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Identification, Characterization, and Prospective Isolation of a Fibroblast Lineage Contributing to Dermal Development, Cutaneous Scarring, Radiation Fibrosis, and Cancer Stroma
Graham G. Walmsley, B.A.1, Yuval Rinkevich, Ph.D.1, Michael S. Hu, M.D.1, Adrian McArdle, MB, BCh, BAO, MRCSI2, Zeeshan N. Maan, MBBs, MS, MRCS2, Hermann P. Lorenz, M.D.1, Irving L. Weissman, M.D.1, Michael T. Longaker, M.D., M.B.A.1.
1Stanford University School of Medicine, Stanford, CA, USA, 2Stanford University, Stanford, CA, USA.
Fibroblasts represent a heterogeneous population of cells with diverse functional and phenotypic features. Such diversity remains largely undefined due to phenotypic drift in vitro and a lack of unique surface markers for functional subclasses of fibroblasts. Here we track the contributions of two distinct fibroblast lineages, defined by embryonic expression of Engrailed-1 (En1) and Wnt1, to connective tissue formation in the context of dermal development, cutaneous wounding, radiation fibrosis, and cancer stroma.
En1Cre and Wnt1Cre transgenic mice were crossed with ROSA26mTmG mice, which harbor a double-fluorescent reporter that replaces the expression of membrane-bound tomato red with membrane-bound green fluorescent protein following Cre-mediated recombination. The resulting offspring were used to trace En1- and Wnt1-derived fibroblasts, defined by their GFP positivity, into the dorsal and oral dermis respectively. Flow cytometry allowed for the isolation of En1-derived fibroblasts from the dorsal dermis of wild type mice on the basis of highly expressed surface molecules. Transplantation methodologies functionally corroborated these surface markers in the context of wounding healing, radiation fibrosis, and cancer stroma formation. Finally, we performed reciprocal transplantation of FACS-sorted fibroblast lineages between the dorsal back and oral cavity to assess whether differences in the outcomes of wound repair between cutaneous and oral dermis are a consequence of cell intrinsic vs. environmental properties.
Here we examined a distinct cellular lineage in the dorsal dermis, defined by embryonic expression of Engrailed-1, and characterized its contribution to connective tissue deposition during dermal development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Using flow cytometry and in silico approaches, we identified CD26 as a surface marker that enables the prospective isolation of this fibrogenic lineage from the dorsal dermis. Microarray analysis of FACS-isolated En1- and Wnt1-derived fibroblasts revealed significant differences in their transcriptional programs that were otherwise masked by conventional harvesting/culturing methodologies. Reciprocal transplantation experiments further identified En1- and Wnt1-derived fibroblasts as functionally distinct lineages in terms of their migratory/secretory programs, and revealed that site-specific differences in scar formation between oral and cutaneous dermis are a consequence of lineage-intrinsic fibrogenic potential rather than extrinsic environmental effects.
These studies demonstrate that distinct tissue-resident fibroblast lineages are responsible for the majority of connective tissue deposition during dermal development, wound healing, radiation fibrosis, and cancer stroma formation. Furthermore, we define unique fibrogenic properties intrinsic to each lineage that explain differences in the scarring/fibrosis of cutaneous vs. oral dermis. These studies demonstrate that distinct fibroblast lineages represent unique cell types and hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.
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