Recapitulating influenza virus infection and facilitating antiviral and neuroprotective screening in tractable brain organoids

Abstract

Human pluripotent stem cell derived brain organoids offer an unprecedented opportunity for various applications as in vitro model. Currently, human brain organoids as models have been used to understand virus-induced neurotoxicity. Methods: The brain organoids were separately challenged by multiple viruses including influenza viruses (H1N1-WSN and H3N2-HKT68), Enteroviruses (EV68 and EV71) and Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) to investigate the impaired effect of these viruses on human brain development. Results: The brain organoids challenged by influenza viruses had decreased overall organoid size, while enteroviruses infected brain organoids displayed the opposite result. Then, we found WSN preferentially infected MAP2+ neurons compared to SOX2+ neural stem cells (NSCs) and GFAP+ astrocytes in brain organoids, and induced apoptosis of NSCs and neurons, and released inflammatory factors (TNF-α, INF-γ, and IL-6), facilitating brain damage. Furthermore, transcriptional profiling revealed several co-upregulated genes (CSAG3 and OAS2) and co-downregulated genes (CDC20B, KCNJ13, OTX2-AS1) after WSN infection for 24 hpi and 96 hpi, implicating target for antiviral drugs development. Finally, we explored compound PYC-12 could significantly suppress virus infection, apoptosis, and inflammatory responses. Conclusions: Collectively, we established a tractable experimental model to investigate the impact and mechanism of virus infection on human brain development.

Keywords: Antiviral screening.; Brain organoids; Human pluripotent stem cells; Influenza virus; RNA transcriptomic profiling.

Figure 1

Organoids as virus infection models. (A) Schematic illustration of experimental design. Day 40 brain organoid was infected with viruses at indicated time. (B, D, F, H, J) Representative bright-field images of day 40-brain organoids infected with viruses at indicated time point, including H1N1-WSN, H3N2-HKT68, EV68, EV71 and SFTSV (n = 3 per group) (Note: The control group was the same in all groups). The infected concentrations for H1N1-WSN were 5x10⁶ pfu, 1x10⁶ pfu and 2x10⁵ pfu; for H3N2 were 5x10⁶ pfu, 1x10⁶ pfu and 2x10⁶ pfu; for EV68 were 4x10⁵ pfu, 8x10⁴ pfu and 1.6x10⁴ pfu; for EV71 were 2x10⁷ pfu, 4x10⁶ pfu and 8x10⁵ pfu. Scale bars, 50 μm. (C, E, G, I, K) Statistical analysis of area (μm²) and diameter (μm) of brain organoid infected with viruses at indicated time point. Measurement were collected till day 11 for the higher dose (red) of EV68 group due to the dissociation of organoid on day 14 and day 16. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Modeling Influenza virus in vitro using brain organoids. (A) Schematic illustration of the experimental design of brain organoids infected with WSN at indicated time points. (B) Immunostaining of neural stem cells and astrocytes infected with WSN. Scale bars, 50 μm. (C, D) Immunostaining of neural stem cells and neurons of day 30 and day 60 brain organoids infected with WSN for 1 day and 4 days. Scale bars, 100 μm. (E, H) The percentage of NP+ cells in infected day 30 and day 60 brain organoid. ~10% of SOX2+ NSCs and ~60% of MAP2+ neurons were infected with WSN. (F, G, I, J) The viral titers of intracellular and supernatants on day 30 and day 60 brain organoids. (K) Immunostaining of neural stem cells, neurons and astrocytes of day 250 brain organoids infected with WSN for 1 day and 4 days. Scale bars, 100 μm. (L) The percentage of NP+ cells in infected brain organoid. ~30% of SOX2+ NSCs, ~60% of MAP2+ neurons and 10% astrocytes were infected with WSN. (M) The viral titers of supernatants on day 250 brain organoids. (N) Schematic illustration of WSN preferentially infected neurons in brain organoid. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

The RNA transcriptomic analysis of human brain organoids after WSN infection. (A) Day-60 organoid was infected with WSN at a multiplicity of infection (MOI) of 1 at 1 dpi and 4 dpi compared to mock-infected organoids (n=3). Hierarchical clustering heatmap of differentially expressed genes derived from the comparison among the group of control, hpi96 and hpi24. (B) Venn diagram of upregulated and downregulated genes of brain organoids infected with WSN at 1 dpi and 4 dpi. (C) Top 10 of enriched GO terms of brain organoids infected with WSN at 1 dpi and 4 dpi. (D) The top 10 of upregulated and downregulated genes at 1 dpi and 4 dpi after WSN infection. (E) The co-upregulated and co-downregulated genes at 1 dpi and 4 dpi after WSN infection. (F) Heatmap of interferon stimulating genes (ISGs).

Figure 4

WSN induced cell apoptosis and inflammation of human brain organoids. (A) Schematic illustration of infection flow. Brain organoid was infected with WSN at a multiplicity of infection (MOI) of 1 at 1 dpi and 4 dpi compared to mock-infected organoids (n=3), then the apoptosis and inflammation were monitored. (B-D) The TUNEL staining and quantification of positive cells on day-30 and day-60 brain organoids infected with WSN at 1 dpi and 4 dpi in a time-dependent increase compared to control group, respectively (Left panel represents SOX2+ NSCs, right panel represents MAP2+ neurons in each figure of C-D). Scale bars, 10 μm. (E) The TUNEL staining of GFAP+ astrocytes of day 250 brain organoids at 4 dpi. (F, G) The secreted inflammatory factors (e.g., TNF-α, IL-6, CCL2, IFN-γ and COX2) of day-30 (F) and day-60 (G) brain organoids at indicated infection timepoint, respectively. (H) Summary of the impairment mechanism of WSN infected brain organoid. Scale bars, 100 μm. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Antiviral drug study of human brain organoids infected with influenza virus. (A) Schematic of the workflow of drug screening. Brain organoids were first treated with compounds for 2 hours, followed by co-treatment with WSN and compounds for 1 hour, then continued to compounds treatment for observed days, respectively. (B) The representative bright field images of organoids cotreated with H1N1-WSN (MOI=1) and several drugs at indicated time points, respectively. Nucleozin was as positive control. Scale bars, 400 μm. (C) Immunostaining of neural stem cells and neurons of day 30 brain organoids co-treated with WSN and PYC-12 for 1 day and 4 days. Scale bars, 10 μm and 50 μm. (D, E) The statistical analysis of NP+ cells on day 30 brain organoids co-treated with WSN and PYC-12 for 1 day and 4 days. (F, G) The statistical analysis of TUNEL+ cells in neural stem cells and neurons of day 30 brain organoids co-treated with WSN and PYC-12 for 1 day and 4 days. Scale bars, 10 μm. (H) The viral titers of intracellular and supernatants on day 30 brain organoids co-treated with WSN and PYC-12. (I) The inflammatory factors release (IL-6 and TNF-α) of day-30 brain organoids co-treated with WSN and PYC-12 at indicated infection timepoint, respectively. (J) The representative bright field images of Microelectrode arrays (MEA) analysis of PYC-12 treated brain organoids. Scale bars, 50 μm. (K) Weighted mean firing rate (Hz) of brain organoids treated with H1N1 or cotreated with H1N1 and PYC-12 at indicated time points. (L) The schematic illustration of antiviral strategy of PYC-12 through anti-apoptosis and anti-inflammation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.