Single-cell and single-nucleus transcriptomic profiling of mouse models and human lung tissues to study pulmonary fibrosis
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE309745 | GEO | 2025/11/03
REPOSITORIES: GEO
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Project description:Single-cell and single-nucleus transcriptomic profiling of mouse models and human lung tissues to study pulmonary fibrosis
| PRJNA1336840 | ENA
Project description:Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease manifested by overtly scarred peripheral and basilar regions and more normal-appearing central lung areas. Lung tissues from macroscopically normal-appearing (IPFn) and scarred (IPFs) areas of explanted IPF lungs were analyzed by RNASeq and compared with healthy control (HC) lung tissues. There were profound transcriptomic changes in IPFn compared with HC tissues, which included elevated expression of numerous immune-, inflammation-, and extracellular matrix-related mRNAs, and these changes were similar to those observed with IPFs compared to HC. Comparing IPFn directly to IPFs, elevated expression of epithelial mucociliary mRNAs was observed in the IPFs tissues. Thus, despite the known geographic tissue heterogeneity in IPF, the entire lung is actively involved in the disease process, and demonstrates pronounced elevated expression of numerous immune-related genes. Differences between normal-appearing and scarred tissues may thus be driven by deranged epithelial homeostasis or possibly non-transcriptomic factors.
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Project description:Mechanical ventilation contributes to diaphragm atrophy and muscle weakness, which is referred to as ventilator-induced diaphragmatic dysfunction (VIDD). The pathogenesis of VIDD has not been fully understood until recently. The aim of this study was to investigate the effects of 24 h of mechanical ventilation on fibro-adipogenic progenitor (FAP) proliferation, endothelial-mesenchymal transition (EndMT), and immune cell infiltration driving diaphragm fibrosis in a rabbit model. The rabbits were anaesthetized and randomly divided into two groups (n = 3 each group): a control group and an experimental group. Diaphragm nuclei for sequencing were prepared by dissociating and filtering muscle tissue. 10X Genomics Platform for single-nucleus RNA sequencing (snRNA-seq) was used to profile the cells. Normalization and clustering were performed by Seurat, and clusters were manually annotated as different cell types. In this study, we performed differentially expressed genes (DEGs) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, pseudotime analysis and high dimensional weighted gene coexpression network analysis (hdWGCNA) to identify the key genes and signaling pathways related to the pathogenesis of VIDD. We further performed quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting to verify the results of snRNA-seq. The snRNA-seq results showed that acute postmechanical ventilation diaphragm cell changes included an increase in the proportion of fibroblasts and a decrease in the proportion of myofibres. The DEGs, KEGG, hdWGCNA and pseudotime analyses demonstrated that fibro-adipogenic progenitor (FAP) proliferation, endothelial-mesenchymal transition (EndMT) and immune cell infiltration are the three main processes involved in early stage of fibrosis development, among which Pdgfd, Sema3a, Cxcr2, are the corresponding regulatory genes. Glycolysis and the gene Pfkfb3 are also important metabolic factors for fibrosis formation. Negr1 and Mef2c are involved in phrenic nerve ending loss and diaphragm fibre atrophy. The qRT-PCR data showed that the mRNA levels of the genes Pdgfd, Cxcr2, Pfkfb3 and Negr1 were significantly greater in the experimental group than in the control group (P < 0.01), and the expression levels of Sema3a and Mef2c were significantly lower (P < 0.01). Despite limitations, including the lack of functional evaluations to confirm ventilator-induced diaphragm dysfunction (VIDD) and the absence of data validating diaphragm unloading during ventilation, our findings suggest that FAP proliferation and immune cell infiltration may play a role in the early stage of driving diaphragm fibrosis during mechanical ventilation. However, future studies are needed to confirm these findings and investigate the potential mechanisms underlying them.
| S-EPMC11682457 | biostudies-literature
Project description:Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.
| S-EPMC6580683 | biostudies-literature
Project description:Idiopathic pulmonary fibrosis (IPF), a devastating, fibroproliferative, chronic lung disorder, is associated with expansion of fibroblasts/myofibroblasts, which leads to excessive production and deposition of extracellular matrix. IPF is typically clinically identified as end-stage lung disease, after fibrotic processes are well-established and advanced. Fibroblasts have been shown to be critically important in the development and progression of IPF. We hypothesize that differential chromatin access can drive genetic differences in IPF fibroblasts relative to healthy fibroblasts. To this end, we performed assay of transposase-accessible chromatin sequencing to identify differentially accessible regions within the genomes of fibroblasts from healthy and IPF lungs. Multiple motifs were identified to be enriched in IPF fibroblasts compared with healthy fibroblasts, including binding motifs for TWIST1 and FOXA1. RNA sequencing identified 93 genes that could be annotated to differentially accessible regions. Pathway analysis of the annotated genes identified cellular adhesion, cytoskeletal anchoring, and cell differentiation as important biological processes. In addition, single nucleotide polymorphism analysis showed that linkage disequilibrium blocks of IPF risk single nucleotide polymorphisms with IPF-accessible regions that have been identified to be located in genes that are important in IPF, including MUC5B, TERT, and TOLLIP. Validation studies in isolated lung tissue confirmed increased expression for TWIST1 and FOXA1 in addition to revealing SHANK2 and CSPR2 as novel targets. Thus, modulation of differential chromatin access may be an important mechanism in the pathogenesis of lung fibrosis.
| S-EPMC8803355 | biostudies-literature
Project description:Chronic obstructive pulmonary disease (COPD) is clinically and molecularly heterogeneous. To investigate COPD heterogeneity, we performed single-nucleus RNA sequencing (snRNA-seq) on lung tissue from individuals with and without COPD spanning the full disease spectrum. We generated 1,516,727 high-quality cell transcriptomes from 146 lung lobes obtained from 141 subjects. The dataset captures broad shifts in cellular composition and disease-associated cell states in epithelial, stromal, and immune populations. These data define the cellular landscape of COPD heterogeneity, revealing molecular drivers and biomarkers that could inform therapeutic strategies.
2025-11-17 | GSE310058 | GEO
Project description:Both single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) can be used to characterize the transcriptional profile of individual cells, and based on these transcriptional profiles, help define cell type distribution in mixed cell populations. However, scRNAseq analyses are confounded if some of the cells are large (>50 µm) or if some of cells adhere more tightly to some adjacent cells than to others. Further, single cell isolation for scRNAseq requires fresh tissue, which may not be available for human or animal model tissues. Additionally, the current enzymatic and mechanical methods for single-cell dissociation can lead to stress-induced transcriptional artifacts. Nuclei for snRNAseq, on the other hand, can be isolated from any cell, regardless of size, and from either fresh or frozen tissues, and compared to whole cells, they are more resistant to mechanical pressures and appear not to exhibit as many cell isolation-based transcriptional artifacts. Here, we describe a time- and cost-effective procedure to isolate nuclei from mammalian cells and tissues. The protocol incorporates steps to mechanically disrupt samples to release nuclei. Compared to conventional nuclei isolation protocols, the approach described here increases overall efficiency, eliminates risk of contaminant exposure, and reduces volumes of expensive reagents. A series of RNA quality control checks are also incorporated to ensure success and reduce costs of subsequent snRNAseq experiments. Nuclei isolated by this procedure can be separated on the 10× Genomics Chromium system for either snRNAseq and/or Single-Nucleus ATAC-Seq (snATAC-Seq), and is also compatible with other single cell platforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and quality control check via RNA Isolation and Analysis Basic Protocol 2: Nuclei Isolation.
| S-EPMC8191490 | biostudies-literature
Project description:UnlabelledBackgroundCombined pulmonary fibrosis and emphysema (CPFE) is characterized by both emphysema of the upper zone and diffuse parenchymal lung disease with fibrosis of the lower zone of the lung on chest computed tomography. The aim of this study was to investigate the mechanism of CPFE regarding gene expressions by comparing the results of microarray sequences between fibrotic and emphysematous lesions in the lungs of CPFE patients.ResultsThe expression profiles of the fibrotic and emphysematous lesions were remarkably different in terms of function. Genes related to the immune system, structural constituents of the cytoskeleton, and cellular adhesion were overexpressed in fibrotic lesions, while genes associated with the cellular fraction, cell membrane structures, vascular growth and biology, second-messenger-mediated signaling, and lung development (all processes that contribute to the destruction and repair of cells, vessels, and the lung) were overexpressed in emphysematous lesions.ConclusionsThe differences in gene expression were detected in fibrotic and emphysematous lesions in CPFE patients. We propose that the development of coexisting fibrotic and emphysematous lesions in CPFE is implemented by these different patterns of gene expressions.
| S-EPMC3541270 | biostudies-literature
Project description:BackgroundIdiopathic pulmonary fibrosis (IPF) is an incurable disease characterized by progressive lung fibrosis ultimately resulting in respiratory failure and death. Recurrent micro-injuries to the alveolar epithelium and aberrant alveolar wound healing with impaired re-epithelialization define the initial steps of the pathogenic trajectory. Failure of timely alveolar epithelial repair triggers hyper-proliferation of mesenchymal cells accompanied by increased deposition of extracellular matrix into the lung interstitium.MethodsWe previously isolated fibrosis-specific mesenchymal stem cell (MSC)-like cells from lung tissue of patients with interstitial lung diseases. These cells produced factors bearing anti-fibrotic potential and changed their morphology from mesenchymal to epithelial upon culture in an epithelial cell (EC)-specific growth medium. Here, we set out to molecularly characterize these MSC-like cell-derived ECs using global gene expression profiling by RNA-sequencing. Moreover, we aimed at characterizing disease-specific differences by comparing the transcriptomes of ECs from IPF and non-IPF sources.ResultsOur results suggest that differentially expressed genes are enriched for factors related to fibrosis, hypoxia, bacterial colonization and metabolism, thus reflecting many of the hallmark characteristics of pulmonary fibrosis. IPF-ECs showed enrichment of both pro- and anti-fibrotic genes, consistent with the notion of adaptive, compensatory regulation.ConclusionsOur findings support the hypothesis of a functional impairment of IPF-ECs, which could possibly explain the poor clinical outcome of IPF that roughly compares to those of advanced-stage cancers. Our study provides a valuable resource for downstream mechanistic investigation and the quest for novel therapeutic IPF targets.
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Project description:Omics analyses are commonly used for identifying pathways and genes responsible for physiologic and pathologic processes. Though sex is considered a biological variable in rigorous assessments of pulmonary responses to oxidant exposures, the contribution of the murine strain is largely ignored. This study utilized an unbiased integrated assessment of high-resolution metabolomic profiling and RNA-sequencing to explore sex- and strain-dependent pathways in adult mouse lungs. The results indicated that strain exhibited a greater influence than sex on pathways associated with inflammatory and oxidant/antioxidant responses and that interaction metabolites more closely resembled those identified as differentially expressed by strain. Metabolite analyses revealed that the components of the glutathione antioxidant pathway were different between strains, specifically in the formation of mixed disulfides. Additionally, selenium metabolites such as selenohomocystiene and selenocystathionine were similarly differentially expressed. Transcriptomic analysis revealed similar findings, as evidenced by differences in glutathione peroxidase, peroxiredoxin, and the inflammatory transcription factors RelA and Jun. In summary, an multi-omics integrated approach identified that murine strain disproportionately impacts baseline expression of antioxidant systems in lung tissues. We speculate that strain-dependent differences contribute to discrepant pulmonary responses in preclincal mouse models, with deleterious effects on clinical translation.
| S-EPMC9612261 | biostudies-literature