Mouse digit AAV gene delivery into fibroblasts regulates regenerative outcome

The distal mouse digit tip regenerates post-amputation, while the proximal digit undergoes fibrosis. This study presents a comparative single-cell RNA sequencing-based analysis of regenerating and non-regenerating digits to computationally identify fibroblast subpopulations and genes associated with fibrosis and regeneration. To test the sufficiency of newly identified candidate genes to alter wound healing outcomes, we developed a robust adeno-associated virus gene delivery technique for digit fibroblasts. We found that overexpression of candidate pro-fibrotic genes Pcolce2 or Prelp in the blastema modifies normal regeneration and overexpression of candidate pro-regenerative factors Ccl2 or Mest in the proximal digit significantly increases bone deposition. These data demonstrate that the computational analysis combined with the AAV delivery approach presented in this study provides a powerful framework for identifying the driving factors of fibrosis and regeneration in the mammalian digit.