Figure 3.

SARS-CoV-2 infects human cerebral organoids and activates expression of innate immunity and neurodegeneration genes in neurons

(A) Representative immunofluorescence image of an 80-day-old organoid shows various cell types including neurons (MAP-2, green), neuronal progenitor (SOX-2, magenta), and nuclear stain (DAPI, blue); merged images are also shown. Scale bars, 100 μm.

(B and C) qRT-PCR analysis shows expression of ACE2 (B) and TMPRSS2 (C) in CORGs, NPCs, neurons, and astrocytes. Mean ± SEM of n = 3 independent organoids and n = 3 independent experiments from NPCs, neurons, and astrocytes. ^∗p < 0.05, ^∗∗p < 0.01 by Student's t test.

(D) Confocal imaging of CORGs showing immunostaining of neuronal marker (MAP-2, red) co-labeled with SARS-CoV-2 receptor (ACE2, green) or TMPRSS2 (red) co-labeled with MAP-2 (green). White boxes were enlarged (right), and arrows show co-labeling of MAP-2/ACE2 and MAP-2/TMPRSS2 in magnified images. Scale bars, 100 μm; for enlarged images, 25 μm.

(E–G) Organoids, NPCs, and neurons were infected with pseudo-SARS-CoV-2-GFP virus at MOI = 2 and analyzed after 24 h of infection. (E) Immunofluorescence and phase-contrast images of an organoid showing SARS-CoV-2 (green). Scale bars, 200 μm. (F) Immunofluorescence confocal images of NPCs stained with antibody against Nestin (red), SARS-CoV-2 (green), and nuclear stain DAPI (blue). Scale bars, 100 μm (merged) and 25 μm (magnified). (G) Immunofluorescence confocal images of NPC differentiated neurons stained with antibody against β-tubulin-III (red), SARS-CoV-2 (green), and nuclear stain DAPI (blue). Scale bars, 100 μm (merged) and 25 μm (magnified).

(H) Organoids were infected with pseudo-SARS-CoV-2-luciferase virus at MOI = 2, and luciferase activities were measured after 24 h of infection. Mean ± SEM of n = 3 independent organoids. ^∗∗∗p < 0.001 by Student's t test.

(I and J) NPCs, neurons, and astrocytes were infected with SARS-CoV-2 USA-WA1/2020 virus at MOI = 2, and viral RNAs from supernatant (I) and cellular (J) fractions were quantified at indicated times of infection. Mean ± SEM of n = 3 independent organoids. ^∗∗∗p < 0.001, ^∗∗p < 0.001, ^∗p < 0.05 by Student's t test.

(K–N) Neurons were infected as above in (I) and (J), and gene expression of indicated genes was quantified by qRT-PCR at 48 h post infection. Bar graph shows expression of innate immune response genes (K), C3 and C1q complement genes (L), synaptic function of neurons (M), and apoptotic pathway genes (N). Mean ± SEM of n = 3 independent organoids. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 by Student's t test.

See also Figure S2.