Fig. 7: Role of SREBF1 in the astroglial commitment/differentiation of hiPSC-NSCs.

A Western blot analyses showing truncated immature (iSREBP1) and mature (mSREBP1) proteins in SREBP1-deficient hiPSCs (clones 8, 15, 34). Full-length iSREBP1 and mSREBP1 were detected in control hiPSCs (Cas9-only and HD1.3) and HeLa cells (expressing high SREBF1 levels). Calnexin (CLX) has been used as a housekeeping protein. B SREBP1 activity evaluated in SREBP1-deficient (SREBP1-def), and Cas9-only treated hiPSCs. Two-tailed Mann-Whitney test: **p < 0.01 (n = 3 biological replicates/clone). C PCA plot of RNA-seq data collected in SREBP1-deficient and Cas9-only treated hiPSCs, hiPSC-NSCs and differentiated cultures (day 7 and day 14 of differentiation) (n = 3 replicates/time-point/clone). D Heatmap showing the expression levels of master regulators of pluripotency and neural commitment in SREBP1-deficient and Cas9-only hiPSCs and hiPSC-NSCs. Colour scale indicates the relative fold change of normalized expression levels in each sample (blue, low; red, high). E Heatmap of sample-to-sample distance among RNA-seq samples of SREBP1-deficient and Cas9-only hiPSCs and hiPSC-NSCs; Embryonic Stem Cells (ESCs; line H1_ESC^202,203 and line ESC⁷⁷); ESC-derived neuroepithelial cells (NE) and early (ERG), middle (MRG), and late (LRG) radial glia cells⁷⁷. F Annexin V/7-AAD FACS analysis to evaluate the percentage of living and apoptotic cells in SREBP1-deficient and Cas9-only hiPSC-NSCs (n = 3 biological replicates/clone). G FACS cell proliferation assay of SREBP1-deficient and Cas9-only hiPSC-NSCs. H Bar plot reporting the quantification of cells expressing the astrocyte (GFAP and S100β) and neuronal (MAP2) markers in mixed glia/neuron cultures (7 and 14 days of differentiation) derived from SREBP1-deficient and Cas9-only hiPSC-NSCs. Two-tailed Mann-Whitney test: **p < 0.01; ***p < 0.001 (n = 3–4 biological replicates/clone). I Representative immunofluorescence images of GFAP⁺ (green) and S100β⁺ (red) astrocytes in mixed glia^/neuron cultures (14 days of differentiation). Nuclei are stained with HOECHST (blue). J Bar plot reporting the quantification of the engrafted cells (hNuclei⁺, hMito⁺ or STEM121⁺) expressing the cell^-specific markers (S100β: astrocytes, GSTπ: oligodendrocytes, β-tubulin III: neurons) at 1.5-months post-transplant in immunodeficient mice treated with SREBP1-deficient and Cas9-only hiPSC-NSCs. Each dot represents one mouse. Two-tailed Mann-Whitney test: **p < 0.01. A–J Data are presented as mean values ± SEM. Source data are provided as a Source Data file.