Kanagarajah, K. | bioRxiv Science

bioRxiv2024

Single-cell RNA-Sequencing Co-Expression Analysis with CFTR in Lung Tissue

1BackgroundWhile cystic fibrosis is caused by loss-of-function variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), other modifier genes have been shown to associate with disease severity. Co-expression of modifiers with CFTR in normal tissue indicates a cooperative relationship and suggests the potential for compensation in the presence of CFTR dysfunction. We examined the co-expression relationships with CFTR in the lung using single cell RNA sequencing to pinpoint cell types and their modifiers involved in the forced expiratory volume in 1 second (FEV1)-based cystic fibrosis lung phenotype and support target cell-type prioritization for therapy 1. MethodsSmartSeq2 single cell RNA sequencing data from non-cystic fibrosis lung tissue was used for evaluation of co-expression with CFTR and modifier genes. Zero-inflated negative binomial model was used to formally test the co-expression association. 10X Chromium based single cell RNA sequencing data from both cystic fibrosis and non-cystic fibrosis studies were assessed graphically to confirm conclusions from the SmartSeq2 primary analysis. ResultsDifferentiating basal, club and alveolar epithelial type 2 cells were found to have high proportions of cells expressing CFTR as well as the greatest number of significant co-expression relationships with the modifiers. In particular, among alveolar epithelial type 2 cells, we observed a strong co-expression trio relationship between CFTR, SLC6A14 and SLC26A9 (p < 0.05). ConclusionsCFTR-modifier gene co-expression suggests basal, club and alveolar epithelial type 2 cells show coordinated expression. Alveolar epithelial type 2 cells showed strong co-expression evidence with two of the most established cystic fibrosis modifier genes.

genetics↗

bioRxiv2023

Early human fetal lung atlas reveals the temporal dynamics of epithelial cell plasticity.

While animal models have provided key insights into conserved mechanisms of how the lung forms during development, human-specific developmental mechanisms are not always captured. To fully appreciate how developmental defects and disease states alter the function of the lungs, studies in human lung models are important. Here, we sequenced >150,000 single single-cells from 19 healthy human fetal lung tissues from gestational weeks 10-19 and identified at least 58 unique cell types/states contributing to the developing lung. We captured novel dynamic developmental trajectories from various progenitor cells that give rise to club, ciliated, and pulmonary neuroendocrine cells. We also identified four CFTR-expressing progenitor cell types and pinpointed the temporal emergence of these cell types. These developmental dynamics reveal broader epithelial cell plasticity and novel lineage hierarchies that were not previously reported. Combined with spatial transcriptomics, we identified both cell autonomous and non-cell autonomous signalling pathways that may dictate the temporal and spatial emergence of cell lineages. Finally, we showed that human pluripotent stem cell-derived fetal lung models capture cell lineage trajectories specifically through CFTR-expressing progenitor cells, that were also observed in the native fetal tissue. Overall, this study provides a comprehensive single-cell atlas of the developing human lung, outlining the temporal and spatial complexities of cell lineage development. HighlightsO_LISingle-cell transcriptomics atlas from 19 human fetal lungs reveals cellular heterogeneity and previously unappreciated cellular plasticity in the epithelial compartment. C_LIO_LIIdentification of novel CFTR-expressing progenitor cells that gives rise to club, ciliated and PNEC. C_LIO_LINovel RNA velocity facilitated the identification of dynamic lineage trajectories in the epithelial compartment. C_LIO_LITemporally regulated cell signaling through promiscuous interactions between sender and receiving cells may dictate cell lineage fates. C_LIO_LIIntegration of human pluripotent stem cell (hPSC)-derived fetal lung cells and organoids with primary lung dataset show hPSC-differentiations captures key developmental trajectories of fetal epithelial cell states. C_LI

developmental biology↗