The Proangiogenic Adipose-derived Stromal Cell (ASC): Cd146+ Ascsenhance The Regenerative Effects Of Grafted Fat
Mimi R. Borrelli, MD, MA, Sandeep Adem, MS, Nestor M. Deleon Diaz, Stephanie Vistnes, MS, Charles Blackshear, MD, Abra H. Shen, SB, Dung Nguyen, MD, Hermann P. Lorenz, MD, Michael T. Longaker, MD, MA, Derrick C. Wan, MD.
Stanford, menlo park, CA, USA.
Purpose: Initially popularized for its ability to restore soft tissue volume and soften contour deformities, fat has become increasingly appreciated for its regenerative potential. The adipose-derived stromal cells (ASCs) are thought to be primarily responsible for these beneficial effects. Recent work has revealed ASCs are a heterogeneous population comprised of distinct subpopulations of cells with differing functional capacities. Given that grafted fat often undergoes significant resorption, and a lack of blood supply is thought to be a contributing factor, we hypothesized the existence of a subpopulation of ASCs with enhanced angiogenic potential that can increase the viability of fat grafts.
Methods: Microfluidic single-cell sorting and linear discriminant analysis of human fat identified CD146 as a surface marker of ASCs with strong expression of proangiogenic genes. Flow cytometry was used to both confirm the existence of a CD146+ ASC subpopulation and to sort CD146+, CD146-, and ‘unsorted’ (US) ASCs (all CD34+) from healthy adult human lipoaspirate (n=3). The expression of proangiogenic growth factors (ANGPT1, VEGF) was compared between populations at the gene and protein level using PCR and ELISA assays, respectively. In vitro angiogenic potential was assessed using endothelial tube-forming assays (Fig.1Ai). In vivo regenerative potential was assessed by grafting the scalps of immunodeficient CD1 Nude mice with 200ul of human lipoaspirate enriched with either CD146+, CD146-, or US ASCs (10,000cells/200ul) (Fig.1Aii). Graft retention was monitored radiographically for 8 weeks, at which point the grafted fat was explanted and processed for histological assessment of graft quality and vascularization.
Results: The CD146+ subpopulation comprised 28.2% of all US ASCs (Fig.1B). CD146+ ASCs exhibited significantly increased expression of VEGF and ANGP1 at the gene and protein level (***p<0.001) (Fig.1C). In vitro, CD146+ ASCs had enhanced tube-forming capabilities; compared to CD146- ASCs, endothelial cells co-cultured with CD146+ ASCs formed endothelial tubes that were denser, longer, and that were more branched (Fig.1D). In vivo, fat grafts enriched with CD146+ ASCs underwent less resorption compared to fat alone or fat supplemented with CD146- ASCs, and this trend became significant 6 weeks post grafting (*p<0.05) (Fig.1Ei-ii). Histological assessment of explanted fat grafts 8 weeks post grafting indicated that supplementation with CD146+ ASCs increased graft integrity, evidence using Hematoxylin and Eosin- and perilipin-staining, and increased graft vascularity, as shown by CD31 immunofluorescent staining (Fig.1F).
Conclusion: We have identified a subpopulation of ASCs positive for CD146 with enhanced angiogenic effects. These effects are likely mediated by increased expression of proangiogenic factors such as VEGF and ANGP1. These findings suggest that enriching lipoaspirate with CD146+ ASCs may enhance fat graft vascularization and retention in the clinical setting.
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