Transcriptional Alterations of Adipose-Derived Stromal Cells in Culture
Paul A. Mittermiller, MD, Melanie Rodrigues, PhD, Ryann E. Shor, High School Graduate, Alexander Y. Li, BS, Geoffrey C. Gurtner, MD.
Stanford University, Palo Alto, CA, USA.
Adipose-derived stromal cells (ASCs) are a valuable source of cells for use in regenerative medicine. Our laboratory has been determining the ideal ASC subpopulation to be used in clinical trials. We have previously identified a subset of ASCs with a favorable gene expression profile that demonstrates improved wound healing in diabetic mice. This subset of cells can be isolated using a unique surface marker combination (CD26+/CD55+). In order to obtain sufficient numbers of these cells for therapeutic purposes, expansion of the cell line is necessary. Therefore, we tested whether expansion would result in changes to their transcriptional profiles.
Adipose tissue was surgically extracted from three wild-type mice. The tissue was digested and single cells were subjected to fluorescence-activated cell sorting (FACS). Using this technique, cells were sorted for non-hematopoietic (CD45-), non-endothelial (CD31-) single cells with the progenitor cell marker (CD34+). This marker profile isolates all ASCs. Cells were also sorted for the ASC subpopulation of interest with the additional markers CD26 and CD55. Two sets of cultures were performed, one with parental ASCs and the other with CD26+/CD55+ ASCs. The cells were passaged every five days for a total of three passages (P0 to P3). Cells were trypsinized after each passage and analyzed by FACS to measure absolute cell numbers. Freshly isolated ASCs were subjected to single-cell transcriptional analysis and compared to the same group in culture after passages P0 and P1.
Using the methods described above, three thousand parental ASCs and three thousand CD26+/CD55+ ASCs were isolated. The number of cells from the parental group increased by two-fold in P0, nine-fold in P1, and over one hundred-fold in P2. The cells from the CD26+/CD55+ ASC group proliferated more than those in the parental group after each passage. Single-cell transcriptional analysis revealed a drastic drift in gene expression profiles between freshly isolated ASCs and those cells in culture. There were notable, statistically significant increases in genes VEGF, FGF2, FGF7, TGFB1, FGFR1, FGFR2, EGFR, CCND1, PCNA, ADAM10, and HB-EGF in the sorted ASCs in culture.
Through single-cell transcriptional analysis, we have identified an ASC subpopulation that improves wound healing in diabetic mice. This study has offered insight into the changes that occur in ASCs with expansion. CD26+/CD55+ ASCs demonstrate an increased rate of proliferation in culture when compared to parental ASCs. Furthermore, during in vitro proliferation ASCs demonstrate an increase in expression of genes responsible for growth factors, growth factor receptors, proliferation and production of extracellular matrix. These findings continue to suggest that the CD26+/CD55+ ASC cell line is favorable for regenerative purposes and can be expanded in vitro for cell-based therapies.
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