Abstract 187: Human Cryopreserved Skin Allografts Recruit M2-macrophages And Induce Angiogenesis In A Murine Xenograft Model

Abstract

Purpose: For over 50 years, human cryopreserved skin allografts (HCAs) have been used for temporary coverage of major burns when autograft donor sites are insufficient. HCAs have also been shown to promote healing of diabetic foot ulcers and prevent fluid loss and infections. Their mechanism of action, however, remains elusive. Methods: HCA and human acellular dermal matrix (ADM) grafts were implanted subcutaneously in C57BL/6 (WT) mice and explanted after 1, 3, 7, 14, and 28 days (n= 5 per group). A sham surgery served as a control. Immunofluorescent staining (IF) of tissue sections was used to determine the immune reaction against HCA and ADM as well as vascularization and pro-angiogenic signaling within the grafts and the overlaying mouse skin. HCA and ADM grafts were also implanted into WT mice parabiosed to GFP-positive mice to analyze the infiltration of circulating cells by IF and flow cytometry. HCA grafts explanted after 14 days were processed for droplet-based microfluidic single cell RNA sequencing (scRNA seq) of infiltrating cells using the 10X Genomics platform. Data were log-normalized and partitioned using UMAP based density mappings. Results: Subcutaneous pockets with implanted grafts healed without clinically apparent rejection. Immune cell infiltration, characterized by F4/80, CD11c, and Myeloperoxidase staining, was greater in HCA compared to ADM on post-implantation day 3, whereas no differences were seen on day 7. CD31 staining showed significantly greater vascularization in HCA on day 7 compared to ADM and sham. HCA also demonstrated higher VEGF expression. A greater number of circulating GFP+ cells were found in HCA compared to ADM and sham by IF and FACS. scRNA seq identified 11 distinct cell clusters out of which 2 were defined as macrophage sub-populations, which highly expressed classical M2 macrophage genes (Mrc1, Arg1, Retnla, Cd163). One M2 subpopulation also highly expressed Col3a1 and pro-angiogenic Hif1a. Conclusion: Our data indicate that the immune reaction to HCA is associated with macrophage polarization towards an M2 phenotype. We have identified a sub-population of pro-angiogenic, collagen-3 expressing macrophages, which may be responsible for increased vascularization after HCA implantation in our xenograft model and may underlie the beneficial clinical effects of HCA transplantation.