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Identification and Isolation of Pro-Angiogenic Stromal Cells for Targeted Improvement of Fat Graft Retention
Monica Chin, B.S.1, Elizabeth R. Zielins, M.D.1, Michael Januszyk, M.D.2, Charles P. Blackshear, M.D.1, Elizabeth A. Brett, M.S.1, Siddarth Menon, B.S.1, Siny Shailendra, B.S.1, William T. Leavitt, B.S.1, Clement D. Marshall, M.D.1, Arash Momeni, M.D.1, Geoffrey C. Gurtner, M.D.1, Michael T. Longaker, M.D., M.B.A.1, Derrick C. Wan, M.D.1.
1Stanford University, Stanford, CA, USA, 2University of California Los Angeles, Los Angeles, CA, USA.

Purpose: Enrichment of lipoaspirate with stromal cells may enhance fat graft retention, but the functional heterogeneity of these supplemental cells complicates this approach. Studies, however, have suggested a paracrine role for stromal cells within fat grafts promoting revascularization, and identification of a subpopulation with enhanced angiogenic potential may yield an optimal subset for use in soft tissue reconstructive strategies.
Methods: Single-cell transcriptional profiling for angiogenic and cell-surface marker genes was performed on human fat-derived stromal cells. Soft partitioning was performed to define an angiogenic cluster of interest and linear discriminant analysis identified correlating surface markers. Cells were then isolated through flow cytometry based on CD248, the strongest correlating marker. Gene transcriptional analysis was performed on CD248+, CD248-, and unsorted cells to confirm angiogenic gene expression patterns. Finally, the pro-angiogenic capacity of CD248+ cells was tested using an endothelial tube formation assay and a mouse model of cell-assisted lipotransfer.
Results: Flow cytometry revealed CD248+ cells to comprise 16% of the overall heterogeneous stromal cell population. qRT-PCR revealed CD248+ cells to express significantly higher levels of VEGFa and HGF compared to CD248- and unsorted cells. CD248+ cells also promoted increased endothelial tube formation and improved fat graft retention in vivo.
Conclusions: Single-cell transcriptional profiling identified multiple markers associated with stromal cell angiogenic capacity, with CD248 being the strongest. CD248+ cells were found to have significantly increased angiogenic potential, making these cells potentially useful to augment fat grafting strategies for soft tissue reconstruction and for other targeted cell-based therapies in hypoxic environments.


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