ASCs derived from burn patients are more prone to increased oxidative metabolism and reactive oxygen species upon passaging

Abstract

Background: Patients with severe burn injury (over 20% of the total body surface area) experience profound hypermetabolism which significantly prolongs wound healing. Adipose-derived stem cells (ASCs) have been proposed as an attractive solution for treating burn wounds, including the potential for autologous ASC expansion. While subcutaneous adipocytes display an altered metabolic profile post-burn, it is not known if this is the case with the stem cells associated with the adipose tissue.

Methods: ASCs were isolated from discarded burn skin of severely injured human subjects (BH, n = 6) and unburned subcutaneous adipose tissue of patients undergoing elective abdominoplasty (UH, n = 6) and were analyzed at passages 2, 4, and 6. Flow cytometry was used to quantify ASC cell surface markers CD90, CD105, and CD73. Mitochondrial abundance and reactive oxygen species (ROS) production were determined with MitoTracker Green and MitoSOX Red, respectively, while JC-10 Mitochondrial Membrane Potential Assays were also performed. Mitochondrial respiration and glycolysis were analyzed with a high-resolution respirometer (Seahorse XFe24 Analyzer).

Results: There was no difference in age between BH and UH (34 ± 6 and 41 ± 4 years, respectively, P = 0.49). While passage 2 ASCs had lower ASC marker expression than subsequent passages, there were no significant differences in the expression between BH and UH ASCs. Similarly, no differences in mitochondrial abundance or membrane potential were found amongst passages or groups. Two-way ANOVA showed a significant effect (P < 0.01) of passaging on mitochondrial ROS production, with increased ROS in BH ASCs at later passages. Oxidative phosphorylation capacities (leak and maximal respiration) increased significantly in BH ASCs (P = 0.035) but not UH ASCs. On the contrary, basal glycolysis significantly decreased in BH ASCs (P = 0.011) with subsequent passaging, but not UH ASCs.

Conclusions: In conclusion, ASCs from burned individuals become increasingly oxidative and less glycolytic upon passaging when compared to ASCs from unburned patients. This increase in oxidative capacities was associated with ROS production in later passages. While the autologous expansion of ASCs holds great promise for treating burned patients with limited donor sites, the potential negative consequences of using them require further investigation.

Keywords: Adipose stem cells; Burn; Glycolysis; Mitochondria; Oxidative phosphorylation; ROS; Respirometry.

Fig. 1

Flow cytometry of ASC markers. Representative flow cytometry readouts for ASC surface marker expression of CD73 (a), CD105 (b), and CD90 (c) for both burned human (BH) and unburned human (UH) patients. The bottom row shows quantification, which shows that while there were no differences in any marker at any passage between BH and UH, there was a significantly lower expression of each marker at passage 2, compared to subsequent passages. Expression of CD73 on UH ASCs was 70.8 ± 9.8, 95.8 ± 3.9, and 97.6 ± 1.3% at passages 2, 4, and 6, respectively, while the expression on BH ASCs was 64.0 ± 8.6, 99.7 ± 0.1, and 99.2 ± 0.4%, respectively. Expression of CD105 on UH ASCs was 52.3 ± 11.7, 83.8 ± 12.9, and 92.9 ± 3.2% at passages 2, 4, and 6, respectively, while the expression on BH ASCs was 47.4 ± 8.2, 94.6 ± 1.8, and 89.5 ± 4.4%, respectively. Expression of CD90 on UH ASCs was 75.3 ± 5.8, 94.4 ± 5.4, and 98.5 ± 0.8% at passages 2, 4, and 6, respectively, while expression on BH ASCs was 69.8 ± 10.5, 99.5 ± 0.3, and 99.2 ± 0.2%, respectively. *P < 0.05, **P < 0.01, ***P<0.001. N = 6 patients in BH ASCs and UH ASCs at each passage

Fig. 2

Mitochondrial characteristics of ASCs in culture. Representative images of staining for MitoTracker (a) and MitoSOX (b) for BH and UH ASCs across passages show relatively consistent quantities of mitochondrial content in green but increasing amount of ROS with passaging in red. Quantification of these fluorescent dyes via flow cytometry shows that nearly all ASCs in both groups stained positive for MitoTracker (c), while MitoSOX (d) expression increased significantly in passage 6 compared to passage 2 in BH ASCs. *P < 0.05. BH ASCs also displayed more MitoSox-positive cells than UH ASCs at passage 2, ^@P < 0.05. e The mitochondrial membrane potential dye, JC-10, was not different between the groups or passages. N = 6 patients in BH ASCs and UH ASCs at each passage

Fig. 3

Oxidative phosphorylation capacity of ASCs in culture. High-resolution respirometry revealed a non-significant increase in routine (a) respiration across passages. Alternatively, both leak (b) and maximal (c) respiration were significantly higher in BH ASCs when taken to passage 6 as compared to passages 4 and 2, respectively. *P < 0.05. N = 6 patients in BH ASCs and UH ASCs at each passage. OCR, oxygen consumption rate

Fig. 4

Glycolytic capacity of ASCs in culture. While no differences were found between the groups or across passage for the non-glycolytic PER (a), a significant decrease in the basal glycolytic rate (b) was found when comparing passage 6 to passage 2 for BH ASCs, but not for UH ASCs. *P < 0.05. N = 6 patients in BH ASCs and UH ASCs at each passage. PER, glycolytic proton efflux rate

Fig. 5

Energy map of BH and UH ASCs across passages. Plotting the oxygen consumption rate (OCR) versus the glycolytic proton efflux (PER) reveals that, when compared to UH ASCs, BH ASCs become more aerobic and less glycolytic as they are passaged