Non-polio enterovirus infection and electrophysiological changes in human iPSC-derived neural networks

Figures

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  Fig. 1. Ngn2-expressing neurons co-cultured with astrocytes form functional neural networks and respond to stimulation and inhibition. hPSC-derived Ngn2-expressing neurons were co-cultured with hPSC-derived astrocytes and matured for 21 DIV. (A) Immunofluorescent staining was performed with astrocytic-specific marker GFAP (magenta) and the neuron-specific marker MAP2 (cyan). Cells were counterstained with the nuclear dye Hoechst at DIV 21. (B) Immunofluorescent staining for functional synapses was performed against MAP2 (cyan), synapsin 1 (green, marker for pre-synaptic density), and Homer 1 (red, marker for post-synaptic density) at DIV 22. (C) Brightfield images of neural co-cultures plated on micro-electrode array plates to measure neural activity at day 21. Data shown are representative pictures of at least 2 independent experiments (Panel A–C). (D) Parameters for neural activity of Ngn2 neural co-cultures between DIV 21 and 31: firing rate, burst frequency, and network burst frequency. Data are depicted as mean ± SEM and are derived from eight independent experiments (n = 6 per experiment, unless datapoints were excluded based on exclusion criteria, see Material and Methods). (E)…

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  Fig. 2. Enterovirus-D68 and Enterovirus-A71 productively replicate in co-cultures and show similarity in their cell tropism. (A) Neural co-cultures were infected with different EV-D68 strains and the EV-A71 strain Sep006 from the C4 genotype at an MOI of 0.1. Viral titres were determined in the supernatant at the indicated timepoints by endpoint dilution, with the first data point being the back-titration of the inoculum. Replication kinetics were performed in technical triplicates in three independent experiments and data represented show the mean with 95% CIs. (B) At 24 hpi, the co-cultures were fixed and stained for the presence of EV-D68 structural antigen VP1 (yellow). MAP2 (cyan) was used as a marker for neurons, and astrocytes were identified by staining for GFAP (magenta). (C) At 24 hpi, EV-A71 inoculated co-cultures were fixed and stained for the presence of dsRNA, a marker for active EV-A71 replication (yellow), astrocytes (GFAP, magenta) and neurons (MAP2, cyan). The immunofluorescence data shown are representative examples from three independent experiments for each culture condition. Maximum intensity projections of Z-stacks are displayed. (D) Quantification of infection…

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  Fig. 3. Enterovirus infection induces cell death in neural co-cultures. Neural co-cultures were infected with EV-D68 or EV-A71 at an MOI of 1. (A) Bar graphs showing percent cell viability after infection with EV-D68 A2/2018, EV-D68 B3/2019 and EV-A71 Sep006 as measured by amount of LDH released into cell culture supernatant at indicated time points. Data are derived from three independent experiments performed in biological triplicates. Statistical significance was determined with a One-Way ANOVA, comparing the LDH release in virus infected samples to untreated mock control at each timepoint. (B) At 72 h post infection (hpi), neural cultures were fixed and stained for double-stranded RNA (dsRNA; yellow) as a marker of viral infection, for the astrocytic marker GFAP (grey) and for cleaved caspase-3 (CC-3; magenta). Cell nuclei were counterstained with Hoechst (blue). (C) At 72 hpi neural cultures were fixed and stained for dsRNA (yellow), for the neuronal marker MAP2 (grey) and for cleaved caspase-3 (CC-3; magenta) as a marker of apoptosis. Immunofluorescent data shown are representative examples from three independent experiments (B,C). (D) Quantification of CC-3 percentage over MAP2…

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  Fig. 4. Heatmap of normalised neural activity data recorded from neural co-cultures inoculated with non-polio enteroviruses. Neural activity was recorded from neural co-cultures, consisting of Ngn2 neurons and astrocytes, on a MEA platform. Neural co-cultures were mock-inoculated or inoculated with EV-D68 A2/2018, B3/2019, or EV-A71 Sep006 with an MOI of 1. Only data that met inclusion criteria for MEA recordings were used (see Material and Methods). Any experiment that contained Not Available values throughout any of the chosen MEA output variables (Table 3) was excluded for PCA analysis, to ensure biological relevance and integrity of the dataset. Data was normalised against baseline recording obtained before inoculation. Data is shown from one (A), three (B), or 10 dpi (C). Rows represent output variables of MEA data and columns represent experimental data from a single well. All MEA data were z-score normalised for each variable across inoculation groups to enable cross-condition comparisons. Colour intensity reflects a positive or negative effect on neural activity, with white to red indicating a more positive effect. Data presented in the heatmap are derived from at least four…

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  Fig. 5. Enterovirus infection impacts the spontaneous activity of neural co-culture. Neural co-cultures were inoculated with EV-D68 A2/2018 (pink), B3/2019 (cyan), or EV-A71 Sep006 (purple) with an MOI of 1, and neural activity was measured between one to three dpi. The following parameters were displayed (A) number of active electrodes, where wells were excluded if they contained 5 or less active electrodes at baseline (indicated with a red line); (B) firing rate; (C) burst frequency, (D) number of spikes per burst, (E) network burst frequency and (F) number of spikes per network burst. Data displayed are derived from at least four independent experiments (n = 48 for control and n = 24 per inoculation group, see exclusion criteria in the Material and Methods section). Statistical significance was calculated with a two-way ANOVA with a Šídák's multiple comparisons post hoc test. Asterisks indicate statistical significance (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). Abbreviations: EV, enterovirus; MOI, multiplicity of infection; dpi, days post inoculation.