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Small molecule-directed differentiation of submerged-cultured human nasal airway epithelia for respiratory disease modelling [scRNA-seq]

ABSTRACT: Submerged cultures of undifferentiated or transformed epithelial cells are widely used in respiratory research due to their ease of use and scalability. However, these systems fail to capture the cellular diversity of the human airway epithelium. Here, we describe a submerged differentiation model using cryopreserved human nasal epithelial cells obtained via minimally invasive brushings. By targeting Notch and BMP signaling with small-molecule inhibitors, we differentiate these cells into complex epithelial cultures containing basal, secretory, and ciliated cell types on standard plastic cultureware. This method supports scalable culturing of both 2D epithelial monolayers and 3D organoids, and is applied to disease modeling in primary ciliary dyskinesia, cystic fibrosis, and respiratory syncytial virus infection. The resulting system enables scalable assessment of disease-relevant epithelial functions in respiratory research

ORGANISM(S): Homo sapiens

PROVIDER: GSE318347 | GEO | 2026/02/05

REPOSITORIES: GEO

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Project description:Background. The growing popularity of cannabis smoking in an era of legalization has prompted concerns about respiratory health. Objective. To investigate clinical and airway epithelial transcriptomic features associated with cannabis smoking. Methods. This cross-sectional study analyzed 139 cannabis-smoking participants categorized by joint-year exposure (low: ≤5; moderate: >5-20; high: >20), and 57 never-smokers. We evaluated respiratory symptom questionnaire scores, lung function measurements, and chest computed tomography and hyperpolarized 129Xe pulmonary magnetic resonance imaging measurements across groups. We compared the expression of immune response signatures and mucin genes in airway epithelial brushings collected from bronchoscopy. Using air-liquid interface (ALI) cell cultures, we quantified epithelial MUC5AC protein and correlated its expression with clinical outcomes. Results. Among cannabis-smoking individuals (48% male and median age of 27 years), 84% reported current or former cigarette smoking or vaping. Cannabis-smoking groups reported worse respiratory symptoms than never-smokers. High joint-year cannabis-smoking participants showed lower pre-bronchodilator FEV1/FVC and FEF25-75, more radiographic emphysema, and more ventilation abnormalities than never-smokers. Airway epithelial brushings from cannabis-smoking individuals demonstrated increased type 2 immune response, decreased type 17 immune response, and higher MUC5AC gene expression than non-cannabis-smoking individuals. Epithelial MUC5AC protein expression in cell cultures correlated with worse clinical outcomes and imaging abnormalities. Conclusions. Cannabis smoking, particularly at high exposures, is associated with worse respiratory symptoms, lower lung function, functional imaging abnormalities, and dysregulated immune responses in the airway epithelium. These observations suggest respiratory harm associated with cannabis smoking and underscore the concerns for future respiratory morbidities related to persistent cannabis use.

Project description:This work presents an exploration of submerged differentiation of the ubiquitous saprophyte and industrially important fungus, Aspergillus niger, in response to a limited availability of a sole carbon and energy source, maltose. In aspergilli and other mold fungi, asexual reproduction through formation of elaborate conidiogenic structures normally requires an aerial interface. This requirement is bypassed in submerged culture in response to severe nutrient limitation. Continuous cultures with cell retention (retentostat cultures) were applied to generate a fundamental physiological state, where the specific growth rate approaches zero, as the density of the cell population adapts to the supply of the limiting energy source. Temporal differentiation of mycelium structure and commitment to asexual reproduction were major phenomena, apparent on biochemical, morphological, physiological, and transcriptomic level. The severe substrate limitation had a rapid negative impact on cytoplasmic processes, and promoted endo- and exogenous nutrient mobilization, and hyphal compartmentalisation. The first conidiogenic structures appeared after one day with little additional differentiation until Day 4 to 6, where a transition to full commitment to reproductive growth took place. Submerged conidiation in A. niger involved transcriptional regulation of homologs of the regulatory pathway, centered around the Bristle gene (brlA), and structural genes previously described in other aspergilli. Comparison of transcriptomes, revealed a number of co-regulated gene clusters, which appear to encode secondary metabolite biosynthetic potential. We discuss the concept of maintenance energy in the context of differentiation, a possible physiological trigger for sporulation and the special physiological adaptations of the starved mycelium. We also present a simple and efficient method for in situ retention of filamentous organisms. The dataset consists of 9 Affymetrix arrays derived from defined growth conditions of lab-scale bioreactor cultures (5L). Total RNA was extracted from biomass harvested at three different growth phases: exponential growth phase, 2 and 8 days of retentostat cultivation. For each of the phases, the data is derived from three biological replicates.

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Small molecule-directed differentiation of submerged-cultured human nasal airway epithelia for respiratory disease modelling [scRNA-seq]

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