Fig. 1.

ZIKV efficiently infects hESC-derived brain organoids of different regional identities. a Schematic representation of hESCs patterning to regional brain organoids in vitro. Without patterning morphogens, human ESCs differentiated into forebrain dorsal (FD) NPCs at day 17. When Shh was applied from day 10 to day 17, hESCs were patterned into forebrain ventral (FV) NPCs. To generate hindbrain & spinal cord (H&S) NPCs, retinoid acid (RA) was added from day 10 to day 17. Light images show typical hESC clone at D0, columnar pNE at D10 (Scale bar, 200 μm), organized neurotube-like rosette of dNE at D14 (Scale bar, 25 μm) and regional organoid at D20 (Scale bar, 100 μm). b Confocal image showing undifferentiated hESCs were Oct4⁺/Sox2⁺. Scale bar, 50 μm. c Confocal image showing day 10 pNE cells were Pax6⁺/Oct4⁻. Scale bar, 50 μm. d FD, FV and H&S organoids were dissociated and plated onto coverslips for immunostaining. FD NPCs were Pax6⁺/Nkx2.1⁻/Foxg1⁺. FV NPCs were Pax6⁻/Nkx2.1⁺/Foxg1⁺. H&S NPCs were Foxg1⁻/Hoxb4⁺/Pax6⁺. Scale bar, 50 μm. e Schematic representation of ZIKV infection of FD, FV and H&S organoids from day 17 to day 23. f Confocal images stained for viral envelope proteins (red), Sox2 (green) and nuclei (blue) of ZIKV-infected FD, FV, H&S NPCs at different viral MOI (0.25, 0.5, 1.0) or mock control at 3 dpi. Sox2 is a hallmark protein expressed in NPCs. Scale bar, 50 μm. g qRT-PCR analyses of ZIKV pre-membrane mRNAs relative to GAPDH extracted from FD, FV, H&S NPCs infected with ZIKV at different MOI (0.25, 0.5, 1.0) or mock control at 2 dpi. Quantification data are presented as mean ± SEM. n = 3. h–j qRT-PCR analyses of regional marker genes Pax6, Nkx2.1 and Hoxb4 mRNAs relative to GAPDH extracted from FD, FV, H&S NPCs infected with ZIKV at 0.5 MOI or mock control at 3 or 6 dpi. Quantification data are presented as mean ± SEM. n = 3