Dataset Information

A Novel Fibroblast Reporter Cell Line for in vitro Studies of Pulmonary Fibrosis.

ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a fatal disease of the lower respiratory tract with restricted therapeutic options. Repetitive injury of the bronchoalveolar epithelium leads to activation of pulmonary fibroblasts, differentiation into myofibroblasts and excessive extracellular matrix (ECM) deposition resulting in aberrant wound repair. However, detailed molecular and cellular mechanisms underlying initiation and progression of fibrotic changes are still elusive. Here, we report the generation of a representative fibroblast reporter cell line (10-4A BFP ) to study pathophysiological mechanisms of IPF in high throughput or high resolution in vitro live cell assays. To this end, we immortalized primary fibroblasts isolated from the distal lung of Sprague-Dawley rats. Molecular and transcriptomic characterization identified clone 10-4A as a matrix fibroblast subpopulation. Mechanical or chemical stimulation induced a reversible fibrotic state comparable to effects observed in primary isolated fibroblasts. Finally, we generated a reporter cell line (10-4A BFP ) to express nuclear blue fluorescent protein (BFP) under the promotor of the myofibroblast marker alpha smooth muscle actin (Acta2) using CRISPR/Cas9 technology. We evaluated the suitability of 10-4A BFP as reporter tool in plate reader assays. In summary, the 10-4A BFP cell line provides a novel tool to study fibrotic processes in vitro to gain new insights into the cellular and molecular processes involved in fibrosis formation and propagation.

SUBMITTER: Nemeth J

PROVIDER: S-EPMC7582034 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A Novel Fibroblast Reporter Cell Line for <i>in vitro</i> Studies of Pulmonary Fibrosis.

Nemeth Julia J Schundner Annika A Quast Karsten K Winkelmann Veronika E VE Frick Manfred M

Frontiers in physiology 20201009

Idiopathic pulmonary fibrosis (IPF) is a fatal disease of the lower respiratory tract with restricted therapeutic options. Repetitive injury of the bronchoalveolar epithelium leads to activation of pulmonary fibroblasts, differentiation into myofibroblasts and excessive extracellular matrix (ECM) deposition resulting in aberrant wound repair. However, detailed molecular and cellular mechanisms underlying initiation and progression of fibrotic changes are still elusive. Here, we report the genera ...[more]

PMID: 33162897

Similar Datasets

Project description:BackgroundRecent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types.MethodsSurgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441.ResultsDNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells.ConclusionsTogether, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

Dataset Information

A Novel Fibroblast Reporter Cell Line for in vitro Studies of Pulmonary Fibrosis.

Publications

A Novel Fibroblast Reporter Cell Line for <i>in vitro</i> Studies of Pulmonary Fibrosis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets