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Identification of Fibroblast Subtypes during Hypertrophic Scar Development
Britta Kuehlmann, MD, Jagannath Padmanabhan, PhD, Melanie Rodrigues, PhD, Geoffrey C. Gurtner, MD, FACS.
Stanford University School of Medicine, Department of Surgery, Hagey Laboratory for Pediatric Regenerative Medicine, Stanford, CA, USA.

Identification of Fibroblast Subtypes during Hypertrophic Scar Development
Britta A. Kuehlmann MD, Jagannath Padmanabhan PhD, Melanie Rodrigues PhD, Geoffrey C. Gurtner MD, FACS
Stanford University School of Medicine, Stanford, CA
Department of Surgery
PURPOSE – The complex biological mechanisms occurring in hypertrophic scarring are still barely understood. However, the identification of (myo)-fibroblasts provides us with clues in determining the pathophysiology of fibrotic changes. Here, we focus on analyzing dermal fibroblast subtypes in murine dorsal skin following hypertrophic scarring in an in vivo model. We aim to understand the role of fibroblast subpopulations in the skin during healing and to determine their involvement in fibrotic scar development.
METHODS – Hypertrophic scars (HTS) were induced by placing a biomechanical loading device onto the dorsum of C57/BL6 mice. On day 4 after causing an incisional wound, which was reapproximated immediately with 6-0 nylon sutures, the mechanical device was placed and tension was applied twice per day for 11 days. On day 11 all devices were removed. The scars of 10 HTS-mice were examined on day 11 and day 18 post-incision. Unwounded skin of C57/BL6-mice served as controls. Hypertrophic scars were identified via staining. The skin/tissue was digested and single cells were subjected to fluorescence-activated cell scanning (FACS) analysis and sorting to isolate cells of interest. Cells were negatively sorted for CD45, CD31 and EpCAM (CD326) and positively sorted for CD34, CD26 and CD55 to isolate fibroblasts and functional fibroblast subpopulations.
RESULTS – FACS analysis revealed a change in the subpopulations of fibroblasts following mechanical stretching of the skin. Specifically, we observed decrease of the stemness marker CD34 in hypertrophic scars compared to unwounded skin. These changes were observed on day 11 as well as day 18 post-incision. CD34 is frequently used as a stemness marker and a marker of adipose-derived stem cells (ASCs). Hypertrophic scars were verified via staining (100% sensitivity).
CONCLUSIONS – For the first time, our results reveal a decrease of the stemness marker CD34 in murine skin cells in hypertrophic scars over time compared to unwounded skin. We are currently conducting single cell transcriptional analyses and functional studies on these fibroblasts to understand the heterogeneity of fibroblasts during hypertrophic scarring and the specific role of CD34+ fibroblasts in skin fibrosis. Our findings have promising therapeutic implications for the treatment of skin fibrosis.


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