Background The combination of gene and cell therapy is a promising therapeutic approach in monogenic lung disorders. This study aimed to identify the best progenitors and airway conditioning technique to develop an autologous cell replacement strategy for primary ciliary dyskinesia (PCD). Methods Airway epithelial cells were differentiated from induced pluripotent stem cell (iPSC) lines from a healthy donor (parental Hy03) and Hy03 in which MCIDAS was knocked out (PCD model) and maintained in air-liquid interface (iALI). The engraftment of GFP+ ventral Anterior Foregut Endoderm (vAFE) cells, differentiated from GFP-expressing Hy03 iPSCs, was assessed after conditioning of the recipient iALI. The efficacy (epithelial cell shedding) and toxicity (cell death) of different conditioning strategies were compared. Cilia functional repair was assessed using microbead motion tracking. Findings GFP+ vAFE cells can successfully integrate and repair trypsin- or EDTA-conditioned airway epithelia derived from the parental and MCIDAS-/- Hy03 iPSC lines. EDTA showed optimal efficacy/safety balance. Progenitor integration and differentiation were confirmed by E-cadherin, tubulin-IV, KRT5 and MUC5AC co-expression in GFP+ engrafted cells at day 35 post-graft (immunofluorescence analysis). The engrafted GFP+ population reached 35-45% of the total epithelial population, as indicated by flow cytometry quantification of EpCAM+/GFP+ cells. Functional analysis demonstrated cilia motion restoration after GFP+ cell engraftment onto MCIDAS-/- iALI. Interpretation Our study shows that vAFE cells can integrate and differentiate to repair epithelial models of PCD. EDTA conditioning is promising for the clinical application of this therapeutic strategy. ### Competing Interest Statement A.B. reports research grants, honoraria and consulting fees from AstraZeneca, GSK and Boehringer Ingelheim, consulting fees and honoraria from Sanofi and Novartis, consulting fees from Chiesi and Celltrion, support for attending meetings and travel from AstraZeneca and Sanofi, participation on a data safety monitoring board for AB Science. J.D.V. reports consulting fees from and shares in Stem Genomics, a research grant and honoraria from AstraZeneca, and is president elected of the French Society for Stem Cell Research (FSSCR). S.A. reports consulting fees from and shares in Stem Genomics. In addition, J.D.V. and S.A. hold a patent EP20150306389 licensed to Stem Genomics. F.F. and C.B. report grants from the Fondation du Souffle-Societe de Pneumologie de langue francaise. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-23-CE52-0011 Fondation du souffle

RATIONALE:Derived iPSCs airway epithelium are challenging given their dependency on the mesenchymal compartment. We hypothesized that growing vAFE cells on well-organized stiff matrix precolonized by adult pulmonary fibroblasts would improve epithelial differentiation yield and maturity. METHODS:Collagen-1/chitosan matrix were engineered to reach stiffness and scaffolding characteristics of subepithelial compartments. Primary fibroblasts derived from human lung samples were seeded for 45 days before the addition of vAFE cells differentiated from iPSCs, and comparisons made with iPSC-derived fibroblasts. Beads tracking was used to assess cilia beating efficiency. RESULTS:Primary human bronchial fibroblasts were able to enrich the CC Matrix with extracellular matrix components such as collagen, decorin and vimentin. In turn, iALI cultures performed in primary fibroblasts enriched CC matrix successfully led to high level of epithelial differentiation including rare cells (club, basal, neuroendocrine, ciliated, secretory). Large apical surfaces were covered by approximately 60% of ciliated cells able to generate mucociliary vortex. CONCLUSION:Primary human bronchial fibroblasts seeded into collagen-chitosan matrix dramatically improved iALI epithelial differentiation from vAFE cells, related to highly specific transcriptomic signatures when compared to iPSC derived fibroblasts.

SARS-CoV-2 causes severe and persistent lower respiratory tract infections, yet human models that recapitulate long-term tissue responses are limited. Here, we used a human induced pluripotent stem cell (hiPSC)-derived bronchial airway models (iALI) to investigate SARS-CoV-2 infection in healthy and COPD-derived tissues. Infection of iALI led to robust viral replication, persistent infection, cilia loss in infected ciliated epithelial cells, increased mucus secretion, and higher inflammatory cytokine release in COPD iALI. Notably, healthy iALI displayed a delayed innate immune response, whereas COPD iALI exhibited an earlier and stronger response, characterized by elevated IL-2, CCL5, G-CSF, and CXCL10 secretion, along with reduced sensitivity to antiviral treatment. These findings reveal donor-specific differences in bronchial epithelial responses to SARS-CoV-2 and establish iALI culture models as a powerful platform for studying long-term respiratory viral infections in both healthy and diseased contexts, especially COPD.

Respiratory infections are a major global health concern, as underscored by the COVID-19 pandemic. To better understand bronchial tissue responses to viral infection, we have developed a preclinical in vitro model mimicking the multiciliated airway epithelium, from induced pluripotent stem cell (iPSC) and cultured in an air-liquid interface (iALI). By using iPSCs reprogrammed from patients with chronic obstructive pulmonary disease (COPD), we successfully generated a fully differentiated and functional bronchial epithelium exhibiting key COPD features with goblet and basal cell hyperplasia and tissue inflammation. SARS-CoV-2 could infected and replicated for several weeks in both healthy and COPD models, with a recurrent peak at 3 days after infection. Infected iALI exhibited cilia destruction and increased mucus secretion. Innate immune response of different infected iALI reveals a differential expression of interferon-stimulated genes (ISGs) and pro-inflammatory cytokine secretion. Notably, COPD iALI displayed an earlier innate immune response to SARS-CoV-2 infection as compared to healthy iALI, suggesting a genetic susceptibility of COPD iALI towards inflammation induced by SARS-CoV-2 infection, and a less efficient response to antivirals. In conclusion, our study demonstrates that the iALI bronchial organoid model is a powerful tool for investigating bronchial tissue responses to long term respiratory viral infections, antivirals, and patients with COPD or other airway pathology. ![Figure][1] HighLights Short Abstract SARS-CoV-2 causes severe lower respiratory tract infection in COVID-19 patients, which can persist over time. Here, we used an in-house developed in vitro airway organoid derived from induced human pluripotent stem cells (iALI) to study SARS-CoV-2 infection over long term in healthy or COPD patients whom respiratory failure is at risk during infection. Our results show that SARS-CoV-2 infection results in high and lethal infection of bronchial epithelial cells, that persist over time, inducing mucus secretion, destruction of ciliated cells and specific cytokine release. A late innate immune response is observed in the healthy iALI, while in iCOPD, it appears earlier and stronger, suggesting a different sensing of SARS-CoV-2 in COPD patients, accompanied by a reduce sensitivity to antivirals. In conclusion, our study demonstrates that the iALI organoid model is a powerful tool for investigating bronchial tissue responses to long term respiratory viral infections, from healthy to pathologic patients. ### Competing Interest Statement The authors have declared no competing interest. CNRS Biologie, VIROCRIB [1]: pending:yes

IntroductionDespite the new biologics to treat inflammation in severe asthma, targeting persistent obstruction of the airways remains challenging. Galectin-10 eosinophil derived crystals, also known as Charcot-Leyden crystals (CLCs) have been described to be present in the mucus plugs in the airways of patients with severe asthma. However, a direct role for CLCs in mucus production has not been established. We hypothesize that plugged airways constitute a unique niche where type 2 immune cells communicate with structural cells to perpetuate disease. We aimed to set up a new model using induced pluripotent stem cells (iPSCs).MethodsThree human iPSCs lines from type 2 severe asthma patients have been derived (MOSAIC study, University Hospital of Montpellier, NCT05616338) and differentiated into airway epithelium in air–liquid interface (i-ALI). The healthy iPSC line UHOMi002-A was used as a control. At day 21 of ALI culture, iPSC derived-airway epithelia were stimulated at the apical side with either IL-13 every two days (10ng/mL) during one week, acute stimulation (24h) with recombinant Gal10 crystals (100ng/mL), both IL-13 and Gal10 crystals or PBS (vehicle). We aimed to evaluate the effect on i-ALI differentiation at day 30.ResultsWe successfully differentiated the iPSC lines generated from the T2 severe asthma patients, and achieving a high purity rate at each developmental stages. The mean cell purity at the definitive endoderm for each cell line was>80% assessed by flow cytometry quantification of C-X-C Motif Chemokine Receptor 4 (CXCR4)/c-KIT double positive cells and immunolabelling of Forkhead Box A2 (FOXA2)+/SRY-box transcription factor 17 (SOX17)+. Purity for ventral anterior foregut endoderm (vAFE) stage was evaluated at 70%, through Transcription Factor NK2 Homeobox 1 (NKX2.1) expression, Carboxy Peptidase M (CPM) by flow cytometry. vAFE cells from the hiPSC lines differentiated into bronchial epithelium in air–liquid interface conditions. Chronic IL-13 challenging and CLC were both able to induce an increasing of MUC5AC+ cells and also an increase of neuroendocrine cells in asthmatic iPSC lines.ConclusioniALI bronchial epithelium can recapitulate T2 severe asthma features in vitro, and highlighted a possible direct effect of the CLC on the airway epithelium.

Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized healthy heavy smoker male donor free from emphysema or tobacco related disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers.The reported UHOMi007-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing.

IntroductionLes maladies respiratoires sévères, dont l’asthme, nécessitent de nouveaux traitements car les traitements actuels sont encore inefficaces chez un certain nombre de patients. Notre projet vise à obtenir un modèle d’épithélium bronchique innervé par une innervation sensitive in vitro à partir de cellules souches pluripotentes induites (iPSC).MéthodesNous avons synthétisé une matrice extracellulaire composée d’un mélange de collagène bovin de type I et de chitosan. Cette matrice est d’abord colonisée par des fibroblastes bronchiques humains primaires et agit comme une muqueuse sous-épithéliale. Des neurones sensitifs dérivés de cellules souches pluripotentes induites (iPSC) sont cultivés sur la face inférieure de la matrice et un épithélium bronchique dérivé d’iPSC provenant des mêmes sujets est cultivé sur la surface, en interface air/Liquide (ALI), selon notre protocole décrit précédemment [1].RésultatsNous avons obtenu un épithélium bronchique dérivé d’iPSC présentant les principaux types cellulaires. Il repose sur une matrice extracellulaire épaissie par le collagène sécrété par les fibroblastes et présentant une lame basale. La coloration HES des coupes en paraffine montre un épithélium pseudostratifié avec différents types cellulaires bronchiques, y compris des cellules ciliées, Club, à mucus et basales. La coloration à l’acide périodique Schiff - Bleu Alcian permet de visualiser les cellules à mucus. Ces résultats sont confirmés par l’immunofluorescence de MUC5AC+. Les cellules ciliées sont TUBULINE ß IV+, les cellules basales Kératine 5+, les cellules club CCSP+, et nous avons également observé des cellules neuroendocrines chromogranineÀ+. Une innervation partielle est démontrée à l’intérieur de la matrice par immunofluorescence des neurones sensoriels dérivés des iPSC (TUBß3+). Pour essayer d’améliorer cette innervation, nous avons différencié et ajouté des cellules de Schwann dérivées d’iPSCS dans ce modèle car elles pourraient permettre le guidage des fibres nerveuses [2]. Les résultats préliminaires montrent une amélioration de l’innervation avec la présence effective de fibres nerveuses PGP9,5+ et TUBß3+ dans la matrice. La maturation de ces fibres sensitives et attestée par la présence de neuropeptides (fibres CGRP +).ConclusionEn conclusion, nous avons obtenu avec succès un épithélium bronchique humain dérivé d’iPSC en 3D reposant sur une muqueuse sous-épithéliale. Des différenciations sont en cours pour confirmer les résultats obtenus avec l’innervation et la fonctionnalité de notre modèle complet.

L’asthme est une maladie respiratoire fréquente, sévère dans 3 à 5 % des cas. L’inflammation chronique de l’épithélium bronchique joue un rôle clé dans sa physiopathologie. Le système nerveux sensitif périphérique contribue à cette inflammation des voies aériennes via une activation par l’environnement bronchique. Cependant les mécanismes d’action mis en jeu ne sont à ce jour pas bien connus, notamment par manque de modèles fiables. Nous avons mis en place, à partir de cellules souches pluripotentes induites reprogrammées à partir de cellules sanguines, un modèle d’épithélium bronchique innervé par des neurones sensitifs. Ce dernier permettra notamment de mieux comprendre les mécanismes d’action sous-jacents à l’inflammation neurogène.

Severe respiratory diseases including asthma need new treatments to prevent severe clinical features in a number of patients. New models are necessary. We constructed an innervated bronchial epithelium model in vitro from iPSCs. We synthesised an extracellular matrix composed of a mixture of collagen and chitosan, and added primary human bronchial fibroblasts to colonize and act as a subepithelial mucosa. Sensory neurons derived from iPSCs were cultured on the underside of the matrix and a bronchial epithelium derived from iPSCs from the same subject was cultured on the surface, in ALI as reported (Ahmed et al., 2022). We obtained an iPSC-derived bronchial epithelium with the main architecture and cell types of pseudostratified epithelium after 60 days culture. It is based on an extracellular matrix thickened with ‘endogenous’ human collagen secreted by the fibroblasts and forming a basal lamina. HES staining showed a pseudostratified epithelium with different bronchial cell types, including ciliated cells, beating according to phase contrast microscopy. The results were confirmed by immunofluorescence of TUBIV+ ciliated cells, MUC5AC+ mucus cells, KRT5+ basal cells, CCSP+ club cells and CHGA+ neuroendocrine cells. Partial innervation was demonstrated within the matrix.To improve this innervation, we added iPSCS-derived Schwann cells to guide newly formed sensory nerve fibres. Preliminary results document the effective presence of longer spreading nerve fibres within the matrix TUBß3+, more mature since presenting the sensory neuropeptide CGRP+ labelling. We successfully obtained a 3D iPSC-derived innervated bronchial epithelium. Functionality test are underway.

Asthma is a frequent respiratory disease, with severe asthma occurring in 3 to 5% of cases. Chronic inflammation of the bronchial epithelium is essential to its pathophysiology. When activated by the bronchial environment, the peripheral sensory nervous system contributes to inflammation of the airways. However, due to a lack of reliable models, the mechanisms of action remain largely unknown. Using induced pluripotent stem cells reprogrammed from blood cells, we have set up a model of bronchial epithelium innervated by sensory neurons. This model will ensure better understanding of the mechanisms of action underlying neurogenic inflammation.

Airway–liquid interface cultures of primary epithelial cells and of induced pluripotent stem-cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models), infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using our own and publicly available bulk and single-cell transcriptome datasets. SARS-CoV-2 infection significantly increased the expression of interferon-stimulated genes (IFI44, IFIT1, IFIT3, IFI35, IRF9, MX1, OAS1, OAS3 and ISG15) and inflammatory genes (NFKBIA, CSF1, FOSL1, IL32 and CXCL10) by day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes (ITGB6, ITGB1 and GJA1) were also altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins was also deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to explain the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments.

Airway-liquid interface cultures of primary epithelial cells and of induced pluripotent stem cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models) infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using publicly available and our own bulk and single-cell transcriptome datasets. SARS-CoV-2 infection significantly increased the expression of interferon-stimulated genes ( IFI44 , IFIT1 , IFIT3 , IFI35 , IRF9 , MX1 , OAS1 , OAS3 and ISG15 ) and inflammatory genes ( NFKBIA , CSF1 , FOSL1 , IL32 and CXCL10 ) at day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes ( ITGB6 , ITGB1 and GJA1 ) also were altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins also was deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to understand the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments.

Asthma is a frequent respiratory disease, with severe asthma occurring in 3 to 5% of cases. Chronic inflammation of the bronchial epithelium is essential to its pathophysiology. When activated by the bronchial environment, the peripheral sensory nervous system contributes to inflammation of the airways. However, due to a lack of reliable models, the mechanisms of action remain largely unknown. Using induced pluripotent stem cells reprogrammed from blood cells, we have set up a model of bronchial epithelium innervated by sensory neurons. This model will ensure better understanding of the mechanisms of action underlying neurogenic inflammation. (c) 2023 SPLF. Published by Elsevier Masson SAS. All rights reserved.

Background The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. Results We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. Conclusions Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

Background: Induced pluripotent stem cells (iPSC) provide a new approach to model COPD. We have generated a functional air-liquid interface bronchial epithelium from human iPSC (iALI) and focus on the characterization of both mesenchymal and epithelial iALI compartments before and after pollution exposure. Objective/Methods: To modelize COPD and study epithelial-mesenchymal crosstalk, we performed single-cell mRNA sequencing before and after exposure to calibrated PM2.5 pollutants (City of Prague 2005) in EpCAM+ vs EpCAM- sorted cells. RT-QPCR and immunofluorescence were used to assess the exposure process. Results: iALI contains all the bronchial epithelial cell subtypes such as ciliated cells, basal cells, neuroendocrine cells, club cells and goblet cells. Furthermore, an EPCAM-COL1A1+DCN+ stromal compartment is identified below the bronchial epithelial layer of iALI. It contains several cell populations such as 1/myofibroblasts characterized by contractile genes such as CCN1 and TAGLN and 2/ a transition population expressing epithelial to mesenchymal transition transcription factor SNAI2 and ZEB1/2. This stromal cell compartment expressed FGF10 while its receptor FGFR2 was expressed on the adjacent epithelium suggesting an epithelial-mesenchymal crosstalk. Under PM2.5 exposure, MUC5B was induced in the EpCAM+ sorted epithelial cells, while CYP1B1 was induced in the EpCAM- mesenchyme, suggesting an adaptive response to exposome. Conclusion: These data suggest that an epithelial-mesenchymal crosstalk is essential to iALI differentiation and maintenance. Furthermore, we show that iALI is a promising tool to model the adaptive response to exposome.

Chronic airway diseases are a common medical condition worldwide and include diseases such as cystic fibrosis, asthma or chronic obstructive pulmonary disease (COPD). COPD is the third leading cause of death in the world, and there is still no curative treatment. Air liquid interface cultures (ALI) mimic the lung development and disease. We and others have recently generated a functional airway multiciliated epithelium in ALI conditions by using human pluripotent stem cells, called “iALI” (Ahmed et al., BioRxiv, 2020). During this differentiation process, a mesenchymal stromal component is consistently described but poorly studied. To comprehensively define the cell types, mechanisms driving iALI model and understand the crosstalk between epithelial and mesenchymal components, we performed single-cell mRNA sequencing. RT-qPCR and immunofluorescence assays were used to confirm the sequencing results. In addition to the epithelial cell subtypes, the iALI cultures also contain a previously poorly documented EPCAM-COL1A1 + DCN+ mesenchymal stromal compartment. Several populations, that partially overlap can be identified : 1/ACTA2+ myofibroblasts including fibromyocytes characterized by high expression of contractile genes such as CNN1 and TAGLN, 2/a transition population closer to epithelial cells expressing MAF and the long non coding mRNA EPB41L4A-AS1 and 3/a highly proliferating PCNA + CDK1+ cells. Moreover, stromal cells expressed growth factors such as FGF10 while its receptor FGFR2 is expressed on the epithelial cells, suggesting that stromal cells could support iALI development. These data provide high-resolution insights into the complexity and plasticity of the iALI and suggest that a crosstalk between epithelium and mesenchyme is essential to iALI development. The functional role of the relevant pathways will be assessed in our iALI model in control condition but also in a pathological context.

An organoid model of innervated bronchial epithelium will help understanding patient9s characteristics in severe asthma. Such a model differentiated from reprogrammed circulating cells of patients into iPSCs will allow the screening and pharmacological studies for ultra-personalized medicine. Our project involves the construction of a subepithelial mucosa including the use of an extracellular matrix composed of a mixture of collagen and chitosan and infiltrated with human bronchial fibroblasts innervated with human iPSC-derived sensory neurons, over which iPSC differentiation into functional bronchial epithelium is performed, using the protocol previously published by the team (E. Ahmed et al, BioRxiv 2020) We obtained a submucosa with a thickened extracellular matrix, and the manufacturing of a basal lamina. Innervation of this sub-mucosa by iPSCs-derived sensory neurons was documented by immunofluorescence, showing the presence of specific markers such as tubulin ß3, and neurofilament M after 28 days. We also successfully produced in parallel an iPSC-derived bronchial epithelium after 40 days of differentiation on the matrix. It comprised keratin 5+ basal cells, and tubulin IV+ multi-ciliated cells that are beating. Tentative characterizations of club cells and mucus cells in the model suggest that a longer differentiation time will be necessary. A human iPSC-derived bronchial epithelium and sensory neurons on collagen-chitosan-fibroblast matrix was obtained. The construction of a 3D innervated bronchial epithelium integrating in one single construct the bronchial epithelium, fibroblasts and sensory neurons is currently underway.

Le projet vise à obtenir un modèle d’épithélium bronchique innervé pour une application à l’asthme sévère, pour lequel les traitements sont souvent inefficaces. En effet, la construction d’un épithélium bronchique innervé devrait permettre la recherche de nouveaux médicaments via la reprogrammation des cellules circulantes de patients en iPSC afin d’obtenir un modèle personnalisé, permettant le criblage et l’étude pharmacologique pour une médecine ultra personnalisée. Le projet comprend en parallèle i) la construction d’une muqueuse sous-épithéliale innervée par des neurones sensitifs dérivée d’iPSC et ii) la différenciation d’iPSC en épithélium bronchique. La construction de la muqueuse sous épithéliale comprend la fabrication d’une matrice extracellulaire composée d’un mélange de collagène et de chitosan, et infiltrée par des fibroblastes bronchiques humains. Des neurones immatures sont ensuite ensemencés sous cette matrice où leur maturation en neurones sensitifs matures est induite pendant 14 jours. La différenciation d’iPSC en épithélium bronchique est faite suivant le protocole publié par l’équipe (E. Ahmed et al., 2020). Les résultats d’ensemencement des fibroblastes bronchiques humains dans les matrices montrent une colonisation effective des matrices collagène-chitosan. Le phénotype des neurones obtenus après différenciation a été validé par immunofluorescence par la présence des marqueurs Tubuline ß3 (neurone), substance P et neurofilament M (neurone sensitif). Les résultats de différenciation d’iPSC en épithélium bronchique sur la matrice montrent l’obtention d’un épithélium bronchique fonctionnel comportant des cellules basales kératine 5+ et des cellules multiciliées Tubuline IV+, et présentant un battement ciliaire. Un épithélium bronchique dérivé d’iPSC sur matrice collagène-chitosan-fibroblastes a été obtenu. Il reste à coupler cette construction cellulaire aux neurones sensitifs matures pour obtenir notre modèle final. Celui-ci pourra par la suite être complexifié en rajoutant des cellules endothéliales et des cellules immunocompétentes.

A bronchial epithelium model innervated by sensory neurons will enable studying several respiratory diseases including severe asthma, for which current treatments are still ineffective in a number of patients with severe clinical features. We used an artificial matrix, composed of a mixture of collagen and chitosan, which was seeded with human bronchial fibroblasts, resulting in a subepithelial mucosa. Induced pluripotent stem cells (iPSC)-derived sensory neurons were then added under the matrix. Finally, iPSC-derived functional bronchial epithelium as described in our previously published protocol (E. Ahmed et al, BioRxiv 2020) was finally added on the other side. After 40 days of culture, we obtained a fully differentiated iPSC-derived bronchial epithelium, laid on a submucosa that was characterized by a thickened extracellular matrix and a basal lamina. Hematoxylin-eosin staining on paraffin sections showed a pseudo-stratified epithelium displaying physiological bronchial cell diversity, including tubulin IV+ ciliated cells, mucin 5AC+ mucus cells and basal Keratin 5+ cells. After 60 days of culture, partial sensory innervation was found within the matrix by immunofluorescent staining of the iPSCs-derived sensory neurons (ß3 Tubulin+; Neurofilament M+). In conclusion, we successfully obtained a 3D human iPSC-derived bronchial epithelium laying on a sub-epithelial mucosa. Developments are underway to obtain a fully innervated epithelial organoid.