Fig. 3

Analysis of mitochondrial respiration of healthy control and patient fibroblasts with a Seahorse XFe96 Extracellular Flux Analyzer. A Representative graph of a mitochondrial oxygen consumption bioenergetics profile of control (black = untreated and grey = MSC-treated) and MT-ND3a (dark red = untreated and red = MSC-treated) patient fibroblasts analysed with Mito Stress Test Assay of one experiment. First six measurements show basal respiration, followed by oligomycin injection that inhibits complex V and induces a leak state. Injections of the uncoupler FCCP stimulates the respiratory chain to increase activity to maximum by disrupting the proton gradient and mitochondrial membrane potential. Rotenone/antimycin A (Rot/AA) injection blocks respiration and enables calculation of the residual oxygen consumption. Treatment with MSCs increases overall oxygen consumption rates (OCRs) of all investigated cell lines. B, C Basal and maximal respiration of untreated MT-ND3- and MT-ND6-deficient fibroblasts compared to control fibroblasts and once (+) or twice (++) treated with MSCs. MT-ND3s show highest reduction in basal and maximal respiratory capacities (BRC and MRC, respectively) compared to control. However, all patients show decreased BRCs and MRCs. Co-cultured patient and control fibroblasts are able to increase their BRC and MRC. Sequential treatment with MSCs enhances the improvement. D, E Spare respiratory capacity (SRC) of MSC-treated (+ or ++) and untreated MT-ND3- and MT-ND6-deficient fibroblasts compared to control fibroblasts. All patients have decreased SRCs compared to control fibroblasts. Treatment with MSCs increases SRCs of all investigated cell lines, but most effectively in patient fibroblasts. Respiration-dependent ATP production is increased in all treated patient fibroblasts, with highest improvement in patient cell line MT-ND3a. F Non-mitochondrial oxygen consumption rates of patient fibroblasts as well as control compared to single (+) or sequential (++) treatment with MSCs show overall improvement for all three patient cell lines. OCR data were obtained in pmol/min and normalised to cell number. In a second step, data were normalised to basal respiration of untreated control (level indicated by dashed red line). Data are presented as mean ± SEM, analysed in n = 3 independent experiments for single and sequential treatment. All samples were checked for significance compared to untreated control and within one group (indicated by respective brackets). Statistical analysis by unpaired t test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001