Additional file 3 of Human midbrain organoids reveal the characteristics of axonal mitochondria specific to dopaminergic neurons

Additional file3. Analysis of mitochondrial membrane potential in axons of midbrain organoids derived from other TH-GFP iPSC lines. (a) Live-cell imaging of axons of GFP-positive dopaminergic and GFP-negative non-dopaminergic neurons in midbrain organoids stained with TMRM. Green and white dotted lines indicate mitochondria in axons of dopaminergic and non-dopaminergic neurons, respectively. “PRKN” represents PRKN-mutant patient. Scale bar, 2 µm. (b) Mean TMRM MFI in all mitochondria per each axon from live-cell images. Data were acquired from GFP-negative (n = 42) and GFP-positive (n = 8) axons from two fields of two organoids in the control B7 T1-3 line, from GFP-negative (n = 21) and GFP-positive (n = 3) axons from two fields of two organoids in the control WD39 T1-2 line, and from GFP-negative (n = 68) and GFP-positive (n = 3) axons from two fields of two organoids in the PRKN-mutant PB2 T1-1 line. The graph shows the relative comparison of the mean TMRM MFI of mitochondria in GFP-negative and GFP-positive neurites. “PRKN” represents PRKN-mutant patient. Values are shown as the mean ± SEM. The differences were evaluated using the nonparametric Mann–Whitney U test. **P < 0.01, ***P < 0.001. The mean TMRM MFI of mitochondria significantly differs between dopaminergic and non-dopaminergic neurons in the control line 201B7 T1-3 (P = 0.0005; effect size, 0.329; 95% CI, 0.207 to 0.622) and WD39 T1-2 (P = 0.0069; effect size, 0.578; 95% CI, 0.372 to 0.772), whereas no significant difference is observable in the PRKN-mutant PB2 T1-1 line (P = 0.486; effect size, 0.124, 95% CI, −0.225 to 0.329). CI: confidence interval, GFP: green fluorescence protein, iPSC: induced pluripotent stem cell, MFI: mean fluorescence intensity, n.s.: not significant, SEM: standard error of the mean, TH: tyrosine hydroxylase, TMRM: tetramethylrhodamine methyl ester