Generation of human iPSC-derived microglia, cortical neurons, and astrocytes. (A–H) Confocal microscopy images of iPSCs and derived astrocytes, microglia, and cortical neurons from a healthy control donor and a pediatric HSE patient heterozygous for the IRF3 R285Q amino acid substitution stained with cell type–specific markers. (A and D) iPSCs: NANOG and OCT4 (both green); (B and E) astrocytes: S100 (red) and GFAP (green); (C and F) microglia: TREM2 and Iba1 (both green); (G and H) cortical neurons: MAP2, β-III tubulin (both green), synaptophysin, and TBR1 (both red); nuclei were identified by DAPI staining. Scale bar, 50 μm. (I and J) Comparison of IFNB response to HSV-1 infection in microglia from two different iPSC lines and two different patient-derived iPSC clones measured by RT-qPCR. Commercially available iPSC lines 015A and BIONC (I) and patient-derived iPSC clones C4 and C6 (J). (K)IFNB expression in microglia after stimulation with polyIC (25 μg/ml) and cGAMP (100 μg/ml). (L and M)ISG15 expression in microglia 4 and 24 h after infection with HSV-1 at MOI 1.0. (N and O)ISG15 expression in cortical neurons infected 24 h with HSV-1 at MOI 1.0 or stimulated for 4 h with polyIC (25 μg/ml) and cGAMP (100 μg/ml). Expression data were normalized to β-actin and shown as fold change compared with the UI control. Statistical analyses of gene expression in CNS cell cultures (K–O) were analyzed by two-tailed two-way ANOVA for difference of means, followed by an unpaired t test of means, error bars; SD. P values <0.05 were considered statistically significant, **P < 0.01, and ***P < 0.001.