The Anti-fibrotic Role Of Cd74+ Ascs In Grafted Fat In The Irradiated And Non-irradiated Setting
Mimi R. Borrelli, Ronak A. Patel, Jan Sokol, Dung Nguyen, Arash Momeni, Michael T. Longaker, MD, MA, Derrick C. Wan, MD
Stanford, Palo Alto, CA
PURPOSE: Fat grafting is gaining popularity as a technique to reconstruct soft tissue deficiencies and reduce soft tissue fibrosis. In the setting of irradiated, hypovascular, and fibrotic recipient sites, however, fat graft retention can be significantly impaired. We have previously shown enrichment of fat grafts with adipose-derived stromal cells (ASC) can enhance fat graft survival and reverse radiation-induced injury to the surrounding soft tissue. Given the inherent heterogeneity of ASCs, however, identifying a subpopulation with enhanced antifibrotic effects may be of therapeutic benefit in the treatment of radiation-induced fibrosis.
METHODS: We performed single cell transcriptional profiling to cluster ASCs based on expression of key antifibrotic genes and then used linear discriminant analysis to identify surface markers correlating with these clusters. Dermal fibroblasts were incubated with conditioned media from CD74+, CD74-, or unsorted ASCs. Ten ng of TGF-β1 was then added to stimulate the fibroblasts and assess for procollagen type-1 production. Adult immunocompromised CD-1 nude mice were treated with 30 Gy external beam irradiation to their scalp, delivered as six fractionated doses of 5Gy over a period of 12 days. After a 5-week recovery period, irradiated mice and non-irradiated control mice were grafted with 200ul human lipoaspirate enriched with CD74+, CD74-, or unsorted ASCs. Fat graft retention was monitored radiographically over 8 weeks, at which point the mice were sacrificed and the grafted fat and overlying skin were processed for histology.
RESULTS: Single cell transcriptional profiling identified multiple surface markers which correlated with expression of antifibrotic genes (Fig. 1A). Subsequent analysis of bulk transcriptional data revealed CD74+ to identify a subpopulation of ASCs with high expression of the antifibrotic genes HGF, FGF2, and TGF-β3 (*p<0.05) (Fig. 1B). Dermal fibroblasts cultured in CD74+ ASC conditioned media exhibited decreased procollagen type 1 production upon stimulation, compared to fibroblasts cultured in media from CD74- or unsorted ASCs (Fig. 1C). Grafted fat enriched with CD74+ ASCs was less fibrotic, contained less vacuoles and cysts, and had increased vascularization compared to fat supplemented with CD74- or unsorted ASCs. The CD74+ ASC-enriched fat also exhibited the greatest retention rates. These effects were more apparent in the irradiated setting.
CONCLUSION: CD74+ ASCs are a subpopulation of ASCs with anti-fibrotic qualities, and supplementation of lipoaspirate with CD74+ ASCs can improve graft retention and enhance fat graft quality. These results suggest that fibrotic remodeling in the recipient site may play an important role in the integration and vascularization of the grafted tissue.
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