Fig. 6: Manipulation of Wnt signaling rescues proliferation of PTHS neural progenitors.
a Ratio of TPM expression abundances for selected Wnt genes between parent and PTHS NPCs in 2D culture. N = 4 parent–child pairs (symbols). b Relative expression of Wnt genes in NPCs. N = 4 pairs (symbols). c Reduced Wnt signaling activity in PTHS NPCs in 2D culture (TOP-Flash assay). N = 4 pairs (symbols). d Relative expression of selected Wnt genes in post-mortem PTHS cortex sample (PTHS #6). N = 3 replicates per group. e Treatment of control NPCs with Wnt pathway antagonists DKK-1 and ICG-001 (yellow bars) phenocopies proliferation deficit in PTHS progenitors. N = 3 pairs (symbols). f Parent CtOs at 4 weeks in vitro after treatment with ICG-001, stained for SOX2 and MAP2. g ICG-001 treatment of CtOs phenocopies low neural progenitor content (SOX2) of PTHS organoids. N = 3 replicates (circles) with pair #4 cells. h Live cell count after treatment of NPCs in 2D culture with Wnt pathway agonist CHIR99021. N = 4 pairs (symbols). i EdU assay in NPCs treated with CHIR99021. Left graph represents data for pair #4, and right graph shows data for pair #1. N = 3 replicates. j Quantification of p16INK4a+ (senescent) cells in NPCs in 2D culture treated with CHIR99021. Data shown are for pair #4 (see graph in Supplementary Fig. 8g for pair #1). N = 3 replicates. k CHIR99021 rescues expression of proliferation genes in treated PTHS NPCs in 2D culture. N = 4 pairs (symbols). See pairwise comparisons in Supplementary Data 1. l PTHS CtOs at 4 weeks in vitro after treatment with CHIR99021, showing marked increase in abundance of NPCs (SOX2; green) and neurons (MAP2) as well as reappearance of neural rosettes (arrowhead). m Quantification of SOX2+ cells after treatment of PTHS CtOs with CHIR99021. N = 3 replicates (circles) with PTHS #4 cells. Symbols in bar graphs indicate parent-patient identities: diamonds, pair #1; squares, pair #2; triangles, pair #3; circles, pair #4; gray dots, post-mortem samples. Colors in bar graphs represent parents (orange), pharmacologically treated parents (yellow), PTHS (blue), pharmacologically treated PTHS (light blue), or control post-mortem sample (black) groups. Bar graphs represent mean + SEM. n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001; two-sample Welch’s t test (in b–d) or one-way ANOVA followed by Tukey’s HSD post-hoc test in remaining panels. Scale bars are 100 μm. See Supplementary Data 1 for sample and effect sizes and exact p-values.
