Fig. 2: EPSC-derived iNSC are a suitable proxy for eNSC in comparative studies with GIC in mice.

a Schematic of experimental workflow. EPSC: expanded potential stem cells, iNSC: induced neural stem cells, eNSC: endogenous neural stem cells, mGIC: mouse glioblastoma initiating cells. b Two dimensional principal component analysis of cell types, representing normalized RNA sequencing data of eNSC, iNSC^Gibco, iNSC^N2B27, two rNSC^syn, one rNSC^{non syn}, two astrocytes, one neuron, and one GMP (granulocyte/monocyte progenitor). Astrocytes, neuron, and GMP are labelled as rOther^syn. e/i/r NSC: (endogenous/induced/reference neural stem cells, Gibco: EPSC neural induction with Gibco commercial protocol. N2B27: EPSC neural induction with a bespoke published protocol³², syn: syngeneic, non-syn: non-syngeneic. c Venn diagrams of overlapping genes (TPM > 1) in iNSC^Gibco and eNSC, rNSC^syn, rNSC^{non syn} or neurons; results are expressed as percentages of genes shared or specific to each type of cells. d Three-dimensional principal component analysis of cell types, representing genome-wide methylation RRBS-Seq data (M-values) of eNSC, iNSC^Gibco, Mueller cells, muscle satellite cells, hematopoietic stem cells (HSC), granulocyte-macrophage progenitor (GMP), and megakaryocyte/erythrocyte progenitors (MEP). e Two-dimensional density plots of DNA methylation profiles (M-values) between iNSC^Gibco and eNSC (top) and between iNSC^Gibco and muscle satellite cells (bottom). A median distribution of M-values has been obtained for each cell type across replicas before performing Spearman’s correlation. The R² value is shown for each plot. f Bar plots showing the relative abundance of genomic regions (distal, exon, intron, promoter, 3′ UTR, 5′ UTR) in methylated (top) and unmethylated (bottom) CpG sites across all cell types. g Left: combined analysis of expression profile and methylation levels of eNSC and iNSC^Gibco (11107 genes are plotted). Genes are considered expressed if log (TPM) > 0 and methylated if M > 0. Concordant genes are depicted in orange and blue, according to the orientation. Right: Venn diagrams showing the overlap of concordant genes between eNSC and iNSC^Gibco for both orientations. h Coexpression of SOX2 (red) and NESTIN (green) in eNSC, iNSC^Gibco, and iNSC^N2B27 with DAPI nuclear counterstaining (representative images, n = 3 for each cell type). The scale bar is 20 μm. i Percentage of genes classified as Differentially Expressed (DE) and not DE in comparisons between GIC vs NSC³⁹ or mGIC vs eNSC, iNSC^Gibco, rNSC^syn, and rNSC^{non syn} respectively.