a hNPCs were challenged with 10 MOI SARS-CoV-2 or SARS-CoV. Viral supernatant samples were harvested at 0, 24, and 48 hpi and viral loads were determined by qRT-PCR. The cell viability of SARS-CoV-2 and mock-infected hNPCs were quantified with CellTiterGlo assays. Bars represent means ± SD of three independent experiments. b Supernatant samples from SARS-CoV-2-inoculated neurospheres were analyzed for viral RNA by qRT-PCR. Bars represent mean ± SD. Infectious virus titer of the supernatant samples from SARS-CoV-2-inoculated neurospheres were determined by plaque assay. Data were obtained from three (n = 3) independent batches of neurospheres in three experiments. c Representative confocal images of SARS-CoV-2- or mock-inoculated neurospheres harvested at 72 hpi. SARS-CoV-2 was identified with a SARS-CoV-2 nucleocapsid (N) protein immune serum (green). Scale bars, 100 µm. d Representative transmission electron microscopy images of SARS-CoV-2-inoculated neurospheres at 72 hpi. Complete SARS-CoV-2 particles (red arrowheads), and nucleus (white N). Scale bars, 2 µm, 500 nm, or 200 nm. e Characterization of 35-day-old brain organoids. Brain organoids were fixed with 4% paraformaldehyde, and then transferred to 30% sucrose solution and embedded for cryosectioning. A representative bright-field image of brain organoid was shown. DAPI-stained 35-day-old brain organoid showing complex inner morphology. Representative images of 35-day-old human brain organoids immunostained for neuronal cell marker TUJ1, radial glial cell marker PAX6, and proliferation neural progenitor cell marker NESTIN. Scale bars, 100 µm. f Representative images of SARS-CoV-2-infected brain organoids. SARS-CoV-2 was detected using a SARS-CoV-2-N immune serum (green). SARS-CoV-2 infected cells were identified at the peripheral region (arrows) and in deeper regions (white arrowheads) of the organoids. Substantial cell-cell fusion was detected (yellow arrowheads). No positive N signals were detected in mock-infected brain organoids. Scale bars, 100 µm. g Viral supernatant samples were harvested at 0, 24, 48, and 72 hpi. Virus replication was detected by qRT-PCR. Infectious virus titer was determined by plaque assay on Vero E6 cells. Statistical significance was determined by one-way ANOVA. Bars represent the means ± SD of three independent experiments. h Representative images of SARS-CoV-2-infected human brain organoids immunostained for SARS-CoV-2-N and TUJ1. Scale bars, 100 µm or 20 µm. i Representative images of SARS-CoV-2-infected human brain organoids immunostained for SARS-CoV-2-N and NESTIN. Scale bars, 100 µm or 20 µm. Confocal images were obtained on a Zeiss LSM880 confocal imaging system. The images were representative of three (n = 3) independent batches of neurospheres or organoids from three experiments. Statistical significance was determined by two-way ANOVA. MOI multiplicity of infection, hpi hours post inoculation.