SARS-CoV-2 targets neurons of 3D human brain organoids (2020)
Figure 1

Figure 1. SARS-CoV-2 targets the cortical region of human brain organoids

A. A positive control experiment. SARS-CoV-2 readily targets ciliated human respiratory epithelial cells (hRECs). Acetylated α-tubulin labels cilia. Arrows point SARS-CoV-2-positive cells labeled by AB4 (green). Figures display scale bars. Bar diagram at right quantifies frequencies of SARS-CoV-2-positive cells in hRECs. At least six hREC sections from three (n=3) independent samples were examined. Data presented as mean + SEM.

B. Mock organoids of two age groups Day-15 (i) and-60 (ii) display typical cytoarchitecture of brain organoids. L, Lumen, VZ, Ventricular Zone is containing compact and palisade-like elongated nuclei of Neural Progenitor Cells (NPCs, blue) and CP, a cortical plate containing TUJ-1-positive neurons (magenta). Note a distinct difference TUJ-1 labeling pattern between younger (Day-15) and older (Day-60) brain organoid. Figures display scale bars. Representative images from eight organoids cultured in at least three independent batches (n=3) derived from donor-1 (IMR90) iPSC line.

C. Compared to mock organoids (i), SARS-CoV-2-exposed Day-15 organoids display SARS-CoV-2-positive cells (AB4, green) in their outer periphery, a region of the cortical plate (ii) that is specified by TUJ-1-positive neurons (magenta). L, the lumen of a VZ, the inner area of an organoid where NPCs are located, is free from SARS-CoV-2-positive cells. Magnified region (dotted while box) is given below. At least ten organoids from five different batches (n=5) are tested. Figures display scale bars.

D. SARS-CoV-2-exposed Day-60 organoids. Compared to Day 15 organoids and mock (i), Day-60 organoids display an increased number of SARS-CoV-2-positive cells (AB4, green) in their cortical plate that is specified by TUJ-1-positive neurons (magenta) (ii). Magnified region (dotted while box) is given below, showing the peri-nuclear location of SARS-CoV-2 in cortical neurons. At least ten organoids from five different batches (n=5) are tested. Figures display scale bars.

E. The bar diagram quantifies frequencies of SARS-CoV-2-positive cells in different brain organoid sections derived from two donor iPSC lines (IMR90 and Crx-iPS, see methods). Please note that each point represents one organoid section. SARS-CoV-2 shows an enhanced tropism for Day-60 organoids. Note, comparative statistics are shown between different age groups and respective days post infection (dpi) of organoids, and the significance is given as Asterisks in Day-60 groups. There is no significant difference in SARS-CoV-2-positive cells between 2- and 4-dpi within each age groups. At least twelve organoids sections from four (n=4) independent batches, from each donor and day post infections (dpi) were analyzed. One-way ANOVA, followed by Tukey's multiple comparisons test, ***P<0.001. Data presented as mean + SEM.

F. Subcellular localization SARS-CoV-2 in cortical neurons. High-resolution imaging and de-convolution show peri-nuclear localization of SARS-CoV-2. SARS-CoV-2 (AB4, green) and nucleus (grey). Figures display scale bars. Representative images from at least 200 cells are examined. White line surrounds perinuclear border and red line encircles the nucleus.

G. Determination of viral progeny. Supernatants of SARS-CoV-2 exposed Vero cells, and brain organoids were analyzed for viral RNA assessed by qRT-PCR. While an increase in viral RNA was detected in the supernatants of Vero cells, no apparent increase was identified in brain organoid supernatants. Data are obtained from five technical replicates from four (n=4) independent batches of organoids. Data presented as mean + SEM.