Fig. 3: A decrease in mitochondrial function was seen in aged iPSCsNs compared to young iPSCsNs, but no evidence of a metabolic shift towards anaerobic glycolysis.

a Cellular ATP level comparing iPSCsNs from aged and young human donors (N = 5 independent experiments, n = 2–3 replicates per experiment for each donor). b MMP level was measured in aged iPSCsNs and young iPSCsNs by staining with TMRM. The fluorescence was detected at ex: 548 nm/em: 574 nm (N = 5 independent experiments, n = 2–3 replicates per experiment for each donor). c Mitochondrial superoxide anion detection using the MitoSOX dye to compare young and aged iPSCsNs. The fluorescence was detected at ex: 485 nm/em: 535 nm (N = 5 independent experiments, n = 3 replicates per experiment for each donor). d Mitochondrial ROS detection in aged and young iPSCsNs. (ex: 485 nm/em: 535 nm). The fluorescence was detected at ex: 531 nm/em: 595 nm (N = 5 independent experiments, n = 3 replicates per experiment for each donor). e Relative gene expression of relevant anti-oxidative stress enzymes, SOD1, CAT, and GPX1. The data are represented as gene expression (2 ^{(−Avg.(Delta(Ct))}) as the Housekeeping gene GAPDH was utilized. f Mito Stress Test profile representing the OCR of young and aged iPSCsNs after sequential injection of oligomycin (O, 2,5 µM), FCCP (F, 2 µM), and lastly combined rotenone (R, 2 µM) with antimycin A (A, 2 µM). g Glycolysis Stress Test profile representing the ECAR of aged and young iPSCsNs after sequential injection of glucose (G, 10 mM), Oligomycin (O, 1 µM), and lastly 2-deoxy-glucose (2-DG, 25 mM). h Bioenergetic parameters of the mitochondria of young and aged iPSCsNs. Basal respiration, ATP-production coupled respiration, maximal respiration, and spare respiration capacity (N = 4 independent experiments, n = 2–3 replicates per experiment for each donor). i Bioenergetic parameters of glycolysis comparing young and aged iPSCsNs. Glycolysis and glycolytic capacity (N = 3 independent experiments, n = 2–3 replicates per experiment for each donor). j Relative gene expression of relevant glycolysis-regulating genes, PFKFB3, PKM, LDHA. The data are represented as gene expression (2 ^{(−Avg.(Delta(Ct))}) as the Housekeeping gene GAPDH was utilized. Cellular NAD⁺ content (k) and NADH content (l) from young and aged iPSCsNs represented as normalized values to the protein concentration (N = 5 independent experiments, n = 3 replicates per experiment for each donor). m, n Mitochondrial network morphology (m) was assessed in iPSCsNs from young and aged human donors by visualizing the mitochondria with TOMM20 and nucleus staining with DAPI. Calculated mitochondrial parameters (n) Form Factor, Area Weighted Form Factor, Aspect Ratio, and Length (N = 5 independent experiments, n = 3 replicates per experiment for each donor). o Relative gene expression of relevant genes involved in mitochondrial dynamics: FIS1, DNM1L, OPA1, MFN2, and MFN1. The data are represented as gene expression (2 ^{(−Avg.(Delta(Ct))}) as the Housekeeping gene GAPDH was utilized. p Mitochondrial Mass comparing young and aged iPSCsNs assessed by using the MitoTracker™ Green FM (ex: 490 nm/em: 516 nm) to stain mitochondria and were normalized to the cell area using Celltracker blue (ex: 353 nm/em: 466 nm) (N = 5 independent experiments, n = 3 replicates per experiment for each donor). Data information: All data are represented as the mean ± SEM of each four different young and aged iNs. Statistical parameters, including the number of values, minimum, maximum, range, mean, standard deviation, and standard error of the mean, are presented in Supplementary Data 2. Only three donors were assessed for gene expression with three technical replicates. Values were normalized on the cell count. Non-parametric Mann–Whitney test was performed to compare young iPSCsNs versus aged iPSCsNs (*p < 0.05, **p < 0.01, ***p < 0.001), or a two-way ANOVA was applied to compare multiple parameters.