the Spp1 promoter, we identified another promoter variant in a putative RUNX2 binding site that increased the DNA protein binding. SPP1 induced dose-dependent mouse lung epithelial-15 cell proliferation. Spp1((-/-)) mice have decreased specific total lung capacity/body weight, higher specific compliance, and increased mean airspace chord length (Lm) compared with Spp1((+/+)) mice. Microarray analysis revealed enriched gene ontogeny categories, with numerous genes associated with lung development and/or respiratory disease. Insulin-like growth factor 1, Hedgehog-interacting protein, wingless-related mouse mammary tumor virus integration site 5A, and NOTCH1 transcripts decreased in the lung of P14 Spp1((-/-)) mice as determined by quantitative RT-PCR analysis. SPP1 promotes pneumocyte growth, and mice lacking SPP1 have smaller, more compliant lungs with enlarged airspace (i.e., increased Lm). Microarray analysis suggests a dysregulation of key lung developmental transcripts in gene-targeted Spp1((-/-)) mice, particularly during the peak phase of alveologenesis. In addition to its known roles in lung disease, this study supports SPP1 as a determinant of lung development in mice.