Definition and Signatures of Lung Fibroblast Populations in Development and Fibrosis in Mice and Men

Abstract

The heterogeneity of fibroblasts in the murine and human lung during homeostasis and disease is increasingly recognized. It remains unclear if the different phenotypes identified to date are characteristic of unique subpopulations with unique progenitors or whether they arise by a process of differentiation from common precursors. Our understanding of this ubiquitous cell type is limited by an absence of well validated, specific, markers with which to identify each cell type and a clear consensus on the distinct populations present in the lung. Here we describe single cell RNA sequencing (scRNA-seq) analysis on mesenchymal cells from the murine lung throughout embryonic (E) development (E9.5 – 17.5), at post-natal day (P1 – 15), as well as in the adult and the aged murine lungs before and after bleomycin-induced fibrosis. We carried out complementary scRNA-seq on human lung tissue from a P1 lung, a month 21 lung and lung tissue from healthy donors and patients with idiopathic pulmonary fibrosis (IPF). The murine and human data were supplemented with publicly available scRNA-seq datasets. We consistently identified lipofibroblasts, myofibroblasts, pericytes, mesothelial cells and smooth muscle cells. In addition, we identified a novel population delineated by Ebf1 (early B-cell factor 1) expression and an intermediate subtype. Comparative analysis with human mesenchymal cells revealed homologous mesenchymal subpopulations with remarkably conserved transcriptomic signatures. Comparative analysis of changes in gene expression in the fibroblast subpopulations from age matched non-fibrotic and fibrotic lungs in the mouse and human demonstrates that many of these subsets contribute to matrix gene expression in fibrotic conditions. Subtype selective transcription factors were identified and putative divergence of the clusters during development were delineated. Prospective isolation of these fibroblast subpopulations, localization of signature gene markers, and lineage-tracing each cluster are under way in the laboratory. This analysis will enhance our understanding of fibroblast heterogeneity in homeostasis and fibrotic disease conditions.

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

“…Several human pulmonary fibroblast cell lines are already available. However, in contrast to the cell line presented here, none of the currently available human cell lines has been characterized in detail with regards to the representation of a specific mesenchymal cell sub-population ( Adams et al, 2019 ; Reyfman et al, 2019 ; Valenzi et al, 2019 ; Habermann et al, 2020 ; Liu et al, 2020 ; Travaglini et al, 2020 ). This might not only affect cellular responses to pro-fibrotic mechanical and chemical cues, but also affect epithelial-mesenchymal crosstalk and fibrotic remodeling when used in complex co-culture in vitro models (e.g., lung-on-a-chip).…”

“…The human lung contains structural and cellular differences to the rodent lung, however, the alveolus is one of the most conserved regions between rodents and human lungs and whether the mesenchymal and epithelial cells believed to be central to development of IPF are different is still a matter of debate. Even lipofibroblasts, a cell that’s presence has been found in rodents but has long been controversial in the human lung ( Tahedl et al, 2014 ), has recently been identified in human lung ( Liu et al, 2020 ; Travaglini et al, 2020 ). Therefore, we believe that the cell line reported here provides a valuable tool for fibrosis research, offering the opportunity to investigate molecular and cellular responses of a representative mesenchymal cell to mechanical and/or chemical stimuli with high resolution and in high throughput formats.…”

Insights Into Development and Progression of Idiopathic Pulmonary Fibrosis From Single Cell RNA Studies

“…Historically, mesenchymal cells were mainly divided into fibroblasts, smooth muscle cells (SMCs) and pericytes, a derivative of fibroblasts with myofibroblast characteristics, that is closely associated with capillaries ( 39 ). Recent studies, including scRNAseq efforts, have vastly increased the heterogeneity of mesenchymal cells found in the human lung ( 12 – 15 , 20 , 22 , 26 , 27 ).…”

“…This was increased in IPF. These authors also found that all mesenchymal subtypes contributed to the excessive ECM production in fibrosis and that there is little evidence for trans-differentiation of fibroblast subtypes and that fibroblast fate is determined early in lung development ( 15 ). This is supported by another recent study that suggested a continuous trajectory toward IPF archetypes co-occurring within fibroblasts and myofibroblasts, but not necessarily across them.…”

“…Apart from ATII cells and alveolar fibroblasts, recent studies also suggested that other cell types may have an important role in IPF initiation. This entails further, recently identified, sub-populations of specific epithelial and/or mesenchymal cells and possible contributions of other cells in the distal lung, most notably macrophages ( 12 – 15 ).…”