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Tracking Phenotypic Drift and Osteogenic Capacity of Fresh Adipose-Derived Stromal Cells
Elizabeth A. Brett, M.S., Elizabeth R. Zielins, M.D., Charles P. Blackshear, M.D., John Flacco, B.S., Anna Luan, M.D., Clement D. Marshall, M.D., William T. Leavitt, B.S., Michael Hu, M.D., Siny Shailendra, B.S., Siddarth Menon, B.S., Arash Momeni, M.D., Natalina Quarto, Ph.D., Michael T. Longaker, M.D., M.B.A., Derrick C. Wan, M.D..
Stanford University, Stanford, CA, USA.

Purpose: Studies have employed various cells surface markers to isolate pro-osteogenic subsets within the larger heterogeneous stromal cell population. This approach, however, has been limited by known phenotypic drift. We thus characterized how individual subpopulations change over time and the impact this may have on bone forming capacity.
Methods: Freshly harvested human adipose-derived stromal cells were analyzed by flow cytometry for markers CD90, CD105, and BMPR-IB. Marker drift was then followed over seven days. Positive and negative fractions for each marker were separated and quantum dot labeled before co-culturing to define subsequent contributions of each subset to the total population. Finally, osteogenic differentiation of individual subsets from freshly harvested and in vitro cultured cells for 36 hours was evaluated.
Results: Contributions from CD90, CD105, and BMPR-IB positive and negative fractions to the total population dynamically changed over seven days. CD90+, CD105-, and BMPR-IB+ cells from freshly harvested lipoaspirate showed enhanced osteogenic differentiation when compared to their counterparts. These differences were more pronounced following 36 hours of culture. Interestingly, a significant number of cells that were CD90+, CD105-, or BMPR-IB+ at 36 hours were actually derived from CD90-, CD105+, or BMPR-IB- cells prior to plating, with CD-negative cells drifting towards positive and CD-positive cells drifting towards negative.
Conclusions: Our observations allude to the volatile nature of surface marker expression with in vitro culture. Furthermore, their changing pattern argues against a direct functional role for specific markers in osteogenesis described in previous studies.


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