Modeling Chronic Obstructive Pulmonary Bronchopathy (COPD) by Induced Pluripotent Cells (iPS)

La Bronchopneumopathie Chronique Obstructive (BPCO), 3ème cause de mortalité dans le monde touche 4 à 16% de la population en France. L'âge, le tabac, les épisodes respiratoires dans la petite enfance en sont les principaux facteurs de risque. Les mécanismes physiopathologiques restent incompris du fait des différentes trajectoires de la maladie notamment pédiatrique. Les modèles actuels utilisés pour mimer la BPCO comportent des inconvénients majeurs (i) le modèle murin présente des discordances fonctionnelles avec la pathologie humaine (ii) les cellules épithéliales primaires bronchiques humaines (HBEC) en interface air-liquide (ALI) ne permettent pas d'investiguer les racines pédiatriques. De plus, la quantité limitée d'épithélium généré est incompatible avec un criblage à grande échelle. En cela, la différenciation des cellules souches pluripotentes induites (iPSC) en épithélium bronchique offre une source cellulaire illimitée permettant la modélisation du développement précoce de la maladie. L'épithélium bronchiolaire étant une des premières barrières aux agressions du tabac, il apparait comme le chef d'orchestre de la maladie. Ainsi, je testerai l'hypothèse d'une « signature » épithéliale spécifique de la BPCO en modélisant l'épithélium bronchique de patients fumeurs sains (Groupe 1) et de patients fumeurs BPCO sévère (groupe 2) à partir d'IPSC. Le modèle HBECs en ALI généré pour chacun des patients sera notre référence. Les modifications induites par le tabac seront étudiés dans les deux modèles par analyse des données de transcriptome. Une analyse bio-informatique permettra de prioriser les candidats à explorer. Notre approche vise ainsi à développer un modèle robuste qui permettra (i) de mieux comprendre les mécanismes en cause dans la susceptibilité au tabac (ii) d'investiguer les racines pédiatriques de la maladie (iii) d'envisager des criblages pharmacologiques à large échelle pour identifier des traitements de la BPCOChronic Obstructive Pulmonary Disease (COPD), the third leading cause of death in the world accounts for 4 to 16% of the population in France. Age, smoking, respiratory events in early childhood are the main risk factors. Pathophysiological mechanisms remain misunderstood because of different trajectories especially pediatric ones. COPD modeling currently used appears perfectible (i) murine model because of functional discrepancies with human disease (ii) air-liquid interface culture (ALI) of human bronchial primary epithelial cells (HBECs) because of inability to mimic pediatric roots. Moreover, limited amount of epithelia generated is not consistent with large scale screening. The differentiation of Induced Pluripotent Stem Cells (iPSC) into bronchial epithelia provide an unlimited cellular source allowing the modelling of early stage development disease. The bronchiolar epithelium is one of the first barriers to tobacco toxicity and appears to be the disease conductor. Our hypothesis holds that there is a specific epithelial COPD "signature". So, I will model bronchial epithelium with IPSCs generated from healthy smoking patients (Group 1) and patients with severe COPD (group 2). The generation of bronchial epithelium from iPSC generated for these two groups will permit to investigate the cigarette extract epithelial toxicity. HBEC model in ALI generated for each patient will be taken as reference. Modifications induced by tobacco will be studied in both models by analysis of transcriptome data. A bioinformatic analysis will prioritize candidates to explore. Our approach aims to (i) better understand mechanisms involved in tobacco susceptibility (ii) investigate the pediatric trend of the disease (iii) permit large scale drug screening in order to identify new treatment

Induced Pluripotent Stem Cells for Primary Ciliary Dyskinesia Modeling and Personalized Medicine

International audiencePrimary ciliary dyskinesia (PCD) is a rare and heterogeneous genetic disorder that affects the structure and function of motile cilia. In the airway epithelium, impaired ciliary motion results in reduced or absent mucociliary clearance that leads to the appearance of chronic airway infection, sinusitis and bronchiectasis. Currently, there is no effective treatment for PCD, and research is limited by the lack of convenient models to study this disease and investigate innovative therapies. Furthermore, the high heterogeneity of PCD genotypes is likely to hinder the development of a single therapy for all patients. The generation of patient-derived induced pluripotent stem cells (iPSC) and their differentiation into airway epithelium as well as genome editing technologies could represent major tools for in vitro PCD modelling and for developing personalized therapies. Here, we review PCD pathogenesis, and then discuss how human iPSC could be used to model this disease for the development of innovative patient-specific biotherapies

Targeted therapy in eosinophilic chronic obstructive pulmonary disease

International audienceChronic obstructive pulmonary disease (COPD) is a common and preventable airway disease causing significant worldwide mortality and morbidity. Lifetime exposure to tobacco smoking and environmental particles are the two major risk factors. Over recent decades, COPD has become a growing public health problem with an increase in incidence. COPD is defined by airflow limitation due to airway inflammation and small airway remodelling coupled to parenchymal lung destruction. Most patients exhibit neutrophil-predominant airway inflammation combined with an increase in macrophages and CD8 + T-cells. Asthma is a heterogeneous chronic inflammatory airway disease. The most studied subtype is type 2 (T2) high eosinophilic asthma, for which there are an increasing number of biologic agents developed. However, both asthma and COPD are complex and share common pathophysiological mechanisms. They are known as overlapping syndromes as approximately 40% of patients with COPD present an eosinophilic airway inflammation. Several studies suggest a putative role of eosinophilia in lung function decline and COPD exacerbation. Recently, pharmacological agents targeting eosinophilic traits in uncontrolled eosinophilic asthma, especially monoclonal antibodies directed against interleukins (IL-5, IL-4, IL-13) or their receptors, have shown promising results. This review examines data on the rationale for such biological agents and assesses efficacy in T2-endotype COPD patients. @ERSpublications Patients with severe COPD and eosinophilic inflammation experience uncontrolled symptoms despite optimal pharmaceutical treatment. The development of new biomarkers is needed for better phenotyping of patients to propose innovative targeted therapy

Generation of four severe early-onset chronic obstructive pulmonary disease (COPD) patient-derived induced pluripotent stem cell lines from peripheral blood mononuclear cells

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Generation of the induced pluripotent stem cell line UHOMi002-A from peripheral blood mononuclear cells of a healthy male donor

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Generation of the induced pluripotent stem cell line UHOMi001-A from a patient with mutations in CCDC40 gene causing Primary Ciliary Dyskinesia (PCD)

Primary Ciliary Dyskinesia (PCD) is a rare heterogeneous genetic disorder affecting motile cilia structure and function leading to lung disease. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a female PCD patient carrying disease-causing variants in the CCDC40 gene. Reprogramming was performed with the human OSKM transcription factors using the Sendai-virus delivery system. The resulting transgene free iPSCs had normal karyotype, expressed pluripotency markers, could differentiate into the three germ layers in vivo and retained the disease-causing CCDC40 mutations. This iPSC line could be useful to model PCD disease and test gene therapy strategies.Resource TableUnlabelled TableUnique stem cell line identifierUHOMi001-AAlternative name(s) of stem cell lineiPCD02.30InstitutionInstitute for Regenerative Medicine & Biotherapy (IRMB), Montpellier, FRANCEContact information of distributorJohn De Vos [email protected] of cell lineiPSCOriginhumanAdditional origin infoAge: 34Sex: FemaleCell SourceDermal fibroblastsClonalityClonalMethod of reprogramminghOCT4, hSOX2, hC-MYC, hKLF4 (CytoTune™-iPS 2.0 Sendai Reprogramming Kit - Invitrogen, Thermo Fisher Scientific Inc.)Genetic ModificationYESType of ModificationSpontaneous mutationAssociated diseasePrimary Ciliary DyskinesiaGene/locusCompound Heterozygous mutations the Coiled-Coil Domain Containing 40 Gene (CCDC40): c.1116_1117delCT (Exon 7) and c.3180 + 1G > A (Intron 19)Method of modificationN/AName of transgene or resistanceN/AInducible/constitutive systemN/ADate archived/stock date2018-02-12Cell line repository/bankN/AEthical approvalThe study was approved by the regional scientific ethical committee (CPP Sud Med IV) under the number ID-RCB: 2013-A00892–43/CILIPS, Promoter University Hospital Of Montpellier) and informed consent was obtained from the patient

Les organoïdes pulmonaires

International audienceAs burden of chronic respiratory diseases is constantly increasing, improving in vitro lung models is essential in order to reproduce as closely as possible the complex pulmonary architecture, responsible for oxygen uptake and carbon dioxide clearance. The study of diseases that affect the respiratory system has benefited from in vitro reconstructions of the respiratory epithelium with inserts in air/liquid interface (2D) or in organoids able to mimic up to the arborescence of the respiratory tree (3D). Recent development in the fields of pluripotent stem cells-derived organoids and genome editing technologies has provided new insights to better understand pulmonary diseases and to find new therapeutic perspectives

Differentiation of Human Induced Pluripotent Stem Cells from Patients with Severe COPD into Functional Airway Epithelium

International audienceBackground: Chronic Obstructive Pulmonary Disease (COPD), a major cause of mortality and disability, is a complex disease with heterogeneous and ill-understood biological mechanisms. Human induced pluripotent stem cells (hiPSCs) are a promising tool to model human disease, including the impact of genetic susceptibility. Methods: We developed a simple and reliable method for reprogramming peripheral blood mononuclear cells into hiPSCs and to differentiate them into air–liquid interface bronchial epithelium within 45 days. Importantly, this method does not involve any cell sorting step. We reprogrammed blood cells from one healthy control and three patients with very severe COPD. Results: The mean cell purity at the definitive endoderm and ventral anterior foregut endoderm (vAFE) stages was >80%, assessed by quantifying C-X-C Motif Chemokine Receptor 4/SRY-Box Transcription Factor 17 (CXCR4/SOX17) and NK2 Homeobox 1 (NKX2.1) expression, respectively. vAFE cells from all four hiPSC lines differentiated into bronchial epithelium in air–liquid interface conditions, with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells, as found in vivo. The hiPSC-derived airway epithelium (iALI) from patients with very severe COPD and from the healthy control were undistinguishable. Conclusions: iALI bronchial epithelium is ready for better understanding lung disease pathogenesis and accelerating drug discovery

Recurrent Genetic Abnormalities in Human Pluripotent Stem Cells: Definition and Routine Detection in Culture Supernatant by Targeted Droplet Digital PCR

International audienceGenomic integrity of human pluripotent stem cells (hPSCs) is essential for research and clinical applications. However, genetic abnormalities can accumulate during hPSC generation and routine culture and following gene editing. Their occurrence should be regularly monitored, but the current assays to assess hPSC genomic integrity are not fully suitable for such regular screening. To address this issue, we first carried out a large meta-analysis of all hPSC genetic abnormalities reported in more than 100 publications and identified 738 recurrent genetic abnormalities (i.e., overlapping abnormalities found in at least five distinct scientific publications). We then developed a test based on the droplet digital PCR technology that can potentially detect more than 90% of these hPSC recurrent genetic abnormalities in DNA extracted from culture supernatant samples. This test can be used to routinely screen genomic integrity in hPSCs