Figure 3

Pathologic accumulation of unesterified cholesterol and GM2 in differentiated hSKIN-MASC derived from NPC patients. (A -B) co-immunostaining for the lysosome marker Limp 1 (red fluorescence) and for unesterified cholesterol (blue fluorescence), revealed lysosomal accumulation of cholesterol after neuronal differentiation (5 days in N2 medium + 48 h in N3 medium, see methods) only in cultures derived from an NPC patient (B), but not in healthy donor derived cells (A). (C-F) Expression and relative quantification of the glycosphingolipid GM2 by immunofluorescence: co-immunostaining for the lysosome marker Limp 1 (green fluorescence) and for GM2 (red fluorescence), revealed accumulation of the ganglioside only in differentiated cultures derived from NPC (D) and Sandhoff (E) patients, but not in healthy donor derived cells (C). F) The percentage of GM2 expressing cells was assayed in undifferentiated cells and after exposure to the neural inductive media (5 days in N2 medium + 48 h in N3 medium, see methods), and compared to cells obtained in the same way but from a patient affected by Sandhoff disease, a specific GM2 gangliosidosis. No GM2 accumulation was detected in both healthy controls (CTRL, n = 3) and NPC (n = 3) undifferentiated cells, whereas a significant fraction of differentiated NPC (n = 3) and GM2 gangliosidosis derived cells showed accumulation of the glycosfingolipid. At least 400 cells have been counted for each cell line. Data are presented as mean ± SD of 3 independent experiments; one-way Anova test followed by Bonferroni post-test were utilized to compare means between groups. P values less than 0.05 were considered significant. *, **, ****, p<0.05 vs. columns 1, 2 and 4, respectively. The Sandhoff patient was omitted from the statistical analysis.