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. Overall design: 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.