Fig 2: ACM prevents the upregulation of ID3 induced by PD901. A, Expression heatmaps of genes which by RNA-seq analysis were found to be commonly affected in WM4265.2 or WM983B cells incubated in the presence versus absence of ACM and PD901. Cells were incubated in ACM for 48 hours and PD901 (10 µmol/L) was added for 8 hours. B, Volcano plot highlighting the top up- and downregulated genes in WM4265.2 and WM983B cell lines treated with PD901 in the presence of AM or ACM. Significance of gene expression changes was defined using FDR threshold of 5%. C, qRT-PCR analysis of RNA levels of ID3, normalized to TBP, using independent samples of WM4265.2 and WM983B. Values shown are mean ± SD of n = 3. ***, P < 0.001; **, P < 0.01; *, P < 0.05 as per two-tailed Student t test. D, Western blot, probed with indicated antibodies, of lysates from WM4265.2, WM983B, and 1205Lu cell lines incubated in AM or ACM, and treated with PD901 (10 µmol/L) for 8 hours.

Fig 3: ID3 regulates expression of genes involved in DNA repair in response to IR. (A) Volcano plots showing differentially expressed genes in ID3-KO cells; untreated (0 Gy, left) and irradiated cells (5 Gy, right). Measured by RNA-seq (n = 4). (B) Gene ontology enrichment analysis of the significantly downregulated genes in irradiated ID3-KO cells. (C) Barcode plot showing gene set enrichment analysis of DNA repair pathways of differentially expressed repair genes in irradiated ID3-KO cells. NES = normalized enrichment score. (D) Heat map showing differentially expressed DNA repair genes with a log2 fold change < -0.5 for downregulated genes and > 0.5 for upregulated genes in irradiated WT, ID3-KO and ID3-rescue cells. n = 4, 4 and 2 for WT, KO and rescue cells, respectively. (E) RT-qPCR expression analysis of BRCA1, EXO1, RBBP8 (CtIP) and RAD51 genes in WT and ID3-KO with IR treatment (5 Gy) at indicated time points, n = 5, data presented as mean ± SEM, one-way ANOVA with Dunnett's multiple comparison test to compare all to the WT untreated. (F) Western blot showing expression of BRCA1, EXO1, RBBP8 (CtIP), and RAD51 proteins in WT and ID3-KO with IR treatment at indicated time points (representative Western blot, upper panel) and a bar plot of the quantification of three independent Western blots (lower panel, mean ± SD), Student's t test was performed. Statistical significance is presented as: * P< 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Fig 4: Astroglia derived from NPC grafted into CNS lesions exhibit features of border-forming astroglia.a Detailed IHC images showing the astrocyte border phenotype of stroke lesions at 4 weeks in untreated stroke only and NPC grafted strokes. b Survey IHC of NPC grafted SCI lesions at 2 weeks showing NPC-derived astrocyte borders formed around Cd13-positive inflammatory cells. c Detailed IHC images showing the astrocyte border phenotype in SCI derived from transplanted NPC and not from host astrocytes (TK-positive). d, e Survey and detailed, higher magnification 3D IHC imaging of healthy mouse cortex and meninges showing higher expression of transcriptional regulator Id3 (inhibitor of DNA binding 3) in host astrocytes (HA+ Gfap-Cre-RiboTag) that form glia limitans at the meninges and penetrating blood vessels. f High magnification 3D IHC images showing astrocyte borders formed at stroke lesions upregulate expression of Id3. g High magnification 3D IHC images showing grafted NPC that remodel stroke lesions also express Id3. h NPC grafted into SCI lesions that form astrocyte borders also express Id3.

Fig 5: Human induced pluripotent stem cell (iPSC)-derived astrocytes confirm a cell-intrinsic vanishing white matter (VWM) phenotype. (A–F) Immunostaining showed expression of astrocyte markers glial fibrillary acidic protein (GFAP; A, D), nestin and Id3 (B, E), and CD44 and SOX9 (C, F) in both control (A–C) and VWM (D–F) human astrocyte cultures. To quantify proliferation of the astrocytes, bromodeoxyuridine (BrdU) labeling was performed. (G, H) Representative control (G) and VWM (H) astrocyte lines are shown. Quantification of proliferation was assessed using the percentage of BrdU-labeled cells of the total population of cells (4,6-diamidino-2-phenylindole [DAPI]) in control human iPSC (hiPSC)-derived astrocytes. (I) VWM astrocytes showed increased proliferation compared to control astrocytes. (J, K) Immunostaining for Olig2 and myelin basic protein (MBP) of primary wild-type embryonic day 18 mouse oligodendrocyte precursor cells cultured for 7 days in media conditioned by hiPSC-derived astrocytes in representative examples of VWM and control cultures is shown. Maturation of the oligodendrocyte precursor cells was assessed using a ratio of MBP-positive cells of the total Olig2-positive population, in the presence of media conditioned by control and VWM iPSC-derived astrocytes. Astrocyte conditioned media were treated with either vehicle or hyaluronidase (HAse). (L) Oligodendrocyte maturation was impaired in VWM compared to control cultures, and HAse rescued oligodendrocyte maturation comparable to control level. Scale bars = 100µm. *p < 0.05, **p < 0.01.