PMC

(A) Direct iN conversion of young and old fibroblasts using Ngn2-2A-Ascl1 and small-molecule enhancers.
(B) Heatmap of 1,118 mitochondrial genes in young (<40years) and old (>40years) purified iNs(n = 12 individuals, one RNA-seq profile each; see also for donor information and ).
(C) Expression of functional gene groups in young and old iNs. Logarithmic variance stabilizing transformation (VST) normalized counts; bars indicate mean values; dots represent genes; significance values were calculated by the non-parametric Wilcoxon test.
(D) Relative expression of ETC complex I, II, III, IV, and V in young and old iNs. VST normalized counts normalized to young; dots indicate mean ± SEM; significance was calculated by Wilcoxon test.
(E) Protein levels of indicated OXPHOS proteins in young and old iNs assessed by quantitative MitoProfile western blot (n = 6 individuals, 2 replicates). Significance values were calculated by t test. Representative blot in .
Significance values in this figure: *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

(A) Heatmap of metabolic genes in human primary fibroblasts and FACS-purified iNs (n = 12 individuals, one iN and one fibroblast RNA-seq each; see also ).
(B) Expression of five representative OXPHOS genes and four canonical glycolysis genes over time during fibroblast-to-iN conversion (four time points and purified iNs from day 18). VST normalized counts were normalized to day 3.
(C–E) Relative ATP levels and schematic drawing of metabolic pathways active in fibroblasts (C), purified iNs (D), and OXPHOS-induced fibroblasts (E) treated with ROT/AA or vehicle for 6 hr (n = 8 individuals per cell type; 4 replicates). Bars represent means ± SD; symbols mark individual measurements. Significance values were calculated by t test.
(F) MMP (JC-1) in young and old OXPHOS-induced fibroblasts (n = 7 individuals; 4 replicates). Bars represent means ± SD. Symbols mark individual measurements. Significance values were calculated by t test.
(G) Total ATP levels in young and old OXPHOS-induced fibroblasts (n = 8 individuals; 4 replicates). Bars represent means ± SD. Symbols mark individual measurements. Significance values were calculated by t test.
Significance values in this figure: *p < 0.05 and ****p < 0.001.

(A) Representative fluorescence images of mitochondria (hSyn::MitoEGFP) in SMI-312-positive axons in young (1 year) and old (71 years) donor-derived iNs (see also and ). Scale bar, 20 μm.
(B) Quantification of axonal mitochondrial morphologies for densities, mean lengths, and coverage in young and old iNs (n = 7 individuals, 6 replicates each). Boxplots show 25th to 75th percentiles, bars indicate medians, and whiskers show ranges. Significance values were calculated by unpaired t tests (see also and ).
(C and D) hSyn::BFP-tagged iNs were gated (D) and analyzed for MMPs using the JC-1 dye (C).
(E) Representative JC-1 FACS density plots showing energized mitochondria in young iNs (1 year) and a loss of MMP in old iNs (71 years).
(F) Quantification of the MMP in young and old hSyn::BFP-positive iNs (n = 7 individuals, 3 replicates). Bar graph shows means ± SD, and triangles indicate individual measurements. Significance values were calculated by Mann-Whitney test.
(G) ATP levels in young and old purified iNs (6 individuals, 3 replicates). Bar graph shows means ± SD, and triangles indicate individual measurements. Significance values were calculated by Mann-Whitney test.
(H) Quantification of oxidized proteins as assessed by quantitative western blot (OxyBlot; 6 individuals, 2 replicates, representative blot in ).
Significance values in this figure: *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

(A) Heatmap of mitochondrial genes in young (<40 years) and old (>40 years) primary human fibroblasts (n = 12 Individuals, one RNA-seq profile each; see also ).
(B) Expression of functional gene groups in young and old fibroblasts. Significance values were calculated by the non-parametric Wilcoxon test (see also for individual ETC genes).
(C) Protein levels of OXPHOS proteins in young and old fibroblasts assessed by quantitative western blot (n = 7 individuals, 3 replicates). Significance values were calculated by t test. Representative blot in .
(D) Representative fluorescence images of mitochondria (MTC02) in young (1 year) and old (71 years) fibroblasts (see also ). Scale bar, 20 μm.
(E) Quantification of mitochondrial morphologies for aspect ratio (length) and form factor (branching) in fibroblasts (n = 7 individuals, 10 replicates). Boxplots show 25th to 75th percentiles; bars indicate medians, and whiskers show ranges. Significance values were calculated by unpaired t tests (see also and ).
(F) Representative MMP (JC-1) FACS density plots of young (0 year) and old (89 years) fibroblasts.
(G) Quantification in young and old fibroblasts (n = 7 individuals, 3 replicates). Bar graph shows means ± SD, and triangles indicate individual measurements. Significance values were calculated by Mann-Whitney test.
(H) Quantification of oxidized proteins as assessed by quantitative western blot (OxyBlot; 6 individuals, a representative blot in ).
(I) Total ATP levels in young and old fibroblasts (6 individuals, 3 replicates). Bar graph shows means ± SD; triangles indicate individual measurements. Significance values were calculated by Mann-Whitney test.
ns, not significant. Significance values in this figure: *p < 0.05, **p < 0.01, and ****p < 0.001.