Figure 3.

Development of Mitochondrial Functions in hiPSC-Derived DA Neurons

(A–G) Mitochondrial membrane potential and ROS levels were measured by TMRM (A and B). Representative images for TMRM staining for hiPSC-derived DA neurons cultured in differentiation media for 5, 10, 15, and 20 days are shown in (A), and quantifications of immunofluorescent intensity for TMRM in (B) (n = 3 independent experiments; mean ± SEM, with 5 cells quantified per experiment). Enzymatic activity of complex I (C), IV (D), and cellular ATP levels (E) were determined in cell lysates from hiPSC-derived DA neurons cultured in differentiation media for 10, 15, and 20 days (n = 3 independent experiments; mean ± SEM in C–E). Mitochondrial ROS levels were measured by MitoSOX staining (F and G). Representative images for MitoSOX staining for hiPSC-derived DA neurons cultured in differentiation media for 5, 10, 15, and 20 days are shown in (F). Quantification of immunofluorescent intensity for MitoSOX is shown in (G) (n = 3 independent experiments; mean ± SEM, with five cells quantified per experiment).

(H and I) EPR for measurement of intracellular ROS levels. Representative EPR images for hiPSC-derived DA neurons cultured in differentiation media for 5, 10, 15, and 20 days are shown in (H), and quantification of EPR signals in (I) (n = 3 independent experiments; mean ± SEM).

Data are expressed as fold increase relative to DA neurons cultured in differentiation media for 10 days. Statistical analysis was performed using StatView version 5.0.1. For (B–E), (G), and (I), one-way ANOVA was used for repeated-measures analysis, followed by Fisher's protected least significant difference for post hoc comparisons. Data are presented as mean ± SEM. Three independent experiments in (A–I); n = 5 per group per experiment in (B) and (G). Scale bars in (A) and (F) represent 5 μm.