E. R. Zielins1, M. Januszyk1, C. Blackshear1, E. A. Brett1, M. Chin1, S. Vistnes1, S. Menon1, S. Shailendra1, G. C. Gurtner1, M. T. Longaker1, D. C. Wan1 1Stanford University,Plastic And Reconstructive Surgery,Palo Alto, CA, USA
Introduction: Adipose-derived stromal cells (ASCs), the stem cell containing population derived from the stromal vascular fraction (SVF) of adipose tissue, has shown much promise as a technique to improve fat graft retention. As ASCs are a significantly heterogenous cell population, identification of cell subpopulations with enhanced secretion of pro-angiogenic growth factors would facilitate their use in strategies to further improve fat graft take.
Methods: Human lipoaspirate was enzymatically digested in order to obtain SVF cells. Individual ASCs were isolated via flow cytometry based on an established surface marker profile. As we have previously described, single cell transcriptional profiling of select angiogenic and adipogenic genes, as well as multiple cell surface markers, was employed. Applying a Fuzzy C-Means algorithm to this data allowed for partitioning of ASCs into multiple, functionally distinct clusters. Linear discriminant analysis was then performed to correlate surface marker expression with cluster definition. We then utilized flow cytometry, a cell proliferation assay, qRT-PCR, and an in vitro endothelial tube formation assay, to evaluate CD248, the most promising of these markers, for the potential to isolate cells with enhanced angiogenic potential.
Results: Using this strategy, we identified multiple markers with the potential capacity to delineate functional subgroups of ASCs based on angiogenic gene expression. Analysis of freshly harvested SVF cells by flow cytometry using CD248, the strongest correlating surface marker, showed 16% of cells were CD248+ and 84% were CD248–. Proliferation, gene analysis, and endothelial tube formation assays were performed, showing differences between CD248+, CD248-, and unsorted cell populations. qRT-PCR showed CD248+ cells to have significantly higher VEGFa secretion (**p<0.01) compared to both unsorted and CD248- cells. CD248+ cells additionally were found to promote increased endothelial tube formation in vitro in comparison to unsorted and CD248- cells.
Conclusion: Our methodology has identified multiple cell surface markers associated with ASC angiogenic capacity. The most highly correlated marker, CD248, characterizes a cell population with significantly enhanced angiogenic potential, suggesting that it may be used in in vivo applications for improvement of fat graft retention.