Bulk RNA sequencing of airway organoids in differentiation medium towards ciliated cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA666459 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12744463_1.fastq.gz | Fastqsanger.gz | |||
| SRR12744463_2.fastq.gz | Fastqsanger.gz | |||
| SRR12744464_1.fastq.gz | Fastqsanger.gz | |||
| SRR12744464_2.fastq.gz | Fastqsanger.gz | |||
| SRR12744465_1.fastq.gz | Fastqsanger.gz |
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Project description:Bulk RNA sequencing was performed on 3D airway cultures in airway organoid expansion medium and differentiation medium (CilM)
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Project description:To identify key genes that define surface airway epithelial (SAE) basal cells, we FACS isolated basal, ciliated, and club cell populations as previously reported (Zhao et al., 2014; PMID: 25043474) and performed microarray analysis on isolated mRNA. For fractionating SAE into basal, club, and ciliated populations, cells were stained with EpCAM-PECy7 (eBiosciences), GSIβ4-FITC (Sigma), SSEA1-Alexa Fluor® 647 (BioLegend), and CD24-PE (BD Pharmingen) for 30 minutes on ice as previously described (Zhao et al., 2014), prior to FACS. Basal cells were considered EpCAM+ and GSIβ4+. Secretory cells were considered EpCAM+ and SSEA1+. Ciliated cells were considered EpCAM+, GSIβ4- and CD24+. Primary SAE cells were harvested from C57BL/6 mice and sorted into basal, ciliated, and club cell populations for the purpose of identifying enrichment of transcripts specific to each cell type population.
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Project description:Zoonoses pose substantial global health risks, highlighting the need to better understand animal-to-human transmission. Reptiles are increasingly recognized as hosts of diverse pathogens, including many viruses. Despite this, reptile pathogens remain poorly understood in terms of their diversity, prevalence, and potential risk to humans. Yet human–reptile contact is increasing, driven in part by the global wildlife trade and pet industry. This underscores the need to better understand reptile-associated pathogens; however, models to study reptile viruses remain scarce. Here, we establish and characterize airway organoids derived from Python regius, providing an in vitro model of the reptile airway. Through de novo assembly of a Python regius reference genome, we characterized airway organoids at single-cell resolution, revealing diverse cell populations including ionocytes, ciliated, goblet, club, endocrine, tuft, and basal cells. The organoids supported productive infection with ball python nidovirus (BPNV) and mounted a robust epithelial antiviral response through induction of interferon-stimulated genes, cytokines and genes involved in chemical defense. As a proof-of-concept, antiviral drug treatment reduced BPNV levels, highlighting the model's utility for drug testing. By providing a reductionist system of the serpentine airway, these organoids constitute a physiologically relevant in vitro model to study reptile viruses and host–pathogen interactions in the native host. This system has potential veterinary applications, ecology of serpentes, and zoonotic disease research.
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Project description:Background: Human basal stem cells, with their self-renewal and multilineage differentiation capacity, are essential tools for modeling airway diseases and advancing regenerative medicine. However, existing culture systems often rely on undefined components like serum, bovine pituitary extract, or feeder cells, limiting reproducibility and clinical translation. To address this limitation, we developed a well-defined culture medium that enables the long-term expansion of human airway basal stem cells while preserving their proliferative and differentiation potential. Methods: We formulated a novel medium (sfBSC) comprising 14 defined components, including basal media, supplements, growth factors (EGF, FGF10), and signaling inhibitors (Y-27632, A-83-01, DAPT, DMH1). Human bronchial and small airway epithelial cells were cultured over multiple passages in sfBSC and assessed for morphology, population doublings, marker expression, and differentiation capacity via air-liquid interface and organoid cultures. RNA-seq was performed to explore molecular changes across passages. Results: Bronchial and small airway epithelial cells were expanded up to 17 and 24 passages, respectively, with stable morphology and consistent cell size (10-15 μm). Cells maintained expression of canonical BSC markers (TP63, KRT5, NGFR) throughout long-term culture. Differentiation assays confirmed the ability to generate ciliated, goblet, and club cells. Optimized concentrations of EGF (1 ng/mL) and FGF10 (0.4 ng/mL) were critical for sustained proliferation. RNA-seq revealed stable marker expression and metabolic changes over time.Conclusions: sfBSC medium offers a defined, reproducible, and scalable platform for basal stem cell culture, enabling applications in disease modeling, regenerative medicine, and clinical-grade cell production.
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