Differentiation of HD-NPC and shHD-NPC into astrocytes.

(A) Astrocytes differentiated from HD-NPC expressing canonical astrocyte-specific markers, GFAP, ALDH1L1, and S100β. Staining with mEM48 showed nuclear and cytoplasmic aggregates of mHTT. (B) Astrocytes differentiated from shHD-NPC expressing canonical astrocyte-specific markers: GFAP, ALDH1L1, and S100β. Staining with mEM48 showed a lower number of cells that are positive for mEM48. (C) Quantification of ICC results showing all three cell lines expressed high percentage of cells with astrocyte specific markers ALDH1L1, GFAP, and S100β. HD cells showed significantly higher percentage of cells with PAX6-positive cells (17.6% vs. 2.20% WT and 9.8% shHD, P < 0.0001). shHD cells showed significantly higher percentage of cells with MAP2-positive cells (8.77% vs. 2.02% WT and 3.42%, P < 0.0001). In HD, mEM48 positive cell counts of HD and shHD astrocytes showing the significant reduced number of mEM48 positive cells in shHD astrocyte population (P < 0.0001, 23.9% and 2.56% respectively). For quantifications, minimum of 5 images and maximum of 24 images were counted in cellSens V2.1 (Olympus) with cell numbers ranging from 181 to 489 for each marker. A total of 2,167 cells of WT, 1,517 cells of HD, 1,727 cells of shHTT cells were counted.