392 Increased Mitochondrial Respiration and ROS Production From Adipose-Derived Stem Cells is Passage Dependent

Abstract

Victims of severe burn injury (over 30% of the total body surface area) experience profound hypermetabolism which prolongs wound healing. Adipose-derived stem cells (ASCs) have been proposed as an attractive solution for treating burn wounds. Moreover, the subcutaneous adipose tissue of severely burned patients normally discarded during surgical debridement is a viable source of ASCs. While previous studies found that severe burn injury alters the metabolic profile of subcutaneous adipocyte, the effect of severe burns on ASCs is largely unknown. The purpose of this study is to compare the metabolic activity of ASCs from burned patients to those from abdominoplasty patients by analyzing mitochondrial respiration and characteristics. Isolated ASCs from severely burned patients (BP, n=6) and abdominoplasty patients (AP, n=6) were grown to 80% confluency, trypsinized, and harvested for analysis at passage 2, 4, and 6. Flow cytometry was used to determine ASC cell surface markers CD90, CD105, and CD73. Mitochondrial abundance and ROS production were determined with MitoTracker Green and MitoSOX Red, respectively. JC-10 Mitochondrial Membrane Potential Assays were used to determine mitochondrial membrane integrity. Mitochondrial respiration and glycolysis were analyzed in technical quadruplicates by a Seahorse XFe24 Analyzer. No significant differences were found in ASC surface markers, mitochondrial abundance or membrane potential among passages or between groups. A two-way ANOVA showed that there was a significant effect (p<0.01) of passages on mitochondrial ROS production. Mitochondrial respiratory states Routine, Leak (uncoupled), and Non-mitochondrial respiration was significantly higher (p<0.05) at passage 6 than passage 2 for BP, but not AP. Glycolysis significantly decreased (p<0.05) from passage 4 to 2 in AP ASCs. Stem cell positive staining and mitochondrial activity were not different between cells from burned or non-burned humans. BP ASCs showed increased mitochondrial respiration with increasing passages. All ASCs showed increased ROS production through multiple passages. Adipose-derived stem cells from burned injured patients remain an exciting potential source for therapeutic purposes. However, increased ROS production and mitochondrial leak may cause oxidative stress that could limit the expansion of these cells ex vivo.