Compositional differences in ECM deposited by Acomys cahirinus and Mus musculus fibroblasts in vitro

The Spiny Mouse (Acomys cahirinus) has the most extensive regenerative capabilities of any known mammal and can regenerate with little to no fibrosis. Key differences between the wounds of Acomys and a scarring counterpart (Mus musculus) include a reduction in fibroblast activation and a less-fibrous ECM composition. We hypothesize that these differences are due in part to reduced stiffness-mediated fibroblast-to-myofibroblast transition (FMT) in Acomys fibroblasts. We sought to investigate the impact of FMT and species differences on ECM composition by fabricating cell-derived matrices (CDMs) from Acomys and Mus fibroblasts.
CDMs were generated by culturing Acomys (N=3) or Mus (N=3) fibroblasts on collagen-functionalized PDMS or glass for 16-21 days in the presence of a macromolecular crowder to promote ECM production. To assess the impact of stiffness on ECM composition, CDMs were generated on both “soft” PDMS (Sylgard 527, ~5kPa) and “stiff” PDMS (Sylgard 182, ~1MPa). The CDMs were decellularized, homogenized, and sent to the University of Florida Mass Spectrometry Research and Education Center for label-free quantitative proteomics.
The comparisons of interest were species-specific (e.g. Acomys CDMs generated on glass vs. Mus CDMs generated on glass) or stiffness-specific (e.g. Acomys CDMs generated on soft PDMS vs. Acomys CDMs generated on stiff PDMS). The most notable differences were observed when comparing between species. Specifically, there were 186 proteins unique to Acomys CDMs and 169 proteins unique to Mus CDMs on soft PDMS. On stiff PDMS, there were 241 proteins unique to Acomys and 159 unique to Mus. On glass, there were 184 proteins unique to Acomys and 181 proteins unique to Mus. When we filtered for secreted proteins, proteins in Acomys CDMs were less associated with fibrosis. Fibrosis-associated ECM proteins including fibrillin 1, ADAMTS1, SPARC, and galectin 1 were significantly reduced or absent in Acomys CDMs compared to Mus CDMs. In addition, proteins that have been connected to fibrosis resolution, including Col12a1 and clusterin, were upregulated in Acomys CDMs.