Fibroblasts are the predominant cell type that synthesizes and remodels the extracellular matrix in organs during both embryonic and adult life and are central to the fibrotic response across a range of pathologic states. Morphologically, they are most commonly defined as elongated, spindle-shaped cells that readily adhere to and migrate over tissue culture substrates. However, fibroblasts exhibit a variety of shapes and sizes, depending on the physiologic or pathologic state of the host tissue, and represent a heterogeneous population of cells with diverse features that remain largely undefined. In cutaneous tissues, fibroblasts display considerable functional variation during wound repair, depending on developmental time, and between anatomic sites. For example, wounds in the oral cavity remodel with minimal scar formation, whereas scar tissue deposition within cutaneous wounds is substantial. The mechanisms underlying this diversity of regenerative responses in cutaneous tissues have remained largely underexplored.

The effective development of treatments for fibrosis depends on a mechanistic understanding of its pathogenesis. The identification and characterization of distinct lineages of fibroblasts, based on functional role, hold potential value for developing therapeutic approaches to fibrosis. We employed a nonselective depletion-based fluorescence-activated cell sorting strategy to isolate fibroblasts from a murine model that labels a particular lineage of cells based on the gene expression of Engrailed-1 (En1) in its embryonic progenitors. Using this reporter mouse, we reveal the presence of at least two functionally distinct embryonic fibroblast lineages in murine dorsal skin and characterize a single lineage that plays a primary role in connective tissue formation.

Genetic lineage tracing and transplantation assays demonstrate that a single somitic-derived fibroblast lineage that is defined by embryonic expression of En1 is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Reciprocal transplantation of distinct fibroblast lineages between the dorsal back and oral cavity induces ectopic dermal architectures that mimic their place of origin rather than their site of transplantation. Lineage-specific cell ablation using transgenic-mediated expression of the simian diphtheria toxin receptor in conjunction with localized administration of diphtheria toxin leads to diminished connective tissue deposition in wounds and significantly reduces melanoma growth in the dorsal skin of mice. Tensile strength testing reveals that, although scar formation is significantly reduced in wounds treated with diphtheria toxin to ablate the En1 lineage, as compared with control wounds, tensile strength in lineage-ablated wounds is not significantly affected. Using flow cytometry and in silico approaches, we identify CD26/dipeptidyl peptidase-4 (DPP4) as a surface marker that allows for the isolation of this fibrogenic, scar-forming lineage. Small molecule–based inhibition of CD26/DPP4 enzymatic activity in the wound bed of wild-type mice during wound healing results in diminished cutaneous scarring after excisional wounding.

We have identified multiple lineages of fibroblasts in the dorsal skin. Among these, we have characterized a single lineage responsible for the fibrotic response to injury in the dorsal skin of mice and demonstrated that targeted inhibition of this lineage results in reduced scar formation with no effect on the structural integrity of the healed skin. Furthermore, these studies demonstrate that intra …