Single cell RNA-sequencing on stimiluated mouse lung lipofibroblast-like cells and control cells.

To achieve the in vitro functions of lipofibroblasts, isolated murine lung fibroblasts were cultured and biochemically stimulated. Stimulated fibroblasts in which a lipofibroblast-like phenotype had been induced using conventional methods displayed pronounced lipid inclusions. scRNA-seq analysis of lipofibroblast-like cells demonstrated that stimulated lipofibroblast-like cells displayed high transcript expression of canonical makers, like Plin2, as reported by others. Murine lungs were dissociated and the entire cell suspension was then plated on appropriately sized tissue culture plasticware. Fibroblasts were cultured in advanced DMEM/F12 (#12634010, Thermo Fisher Scientific), with 10 % vol/vol FBS (HyClone) or to induce a lipofibroblast like phenotype with the addition of 1 % vol/vol ITS+3 liquid media supplement (#I2771, Merck Milipore, Burlington, MA, USA) to the culture media. Media was changed every other day and the cells sub cultured when 80 % confluent. Fibroblasts cultured to passage three (P3) were considered free of contaminating cells based on previous experience in the laboratory. At P3 the cultured fibroblasts were then stimulated with one or a combination (as indicated) of 10 μM rosiglitazone (#72622, Stem Cell Technologies, Cambridge, MA, USA), 1 μM SB4315442 (#1614, R&D Systems, Minneapolis, MO, USA), 4 μM, rhBMP4 (#314-BP, R&D Systems) for 14 days. Matched control cells isolated from the same lung were stimulated in an identical media, for an identical duration, with the appropriate vehicle.