Evaluation of 3D Human Intestinal Organoids as a Platform for EV-A71 Antiviral Drug Discovery

Figures

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  Figure 1. Characterization of the differentiation of human induced pluripotent stem-cells-derived small intestine organoids (HIOs) and Enterovirus A71 (EV-A71) replication kinetics in the derived HIOs and viability post infection. (A) Relative expression of total RNA of adult small intestine as a control and HIOs. The expression level of each gene was calculated relative to EEF1A1 gene expression as housekeeping gene. (B) Images represent differentiated HIOs: DAPI (blue) for nucleus, phalloidin (red) for F-actin, lysozyme (white) for Paneth cells, and mucin 2 (green) for goblet cells. Scale bar, 20 µM. (C) HIOs were infected with EV-A71 virus at multiplicity of infection (MOI) 0.01, 0.1, and 1. At the indicated time points, total culture was collected by combining the triplicates to measure the levels of viral RNA yield by RT-qPCR. (D) Brightfield images of mock HIOs and MOI 0.1 infected HIOs at 24 h post-infection (hpi) and 72 hpi show a difference in morphology indicating cytopathic effect (CPE) due to infection. All images were acquired using the same objective. Scale bar, 50 µM. (E) To measure viability of HIOs, Cell Titer-Glo 3D was used at the indicated time points post-infection.…

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  Figure 2. Comparison of EV-A71 replication kinetics between HIOs and RD cells. (A) EV-A71 replication kinetics 24 hpi. HIOs and RD cells were infected with EV-A71 virus at 0.01, 0.1, and 1 MOI. Total culture of HIOs and supernatant of RD cells were collected by combining triplicates and measured for viral RNA yield by RT-qPCR, and fold change is calculated based on mock infection. (B) Viability 24 h post EV-A71 infection. Viability in HIOs and RD cells was measured using Cell Titer-Glo 3D or Cell Titer-Glo, respectively. Mock-infected HIOs/cells were used to normalize the viability as a percentage. Data are the mean ± SEM of three independent experiments, each carried out in triplicate; ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001; ns indicates not significant.

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  Figure 3. Compounds’ cytoxicity in HIOs and RD cells. To measure the viability of HIOs and RD cells, Cell Titer-Glo 3D and Cell Titer-Glo, respectively, were used 72 h post-treatment. DMSO control was used to normalize the viability as a percentage. The half maximal cytotoxic concentration (CC50) was calculated using Prism nonlinear regression (GraphPad Prism 9). Data are the mean ± SEM of at least four independent experiments, each carried out in triplicate.

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  Figure 4. Comparison of antiviral activity determined by CPE inhibition and viral RNA yield between HIOs and RD cells. (A) Viability of HIOs and RD cells was measured, using Cell Titer-Glo 3D or Cell Titer-Glo, after 72 h of EV-A71 infection at MOI 0.1 and incubation with compounds at indicated concentrations. CPE inhibition is calculated based on percentage of mock-infected control. The half maximal effective concentration (EC50) was calculated using Prism nonlinear regression (GraphPad Prism 9). (B) For viral RNA yield, HIOs and RD cells were infected with EV-A71 at an MOI of 0.1 and treated with compounds at indicated concentrations for 72 h before total culture of HIOs or supernatant of cells were collected by combining triplicates and determined by RT-qPCR. Viral RNA yield is calculated based on percentage of mock-infected control. The EC50 was calculated using Prism nonlinear regression (GraphPad Prism 9). (C) Brightfield images of mock control, infection treated with 1.25 µM rupintrivir, and infected control HIOs and RD cells at 72 hpi. All images were acquired using the same objective. Scale bar, 100 µM. (D) Images represent mock-, EV-A71-infected HIOs (0.1 MOI), and…