Epithelial- IPF Fibroblast Crosstalk and its Effects on Gene Expression of Airway Epithelial Differentiation Markers In 3D Organoids
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Date
2023-11
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Abstract
Introduction: Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive disease characterized by a loss of alveolar epithelial type 2 cells (AEC2s) and a concurrent and abnormal rise of KRT5+ basal cells in the alveoli. This phenomenon is a contributing factor to mortality in patients with this condition. We hypothesized that the interaction between IPF human lung fibroblasts (HLF) with KRT5+ basal cells in an in vitro 3D organoid model may alter the airway epithelial cells differentiation.
Aim: To compare the differentiation potential of KRT5+ basal cells cocultured with IPF versus healthy human lung fibroblasts into airway organoids.
Methods: A transcriptomic method, specifically single-cell RNA sequencing (scRNA-seq), was conducted in an in vitro 3D organoid model. Basal cells were isolated from the bronchus and were called human bronchial epithelial cells (HBECs). HBECs from healthy donors were co-cultured with HLF from healthy and/or IPF donors. Two co-cultures were established for the subsequent comparisons, healthy HBEC & healthy HLF and healthy HBEC & IPF HLF and a monoculture of HBECs. Organoids were dissociated to obtain a whole cell suspension for scRNA-Seq. HBECs were segregated based on KRT5 expression and fused for UMAP. The cells were subsequently grouped into distinct basal subsets as well as differentially expressed genes (DEG) in each HBECs subset by bioinformatics analysis.
Results: Five different clusters were identified by single-cell RNA-seq analysis from KRT5+ cell population: multi-potent basal, ciliated, club, club-ciliated and secretory primed basal. We also identified a cluster of PDGFRA+ fibroblasts in the co-cultures. Club and club-ciliated clusters were decreased in the healthy BC & IPF HLF co-culture compared to the co-culture with healthy HLF and BC monoculture. Our results also showed that the number of cells in the ciliated cluster increased and, conversely, the club cluster decreased in co-cultures compared to the monoculture of BC. Additionally, the results showed that the interaction of BC & IPF HLF decreased the alveolar fibroblast cluster compared to the co-culture with healthy HLF.
Conclusion: The results showed that BC and IPF HLF co-culture compared to the Healthy HLF co-culture, has a lower differentiation potential in 3D airway organoids.
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Gene expression in 3D Airway Organoids