Extended Data Figure 5. The old NSC lineage exhibits a heterogeneous response to IFNγ.

a) Principal component analysis (PCA) of 562 old cells in the neural stem cell lineage (astrocytes/qNSCs and aNSCs/NPCs) performed only using genes in the IFNγ Response Hallmark from MSigDB (Supplementary Table 8). IFNγ-high cells (dark red with black ring) are defined as old cells exhibiting an average expression of genes in the IFNγ Response Hallmark pathway in the top 5% of old cells; b) PCA as in (a), but with a separate PCA performed for each of three 10x Genomics replicates (n=162, n=315, n=85 cells respectively) and for the dataset generated with Fluidigm C1 technology (n=137 cells); c) Average of normalized expression values of genes in IFNγ Response Hallmark for young and old cells in cells of the NSC lineage (Astrocytes/qNSCs and aNSCs/NPCs). Cells are grouped by age. IFN-high cells (in black) are defined as cells exhibiting an average expression of genes in the IFNγ Response Hallmark pathway in the top 5% of the cells analyzed within each 10x Genomics replicate. Note that replicate three contains ~2 fold more young cells than old cells. Horizontal lines in violin plots denote median IFNγ Response Pathway expression. See Supplementary Table 3 for exact cell counts; d) FACS analysis of STAT1 positive cells in the young and old NSC lineage (PROM1+CD45−CD31−CD24−O4−). Left: FACS histograms of STAT1 fluorescence in PROM1+ cells isolated from the SVZ from two representative young (3 months) and old (20 months) male mice. Inset: Quantification of the percentage of STAT1-high cells in 15 young (3-5 months) and 14 old (19-26 months) mice. Mean +/− s.e.m. of percentage of cells that are STAT1-high of the ~500 cells analyzed for each mouse. Each dot represents ~500 cells from one mouse. The combined results from five independent experiments are shown (for independent experiments, see Supplementary Table 12). ***P=5.08 × 10⁻⁶, two-sided Wilcoxon rank sum test; e) The gene encoding the surface marker BST2/Tetherin is expressed in the old NSC lineage and is correlated with genes that belong to IFNγ signaling. Data is shown as heatmap with log-normalized expression of Bst2 and other select genes in the IFNγ Response Hallmark pathway. Cells are clustered based on expression of this gene set. The age of the mouse from which the cells are isolated is indicated in a bar above the heatmap; f) Live FACS analysis for BST2 in cultured NSCs following IFNγ treatment for 48 hours; g) Abundance of total STAT1 protein in BST2-positive versus BST2-negative aNSCs/NPCs isolated from 9 old (25 months) mice, as measured by intracellular FACS. Mean +/− s.e.m. of total STAT1 fluorescence. Each dot represents cells from one mouse. The combined results from two independent experiments are shown (for independent experiments, see Supplementary Table 12). *P=0.04, two-sided Wilcoxon rank sum test; h) FACS quantification for KI67, a marker of cycling cells, in BST2-positive and BST2-negative aNSCs/NPCs from 15 old (23-25 months) mice. Mean +/− s.e.m. of percentage of cells that are KI67 positive of the ~100 cells analyzed for each mouse. Each dot represents ~100 cells from one mouse. The combined results from three independent experiments are shown (for independent experiments, see Supplementary Table 12). ***P=7.80 × 10⁻⁴, two-sided Wilcoxon rank sum test.