In Figure 4, we demonstrate mitochondrial damage and increased oxidative stress in SZ hiPSC NPCs. MMP (ΔΨm) is the voltage difference across the inner mitochondrial membrane. MMP and cytosolic reactive oxidative stress (ROS) are intricately and inversely related: increased ROS can decrease MMP through various mechanisms, whereas mitochondrial dysfunction (decreased MMP) can increase ROS.16 We assayed MMP using JC-1 lipophilic cationic dye. The fluorescence of JC-1 dye changes from green to red as MMP increases, an inverse measure of oxidative stress (Figure 4a). We observed significantly decreased MMP (median intensity of JC-1 red/green fluorescence), indicative of increased oxidative stress, in SZ hiPSC NPCs (2.20±0.07) relative to control hiPSC NPCs (1.09±0.03) (P<0.00001; Figure 4a and b). Immunohistochemical staining for the mitochondrial marker MTC02 revealed that mitochondria in SZ hiPSC NPCs tended to be smaller, disconnected and distally distributed, whereas mitochondria in control hiPSC NPCs tended to be more connected, tubular and highly packed near the perinuclear regions (Supplementary Figure 9A). Exceptions were that some mitochondria from Patient 3 were connected and/or packed, whereas some from patient 4 were tubular (Supplementary Figure 9A). Transmission electron microscopic analysis confirmed these features (Supplementary Figure 9B).
