Table 1.
Antibodies used for immunocytochemical staining.
Table 2.
Antibodies used in Western Blot.
Table 3.
Primers sequences.
Fig 1.
Expression of stem cell markers by undifferentiated ebiNSc.
Double staining with antibodies against Nestin (red) and SOX2 (green) showing expression of neural stem cell markers in the same cells. Each image was taken at magnification 400x; scale bars mark 50μm.
Fig 2.
(A) Endogenous wild type IDH1 expression in the non-transuded ebiNSc controls. Arrows mark the characteristic punctuate expression pattern. (B) Lack of the endogenous IDH1R132H expression in the non-transuded ebiNSc controls. (C) Expression of wild type IDH1 in ebiNSc transduced with wild type gene, ebiNScIDH1wt. Arrows mark the punctuate pattern characteristic for the endogenous IDH1. Arrowheads mark the strong, diffuse pattern of the induced expression. (D) Expression of IDH1R132H in ebiNSc transduced with the mutant gene, ebiNScIDH1R132H. Arrows mark cells lacking the induced expression. Arrowheads mark the strong, diffuse pattern of the induced expression. (E) Expression of endogenous wild type IDH1 in ebiNSc transduced with the empty vector, ebiNScempty. Arrows mark the characteristic punctuate expression pattern. Each image was taken at magnification 400x; scale bars mark 50μm. (F) IDH1 (non-mutation-specific) expression at the mRNA level in the four cultures Error bars indicate SEM. Statistical significance calculated by One-way ANOVA with Tukey's post-comparisons test. ***, p<0.005; ns, not significant.
Fig 3.
Expression of wild type IDH1 protein in different cell types.
Endogenous wild type IDH1 expression in the ebiNSc, neurospheres derived from glioblastoma primary culture, neurons and astrocytes. Each image was taken at magnification 400x; scale bars mark 50μm.
Fig 4.
Cultures of ebiNSc expressing IDH1R132H retained morphology of undifferentiated cells.
(A) Micrographs showing morphology of ebiNSc expressing IDH1R132H along with control cultures before differentiation (day 0). No evident morphological differences between ebiNScIDH1R132H and control cultures were visible on day 0. Cells were incubated in differentiation medium and photographed every 7 days in culture. (B) Morphology after 7 days of differentiation. Early cluster formation is visible in ebiNSc, ebiNScempty, ebiNScIDH1wt. (C) Morphology after 14 days of differentiation. More advanced cluster formation is visible in ebiNSc, ebiNScempty, ebiNScIDH1wt, but not in ebiNScIDH1R132H. Photomicrographs taken at magnification 40x. (D) Morphology after 14 days at higher magnification (magnification 100x, scale bars mark 50μm). The characteristic features of the differentiating cells are marked with arrows (cell clusters) and arrowheads (network-like connections between clusters). For better readability, the light microscopy images in this figure were contrast-enhanced.
Fig 5.
Expression of glial and neuronal markers by differentiated ebiNSc.
Double staining with antibodies to GFAP (red) and MAP2 (green) after 14 days of differentiation of non-transduced ebiNSc showing populations of astrocytic and neuronal cells. Photomicrographs demonstrate (A) Merged image of GFAP (red), MAP2 (green) and DAPI (blue) showing populations of GFAP and MAP2 positive cells with some overlap between the two markers; DAPI was used to estimate the percentage of cells positive for GFAP and MAP2; (B) GFAP (red); (C) MAP2 (green). The images at 200x magnification, scale bars mark 100μm.
Fig 6.
Impaired astrocytic differentiation of ebiNSc expressing IDH1R132H.
(A) Immunocytochemical characterization of GFAP expression after 0, 7 and 14 days of differentiation in ebiNSc cultures expressing IDH1R132H compared to control cultures (magnification 200x, scale bars mark 100μm). GFAP-positive cells are present after 7 days in ebiNSc, ebiNScempty and ebiNScIDH1wt cultures. No GFAP-positive cells are visible in ebiNScIDH1R132H after either 7 or 14 days. (B) Graph demonstrating the percentage of GFAP-positive cells after 7 days of differentiation in ebiNSc expressing IDH1R132H and control cultures. Error bars indicate SEM. Statistical significance calculated by Kruskal-Wallis with Dunn’s multiple comparison test. ***, p<0.005. (C) Graph demonstrating the quantitative analysis of GFAP expression at the mRNA level after 0, 7 and 14 days of differentiation (d0, d7, d14) in ebiNSc expressing IDH1R132H and control cultures. Error bars indicate SEM. Statistical significance calculated by Two-way ANOVA with Bonferroni’s post-comparison test. *, p<0.05; **, p<0.01; ***, p<0.005; ns, not significant.
Fig 7.
Impaired neuronal differentiation of ebiNSc expressing IDH1R132H.
(A) Immunocytochemical characterization of MAP2 expression after 0, 7 and 14 days of differentiation in ebiNSc cultures expressing IDH1R132H compared to control cultures (magnification 200x, scale bars mark 100μm). Fraction of MAP2-positive cells is present before the differentiation. The early clusters of MAP2-positive cells are visible after 7 days in ebiNSc, ebiNScempty and ebiNScIDH1wt controls. No MAP2-positive clusters and a low fraction of MAP2-positive cells are visible in ebiNScIDH1R132H after either 7 or 14 days. (B) Graph demonstrating the percentage of MAP2-positive cells with the elongated morphology after 7 days of differentiation in ebiNSc expressing IDH1R132H and control cultures. Error bars indicate SEM. Statistical significance calculated by Kruskal-Wallis with Dunn’s multiple comparison test. ***, p<0.005. (C) Graph demonstrating the quantitative analysis of MAP2 expression at the mRNA level after 0, 7 and 14 days of differentiation (d0, d7, d14) in ebiNSc expressing IDH1R132H and control cultures. Error bars indicate SEM. Two-way ANOVA analysis did not reveal any statistical significance between samples, however, the trend can be appreciated.
Fig 8.
Immunocytochemical characterisation of Synapsin I expression in differentiating cells.
Synapsin I expression after 14 days of differentiation in ebiNSc cultures expressing IDH1R132H compared to control cultures. (A) Synapsin I (SYN1) expression in ebiNSc. (B) Synapsin I expression in ebiNScIDH1wt. (C) Synapsin I (SYN1) expression in ebiNScempty (D) Staining with antibodies to Synapsin I (green) and IDH1R132H (red) in ebiNScIDH1R132H. No SYN1-positive cells are visible. All images at magnification 200x, scale bars mark 100μm. (E) Graphs demonstrating the percentage of SYN1-positive cells after 14 days of differentiation in ebiNSc expressing IDH1R132H and control cultures. Error bars indicate SEM. Statistical significance calculated by Kruskal-Wallis with Dunn’s multiple comparison test. *, p<0.05; **, p<0.01; ***, p<0.005. (F) Graph demonstrating SYN1 expression at the mRNA level after 0, 7 and 14 days of differentiation (d0, d7, d14) in ebiNSc expressing IDH1R132H and control cultures. Error bars indicate SEM. Statistical significance calculated by Two-way ANOVA with Bonferroni’s post-comparison test. **, p<0.01; ***, p<0.005; ns, not significant.
Fig 9.
IDH1R132H increases apoptosis susceptibility of induced neural stem cells and their derivatives.
(A) Micrographs showing apoptotic cells in control ebiNSc and ebiNScIDH1R132H cultured in non-differentiating medium with synthetic reporter of Caspase 3/7 activity. Increased apoptosis visible in IDH1R132H-expressing cells. (B) Apoptotic cells in control ebiNSc and ebiNScIDH1R132H after 7 days of differentiation. Increased number of apoptotic cells was observed in ebiNScIDH1R132H cells compared to differentiating control ebiNSc. Experiment was conducted in a manner similar to that described in A. Each image was taken at magnification 100x, scale bars mark 50μm. (C) Western Blot analysis for PARP in non-transduced ebiNSc and ebiNScIDH1R132H under differentiating and self-renewing conditions. In case of undifferentiated cells (0d), more cleaved form of PARP was observed in ebiNScIDH1R132H than in control. The decrease in PARP level during differentiation of ebiNScIDH1R132H may be correlated with reduced total protein amount as indicated by actin.