Fig 1: In vitro differentiation of NSCs into astrocytes fails to recapitulate in vivo maturation.a NSCs maintained in EGF/FGF2-containing medium (NSC_EF) are differentiated into astrocytes by culture in BMP4-containing medium for 14 days (Astr_BMP); maturity of astrocytes in these cultures is assessed by RNA-Seq and ATAC-Seq analysis and comparison with datasets from P4 and adult cortical astrocytes (Astr_P4/Astr_2m from Figs. 2 and 3). b Immunolabeling for the proliferation marker Ki67 and the astrocyte marker GFAP after 3 days of differentiation; scale bar 50 µm. Representative images from 3 biological replicates. c Expression of mature astrocyte-specific genes (from Fig. 2c) in cultured astrocytes and brain astrocytes. Heatmap of selected genes and number of genes, grouped by their expression patterns in cultured astrocytes. ‘NSC_EF/Astr_BMP low’ refers to significantly lower expression compared to Astr_2m, ‘high’ to equal or higher expression. d ATAC-Seq genome tracks show chromatin accessibility in cultured astrocytes vs brain astrocytes around the transcriptional start site (TSS) of Slc1a2 and Ntrk2 (expression highlighted in (c)). e Mature astrocyte-specific genes with low expression in vitro (Astr_BMP) lack accessibility at sites open in vivo (Astr_2m), more than genes with high expression. f Transcription factor binding motifs enriched in DNA regions where ATAC-Seq analysis shows lower chromatin accessibility in cultured astrocytes compared to adult brain astrocytes (de novo motif enrichment analysis). g Expression of selected transcription factors induced during astrocyte maturation in vivo and expressed at low levels in cultured astrocytes. Highlighted are factors that might bind the motifs in (f). Heatmap of cortical astrocyte bulk RNA-Seq data from Fig. 2; the factors displayed are differentially expressed in cortical and striatal astrocytes (except Lhx2 and Pou3f3 only in striatal astrocytes). Expression of the factors in striatal astrocytes is shown in Supplementary Fig. 6e. Statistical analysis and data presentation: c, g Differential genes/peaks: DESeq2 analysis; each n = 3 (n = 4 for ATAC-Seq Astr_2m data); two-sided Wald test with Benjamini–Hochberg correction; significance threshold: adjusted p-value = 0.05, absolute log2(fold change) = 1; Heatmaps show log2-transformed mean centred, normalized expression values; d Tracks represent merged reads of 3 replicates (4 replicates for Astr_2m). e Two-sided Wilcoxon Rank Sum test (n = 211 with low vs 143 genes with high expression in vitro). Boxplots show: centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. See also Supplementary Fig. 6, Supplementary Data 6.