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. 2022 Nov 22;11:e77335. doi: 10.7554/eLife.77335

Figure 2. Hypo-methylated differentially methylated regions (DMRs) from bias-glioblastoma initiating cells (B-GICs) are enriched for transcription factor binding sites linked to glial differentiation.

(A) Top five motifs enriched in all hypo-methylated DMRs from B-GICs as compared to all other DMRs from all GICs. (B) Top five motifs enriched in all hypo-methylated DMRs from B-GICs as compared to all the hypo-methylated DMRs from all other GICs. (C) Top five motifs enriched in all hyper-methylated DMRs from B-GICs as compared to all other DMRs from all GICs. (D) 3D principal component analysis (PCA) of methylation data from patient-derived fibroblasts, induced pluripotent stem cells (iPSCs), induced neural stem cells (iNSCs), induced astrocyte progenitor cells (iAPCs), induced oligodendrocyte progenitor cells (iOPCs), and publicly available reference datasets of NSCs, astrocytes, and oligodendrocyte precursor cells. (E) Unsupervised hierarchical clustering based on the top 5000 variable methylation probes of iAPCs, iNSCs, iOPCs (purple bar), and publicly available reference datasets (yellow bar). (F) Single sample gene set enrichment analysis (ssGSEA) enrichment scores for the astrocyte composite signature (ACS) of iAPCs (N=10), iNSCs (N=10), and publicly available reference datasets, statistical differences tested with one-way ANOVA. ssGSEA enrichment scores of iOPCs (N=10) and iNSCs (N=10) for the Oligodendrocyte Specific-300 (G) and Oligodendrocyte Enriched-300 (H) gene signatures, statistical significance tested using Mann-Whitney t-test. Top five de novo (I) and known (J) motifs enriched in all hypo-methylated DMRs in iAPCs, from each iAPC versus iNSC comparison.

Figure 2—source data 1. Source data for Figure 2.

Figure 2.

Figure 2—figure supplement 1. Characterisation of induced astrocyte progenitor cell (iAPC) and induced oligodendrocyte progenitor cell (iOPC) obtained from induced neural stem cell (iNSC).

Figure 2—figure supplement 1.

(A) Flow cytometry results of iAPCs analysed after 10 days of differentiation (top panel) and 20 days (bottom panel). iAPCs (red) (N=5) and iNSCs (teal) (N=3) were analysed for the markers NESTIN, GFAP, PDGFRA, and CD44. Results presented as bar charts of percentage of positive cells for respective markers, and for each timepoint representative dot plots of NESTIN versus CD44 of iNSC and iAPCs, to show co-expression of the two markers. (B) Immunostaining of iAPCs, for GFAP (top panel, green) and CD44 (bottom panel, green) with DAPI nuclear staining (blue), after 30 days of differentiation, 50 µm scale bars. (C) Concentration of interleukin-6 (IL-6) produced by iAPCs and iNSCs stimulated with 50 µg of LPS for 24 hr (turquoise) and unstimulated (burgundy). Statistical significance was tested using a two-way ANOVA (N=5–7). (D) Cell counts of iNSCs (maroon) and iAPCs differentiated for 20 days (iAPC D20) (orange). Cell counts at days 2, 3, and 4 were statistically compared using a t-test (N=5). (E) Fluorescent activated cell sorting (FACS) results, based on PDGFRA sorting, for Batch 1 (top panel) and Batch 2 (bottom panel) of iOPC differentiation. (F) FACS results summary: the percentage PDGFRA+ cells for each cell line from each batch. Top five de novo (G) and known (H) motifs enriched in all hypo-methylated differentially methylated regions (DMRs) in iOPCs, from each iOPC versus iNSC comparison.
Figure 2—figure supplement 1—source data 1. Source data for Figure 2—figure supplement 1.