Integrated Single-Cell Analysis Reveals Spatially and Temporally Dynamic Heterogeneity in Fibroblast States During Wound Healing.

ABSTRACT

Wound healing is a dynamic process over temporal and spatial scales. Key to repair outcomes are fibroblasts, yet how they modulate healing across time and in different wound regions remains incompletely understood. By integrating single-cell RNA-sequencing datasets of mouse skin and wounds, we infer that fibroblasts are the most transcriptionally dynamic skin-resident cells, evolving during postnatal skin maturation, and rapidly after injury towards distinct late scar states. We show that transcriptional dynamics in fibroblasts are largely driven by genes encoding extracellular matrix and signaling factors. Lineage trajectory inference and spatial gene mapping reveal that Prg4-expressing fibroblasts transiently emerge along early wound edges. Within days, they become replaced by long-lasting and likely non-interconverting fibroblast populations, including Col25a1-expressing and Pamr1-expressing fibroblasts that occupy subepidermal and deep scar regions, respectively, where they engage in reciprocal signaling with immune cells. Signaling inference shows that fibroblast-immune crosstalk repeatedly uses some signaling pathways across wound healing time, while use of other signaling pathways is time- and space-limited. Collectively, we uncovered high transcriptional plasticity by wound fibroblasts, with early states transiently forming distinct micro-niches along wound edges and in the fascia, followed by stable states, that stratify scar tissue into molecularly dissimilar upper and lower layers.