Astrocytes generated from the D-HD-iPSCs with increased cytoplasmic vacuolation. A: Vacuoles were detected in the cytoplasm of astrocytes (arrows) under phase contrast microscopy. The number of vacuoles varied depending on the iPSC line used to generate the astrocytes. B: Quantification of cytoplasmic vacuolation. Values represent mean + SEM (n = 4; *: P < 0.0001; ANOVA). C: Cytoplasmic vacuoles appeared empty and were negative for GFAP staining. D: The autophagosomal marker LC3 was weakly detected in astrocytes grown under normal culture conditions and rarely colocalized with cytoplasmic vacuoles. Vacuoles often appeared empty (arrows). Scale bars: 50 μm.