FIG 3.

CNS-adapted MeV variants spread efficiently in human pluripotent stem cell (hiPSC)-derived brain organoids. (A) Two separate sets of 90-day-old human brain organoids (derived from two hiPSCs, FA10 and FA11) were infected with recombinant MeV viruses (with either EGFP or tdTomato fluorescent protein) bearing the indicated MeV fusion (F) proteins. For each virus, 3 separate wells each containing 2 to 4 organoids were infected (5,000 PFU/well). The brain organoids were monitored over time, and the fluorescence shown here reflects the infection after 10 days. Bar = 1,000 μm. (B) Viral titer of the inoculum used for infection was assessed on Vero CD150 cells (PFU/ml, log). (C) Total RNA was harvested from the human brain organoids at 10 days postinfection, and the level of MeV N gene expression was quantified by RT-qPCR. (D) RNA-Seq analysis of WT versus L454W F-bearing virus infection in brain organoids (data from three separate experiments). Seven replicates of uninfected and MeV-infected brain organoids were transcriptionally profiled (n = 6 uninfected, n = 5 WT, n = 2 L454W F-bearing virus). (E) RPM values for MeV for each sample are depicted in the same order as for the heatmap in panel D. Raw counts were normalized across all samples, and differential expression analysis was performed. The 50 genes with the lowest adjusted P value between MeV IC323-EGFP-F L454W and uninfected are depicted in the heatmap, colored by log₂ fold change of each sample relative to the mean normalized counts for each gene.