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Development Of A Novel Murine Xenograft Model Of Human Foreskin As A Platform For Investigating Skin Fibrosis
Abra H. Shen, SB, Mimi R. Borrelli, MBBS, MSc, Shamik Mascharak, BS, Nestor M. Diaz Deleon, Sandeep Adem, MS, Derrick C. Wan, MD, Michael T. Longaker, MD, MBA, H. Peter Lorenz, MD, FACS.
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University, Stanford, CA, USA.

PURPOSE: Human cutaneous skin scarring is difficult to accurately capture using animal models as there are significant differences between human and mouse skin. For example, human skin has a thicker epidermis and dermis. To investigate the complex cellular interactions underlying cutaneous wound healing, several humanized mouse models have been proposed. However, current models that utilize full-thickness human adult skin are limited by poor engraftment, and variability of skin samples across age, gender, body site, and sun-exposure. We describe a novel xenograft model using full-thickness human neonatal foreskin to investigate the mechanisms mediating fibrosis in human skin.

METHODS: Human foreskin was collected from routine neonatal circumcisions, cut into 8 mm circular full-thickness grafts, and transplanted subcutaneously on the dorsum of NOD.CgPrkdcscidIl2rgtm1Wjl (NSG) pups five to seven days after birth. After pup weaning three weeks later, the overlying mouse skin was excised to expose the human foreskin xenograft to air. Xenografts healed over the subsequent 30-60 days (Figure 1A). To explore the baseline response to wounding, linear incisions were made in the xenograft. Wounds were followed grossly and harvested on post-operative day (POD) 14. To induce fibrosis, bleomycin was injected daily into the dermis of unwounded xenografts until harvest one week later. To reduce fibrosis, fibroblast growth factor 2 (FGF2) was injected into a xenograft on POD 1, 2, 3 and 4 after wounding and was harvested two weeks later. Harvested tissues were processed for histology, immunofluorescent, and Picro Sirius Red stain.

RESULTS: Human foreskin successfully engrafted with mouse dorsal skin. On H&E staining, xenografted human skin was visualized as distinct from mouse skin by a thicker dermis and lack of hair follicles, consistent with the microscopic differences seen between ungrafted foreskin and mouse skin (Figure 1B). This was confirmed by mouse-specific CD90 immunofluorescence staining (Figure 1C). Wounded xenografted skin at POD14 (Figure 1Di) displayed a higher density of collagen on Trichrome staining (Figure 1Dii) and immunostaining with human- and mouse-specific Collagen I (Figure 1Diii). Grossly, unwounded bleomycin-injected skin appeared fibrotic while FGF2-injected wounds appeared more similar to unwounded skin (Figure 1Ei). These findings were confirmed histologically using Trichrome and Picro Sirius Red staining (Figure 1Eii). FGF2-injected wounds also had a marked increase in vascularity as measured by CD31 staining (Figure 1F).

CONCLUSIONS: This is a novel xenograft model of human foreskin that serves as a platform to explore wound healing and fibrosis. Future work will include using the model to further investigate human fibroblast heterogeneity and potential therapeutics to reduce human scarring.

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