Half of the Chronic Obstructive Pulmonary Disease (COPD) patients started in early adulthood from a low lung function. In utero maternal smoking is associated with increased adult COPD risk. iPSCs offer an opportunity to model human development of the lung system. This project aimed to model COPD trajectories related to a developmental origin through two extreme groups i) patients with an early onset of COPD, and ii) patients free of COPD despite heavy smoking. Paired cultures of airway epithelium were established at air-liquid interface (ALI) from hiPSC (iALI) and bronchial primary epithelial cells (ALI) of one healthy control, one healthy smoker, and 4 COPD patients [NCT03181204]. We characterized cigarette smoke extract (CSE) effects on human airway epithelial cell cultures using bulk RNA and DNA methylome sequencing. Unsupervised clustering analysis and Lasso regression showed that iALI samples formed a distinct cluster from ALI samples. GO biological process and function analysis showed upregulation of morphogenesis, development, and extracellular matrix processes in iALI versus epithelial differentiation, cilium organization, and innate immune response in ALI system. We found that CSE exposure induced a specific signature in iALI but not in paired ALI, suggesting a higher susceptibility of iALI to CSE. iALI culture upregulated reactive oxygen species, and xenobiotic metabolism genes in response to CSE (cytochrome P450 [CYP]1B, NADPH oxidase-1) and aryl hydrocarbon receptor signaling. Our results demonstrate that iALI cultures are less mature than paired ALI culture, and enables modeling early exposure of human fetal airway epithelium in response to environmental exposures.

La production in vitro, de manière reproductible et en larges quantité, d’un épithélium bronchique à partir de cellules souches pluripotentes est un objectif clé pour le criblage de médicaments à haut débit pour identifier de nouvelles molécules et la production de cellules pour une thérapie cellulaire en pneumologie (Pharmacol Ther 2018 :183, 58-77). Nous avons conçu une méthode simple et reproductible qui conduit un simple échantillon de sang en hiPSC par reprogrammation, puis différencié en 45 jours en épithélium bronchique en interface air-liquide (iALI), en suivant successivement les étapes clés du développement que sont la formation de l’endoderme définitif (DE) et de l’endoderme antérieur ventralisé (vAFE). La reprogrammation des cellules sanguines d’un patient sain et de 3 patients atteints de BPCO, ainsi que de fibroblastes dérivés de la peau obtenus chez un patient PCD, a été obtenue en utilisant des virus Sendai. La pureté moyenne des cellules aux stades DE et vAFE évaluée par l’expression de CXCR4 et NKX2.1, respectivement, était supérieure à 85 %, évitant ainsi la nécessité d’un tri cellulaire. Lorsqu’elles sont transférées dans des conditions de culture d’interface air-liquide (ALI), les cellules vAFE se différencient en 4 semaines en épithélium bronchique avec de grandes zones couvertes par les principales sous-populations cellulaires de l’épithélium bronchique : cellules basales (KRT5 + ), ciliées (TubIV + ), à mucus (Muc5AC + ), Club (CCSP + ), ainsi que des cellules neuroendocrines (CHGA + )(épithélium bronchique iALI). La microscopie électronique confirme la présence de cils motiles et leur fréquence de battement est similaire à celui observé dans des HBEC. Certains iALI ont été maintenus pendant plus de 400 jours. La reprogrammation et la différenciation fiables des hiPSC dérivés du sang, quel que soit leur contexte clinique, en épithélium bronchique iALI mature et fonctionnel est maintenant accessible à une utilisation plus large, ce qui permettra de mieux comprendre la pathogenèse des maladies pulmonaires et d’accélérer le développement de nouvelles thérapies géniques et la découverte de médicaments.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease leading to irreversible destruction of the terminal bronchioles. Although the precise patho-physiological mechanisms remain to be elucidated, the bronchial epithelium seems to play a pivotal role in the disease. Recent studies have highlighted a great heterogeneity among COPD patients, with various disease courses including, in about half the cases, an origin in childhood. Modelling of COPD is a major goal but currently available models are imperfect. Our work aims to create a new in vitro cellular model to study the pathology of the disease. The differentiation of human induced pluripotential stem cells (hiPSCs) in bronchial epithelium is a step towards a better understanding of the developmental origin and the identification of new therapeutic targets. (c) 2020 SPLF. Published by Elsevier Masson SAS. All rights reserved.

Background & Aim Genetic integrity of human pluripotent stem cells (hPSCs) is essential for the accuracy of the disease models and for the security of cell therapies that are use those cells. Because genetic abnormalities can accumulate during the generation of hPSC, their long term maintenance or during genome editing, it is mandatory to regularly check the genome of hPSCs. However, the current methods to assess genomic integrity of hPSC are not fully suitable for such regular screening. These approaches can be limited in term of resolution, flexibility, cost and time to process. Our aim is improving genetic screening of hPSC. Methods, Results & Conclusion Based on a large meta-analysis of all hPSC genetic abnormalities reported in more than 100 publications, we were able to exhaustively identify recurrent genetic abnormalities accumulating in hPSCs. We then developed a test (iCS-digital test) based on the droplet digital PCR technology that can detect more than 90% of these hPSC recurrent genetic abnormalities in DNA extracted from culture supernatant samples. This test was successfully used to detect copy number variations that arose during cell culture or gene editing. Hence, the iCS-digital test can be used to routinely screen genomic integrity in hPSCs, contribute to secure genome editing workflow, and reduce time and financial losses induced by the use of an abnormal hPSC lines. Genetic integrity of human pluripotent stem cells (hPSCs) is essential for the accuracy of the disease models and for the security of cell therapies that are use those cells. Because genetic abnormalities can accumulate during the generation of hPSC, their long term maintenance or during genome editing, it is mandatory to regularly check the genome of hPSCs. However, the current methods to assess genomic integrity of hPSC are not fully suitable for such regular screening. These approaches can be limited in term of resolution, flexibility, cost and time to process. Our aim is improving genetic screening of hPSC. Based on a large meta-analysis of all hPSC genetic abnormalities reported in more than 100 publications, we were able to exhaustively identify recurrent genetic abnormalities accumulating in hPSCs. We then developed a test (iCS-digital test) based on the droplet digital PCR technology that can detect more than 90% of these hPSC recurrent genetic abnormalities in DNA extracted from culture supernatant samples. This test was successfully used to detect copy number variations that arose during cell culture or gene editing. Hence, the iCS-digital test can be used to routinely screen genomic integrity in hPSCs, contribute to secure genome editing workflow, and reduce time and financial losses induced by the use of an abnormal hPSC lines.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease leading to irreversible destruction of the terminal bronchioles. Although the precise patho-physiological mechanisms remain to be elucidated, the bronchial epithelium seems to play a pivotal role in the disease. Recent studies have highlighted a great heterogeneity among COPD patients, with various disease courses including, in about half the cases, an origin in childhood. Modelling of COPD is a major goal but currently available models are imperfect. Our work aims to create a new in vitro cellular model to study the pathology of the disease. The differentiation of human induced pluripotential stem cells (hiPSCs) in bronchial epithelium is a step towards a better understanding of the developmental origin and the identification of new therapeutic targets.

La bronchopneumopathie chronique obstructive (BPCO) est une atteinte chronique des voies aériennes distales caractérisée par une destruction irréversible des bronchioles terminales. Bien que les mécanismes physiopathologiques demeurent incompris à ce jour, l’épithélium bronchique semble être le chef d’orchestre de la maladie. De récentes études ont montré l’existence de différentes trajectoires de la maladie incluant dans la moitié des cas une origine pédiatrique. La modélisation de la BPCO constitue donc un enjeu majeur mais les modèles actuels sont imparfaits. La différenciation des cellules souches pluripotentes induites humaines (hiPSC) en épithélium bronchique représente un nouvel outil pour étudier les racines pédiatriques de la maladie et identifier de nouvelles cibles thérapeutiques.