Integrin α8 is a useful cell surface marker of alveolar lipofibroblasts.

Abstract

Background: Recent advances in comprehensive gene analysis revealed the heterogeneity of mouse lung fibroblasts. However, direct comparisons between these subpopulations are limited due to challenges in isolating target subpopulations without gene-specific reporter mouse lines. In addition, the properties of lung lipofibroblasts remain unclear, particularly regarding the appropriate cell surface marker and the niche capacity for alveolar epithelial cell type 2 (AT2), an alveolar tissue stem cell.

Methods and results: Using cell surface markers applicable even into wild-type mouse lungs, we could classify PDGFRα⁺ total lung resident fibroblasts into at least two major distinct subpopulations: integrin α8 (ITGA8)⁺ and SCA-1⁺ fibroblasts. We analyzed their characteristics, including lipid content, transcriptome profiles, and alveolar stem cell niche capacity. ITGA8⁺ fibroblasts showed higher positivity of intracellular lipid droplets compared to SCA-1⁺ fibroblasts (91.0 ± 1.5% vs. 5.0 ± 0.5% in LipidTOX staining; 91.3 ± 1.4% vs. 7.1 ± 1.7% in Oil Red O staining). The fluorescence intensity of LipidTOX in the ITGA8⁺ fibroblasts was highest in newborn compared to adult or aged lungs. The transcriptome profile of ITGA8⁺ fibroblasts in adult mouse lungs, evaluated through two independent single-cell RNA-seq datasets, consistently showed higher expression of Tcf21 and Plin2, which are canonical markers of lipofibroblasts. ITGA8⁺ fibroblasts were primarily located in the alveolar area, particularly in the neighborhood of AT2. Compared to SCA-1⁺ fibroblasts, ITGA8⁺ fibroblasts showed higher mRNA expression of potential AT2-supportive factors, Fgf10, Fgf7, and Wnt2, but unexpectedly, exhibited lower efficiency in alveolar organoid formation.

Conclusions: ITGA8⁺ lung fibroblasts correspond to alveolar lipofibroblasts, but the alveolar niche capacity may be lower than SCA-1⁺ lung fibroblasts. Further studies are necessary for the functional distinction between lung fibroblast subpopulations.