Biology subjects

Assou, S.

Publications and source records attributed to Assou, S..

4 recordsLinked to original sources

bioRxiv2026

Ventral anterior foregut endoderm cells as progenitors for airway epithelial cell replacement in primary ciliary dyskinesia

BackgroundLung transplantation remains the ultimate treatment option for patients with end-stage lung disease, but has many limitations. This underlines the urgent need of developing alternative approaches to treat lung disorders. Among the emergent strategies, gene therapy holds great potential for the treatment of monogenic lung diseases. However, so far, aerosolized viral vector-based delivery for gene therapy has failed likely because of difficulties in accessing the target cells. Combined gene and cell therapy approaches could be a promising alternative. Trials using basal cell transplantation already showed encouraging results. Moreover, the induced pluripotent stem cell (iPSC) technology broadens the scope of personalized therapies by paving the way for autologous approaches. Our group previously derived iPSC lines from patients with Primary Ciliary Dyskinesia (PCD) and found that their correction by gene conversion allows functional recovery. This study aimed to identify the best progenitors and airway conditioning technique to develop an autologous cell replacement strategy for PCD. MethodsAirway 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. ResultsGFP+ 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. ConclusionsOur 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.

cell biology↗

bioRxiv2023

Human airway ex vivo models: new tools to study the airway epithelial cell response to SARS-CoV-2 infection

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.

cell biology↗

bioRxiv2020

Differentiation of human induced pluripotent stem cells into functional airway epithelium

RationaleHighly reproducible in vitro generation of human bronchial epithelium from pluripotent stem cells is an unmet key goal for drug screening to treat lung diseases. The possibility of using induced pluripotent stem cells (hiPSC) to model normal and diseased tissue in vitro from a simple blood sample will reshape drug discovery for chronic lung, monogenic and infectious diseases. MethodsWe devised a simple and reliable method that drives a blood sample reprogrammed into hiPSC subsequently differentiated within 45 days into air-liquid interface bronchial epithelium (iALI), through key developmental stages, definitive-endoderm (DE) and Ventralized-Anterior-Foregut-Endoderm (vAFE) cells. ResultsReprogramming blood cells from one healthy and 3 COPD patients, and from skin-derived fibroblasts obtained in one PCD patient, succeeded in 100% of samples using Sendai viruses. Mean cell purity at DE and vAFE stages was greater than 80%, assessed by expression of CXCR4 and NKX2.1, avoiding the need of cell sorting. When transferred to ALI conditions, vAFE cells reliably differentiated within 4 weeks into bronchial epithelium with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells as found in vivo. Benchmarking all culture conditions including hiPSCs adaptation to single-cell passaging, cell density and differentiation induction timing allowed for consistently producing iALI bronchial epithelium from the five hiPSC lines. ConclusionsReliable reprogramming and differentiation of blood-derived hiPSCs into mature and functional iALI bronchial epithelium is ready for wider use and this will allow better understanding lung disease pathogenesis and accelerating the development of novel gene therapies and drug discovery.

bioengineering↗

bioRxiv2019

Recurrent genetic abnormalities in human pluripotent stem cells: definition and routine detection in culture supernatant by targeted droplet digital PCR

Genomic 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 routinely monitored, but the current assays to assess hPSC genomic integrity are not fully suitable for suchregular 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 94.3% 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.

cell biology↗