Fig. 2: Aged iNs represented aging-associated phenotype from the donor on the mitochondrial level and the correlated metabolic shift toward anaerobic glycolysis.

a Cellular ATP level comparing iNs from aged and young human donors (N = 3–4 independent experiments, n = 2–3 replicates per experiment for each donor). b MMP level was measured in aged iNs and young iNs by staining them with TMRM. The fluorescence was detected at ex: 548 nm/em: 574 nm (N = 4–5 independent experiments, n = 2–3 replicates per experiment). c Mitochondrial superoxide anion detection using the MitoSOX dye to compare young and aged iNs. The fluorescence was detected at ex: 485 nm/em: 535 nm (N = 4–5 independent experiments, n = 2–3 replicates per experiment for each donor). d Mitochondrial ROS detection in aged and young HFs. (ex: 485 nm/em: 535 nm). The fluorescence was detected at ex: 531 nm/em: 595 nm (N = 4–5 independent experiments, n = 2–3 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 iNs 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 iNs 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 iNs. Basal respiration, ATP-production coupled respiration, proton leak, maximal respiration, and spare respiration capacity (N = 4–5 independent experiments, n = 2–3 replicates per experiment). i Bioenergetic parameters of glycolysis comparing young and aged iNs. Glycolysis, glycolytic capacity, and glycolytic reverse (N = 3–5 independent experiments, n = 2–3 replicates per experiment for each donor). j Relative gene expression of relevant glycolysis-regulating genes, PFKFB3 and LDHA. The data are represented as gene expression (2 ^{(−Avg.(Delta(Ct))}) as the Housekeeping gene GAPDH was utilized. k, l Cellular NAD⁺ content (L) and NADH (M) content from young and aged iNs represented as normalized values to the protein concentration (N = 4–5 independent experiments, n = 2–3 replicates per experiment for each donor). m, n The mitochondrial network morphology (m) visualization was assessed in iNs from young and aged human donors by staining the mitochondria with TOMM20. Calculated mitochondrial parameters (n) Form Factor, Area Weighted Form Factor, Aspect Ratio, and Length (N = 4–5 independent experiments, n = 12–33 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 iNs 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 = 4–5 independent experiments, n = 2–3 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 using three technical replicates. Values were normalized on the cell count. The representative images were chosen for visualization purposes. Non-parametric Mann–Whitney test was performed to compare young iNs versus aged iNs (*p < 0.05, **p < 0.01, ***p < 0.001), or a two-way ANOVA was applied to compare multiple parameters.