Contemporary enterovirus-D68 isolates infect human spinal cord organoids

Figures

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  Fig 1. iPSCs can be differentiated into human spinal cord organoids. (A and C) Schematic showing differentiation protocol for spinal motor neuron (SMN) organoids (A) and 3-dimensional spinal cord (3-DiSC) organoids (C). (B and D) Morphologic evaluation of transition from iPSC aggregates to hSCO, SMN (B) and 3-DiSC (D). The day post-differentiation is marked in the upper left. (E) Schematic describing the expression pattern of progenitor markers in the spinal cord during development. Adapted from Ogura et al. (20). (F) qRT-PCR on day 14 comparing relative marker expression of SMN or 3-DiSC hSCO (*P < 0.05, **P < 0.01). Scale bar, 250 µm.

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  Fig 2. Contemporary EV-D68 infects human spinal cord organoids. (A and D) SMN (A) and 3-DiSC (D) organoids at 14 days post-differentiation in pools of 8–12 structures were either mock-infected or infected with 8.5 × 105 PFU/well of US/KY/14-18953. At 48 h post infection (hpi), lysates were collected for qRT-PCR. (B and C, E and F) SMN (B and C) and 3-DiSC (E and F) hSCOs in pools of 8–12 were either mock-infected or infected with 105 PFU/well US/KY/14-18953 and processed for immunofluorescence at 24 hpi. 4′,6-Diamidino-2-phenylindole (DAPI) in blue, neuron-specific beta III tubulin (tuj1) in green, and EV-D68 VP1 in red. Mean fluorescence intensity (MFI) for VP1 staining across multiple Z-stacks and individual organoids (n ≥ 3) is quantified for SMN (C) and 3-DiSC (F) (*P < 0.05, **P < 0.01). Scale bars, 100 µm.

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  Fig 3. 3-DiSC organoids maintain consistent cell number and viability. 3-DiSC hSCOs were differentiated per protocol and evaluated at multiple time points post-infection for the number of cells per structure (A), percent viability (B), and morphology (C and D). Structures were dissociated into single cells using Accumax and enumerated. Trypan blue staining was used to assess viability (*P < 0.05). Scale bar, 500 µm. Pix refers to pixels.

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  Fig 4. Productive EV-D68 infection does not alter hSCO morphology. 3-DiSC hSCOs 14 days post-differentiation were infected with 105 PFU/mL of either Fermon or US/KY/14-18953 in pools of 12. Supernatants were collected for titer determination between 0 hpi and 14 days post infection (dpi). Terminal cellular lysate was collected at 14 days post infection (14 dL) (A). hSCOs were monitored daily for morphology for 10 days (B and C). Scale bar, 500 µm. Pix refers to pixels.

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  Fig 5. 3D hSCOs can be infected with multiple contemporary strains of EV-D68. 3-DiSC hSCOs were infected with a panel of EV-D68 viruses as seen in the phylogenetic tree (A) representing multiple clades. These viruses all replicated robustly in RD cells (B), but more variably in 21-day post-differentiation 3-DiSC hSCO as demonstrated by titer (hpi) and terminal cellular lysate (96L hpi) (C). Fourteen-day post-differentiation 3-DiSC hSCOs in pools of 8–12 were either mock-infected or infected with 105 PFU/well EV-D68 and processed for immunofluorescence at 24 hpi. DAPI in blue, actin in red, and EV-D68 VP1 in yellow. Arrowheads highlight VP1 signal (D). Mean fluorescence intensity (MFI) for VP1 staining is quantified for each strain across multiple Z-stacks and organoids (N ≥ 3) (E) (**P < 0.01, ****P < 0.0001). Scale bars, 100 µm.

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  Fig 6. 3-DiSC hSCOs are infected beyond the surface layer of cells. Pools of 8–12 3-DiSC hSCOs were infected for 24 h with US/MA/18/23089 and fixed and processed for confocal microscopy (A). Selections from the Z-stack are shown up to 85 µm in depth, as shown by the schematic (B). VP1 in red, with several infected cells identified with arrows measuring the distance from the structure surface. Scale bar, 100 µm. Pools of 8–12 3-DiSC hSCOs were infected for 24–72 h with US/MA/18/23089 and fixed and processed for confocal microscopy. The number of infected cells visualized was counted and grouped by distance from surface across multiple Z-stacks (17.5 µm or deeper into the stack) and individual organoids (N3). The data are shown as individual fields (C) and as total percentage over time (D) (**P < 0.01, ****P < 0.0001).

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  Fig 7. EVD-68 infection of hSCO causes less apoptosis than infection with E11. (A and B) 3-DiSC hSCOs at 14 days post-differentiation were infected with 105 PFU/mL of echovirus 11 (E11). Morphology was evaluated at 24 and 48 hpi. Arrowheads mark cellular debris. (C and D) 3-DiSC hSCOs at 14 days post-differentiation were infected with 105 PFU/mL of either US/KY/14-18953 or E11. hSCOs were fixed for immunofluorescence at 24 hpi and stained for VP1 (red), cleaved caspase 3 (green), and DAPI (blue). (D) MFI was quantified for cleaved caspase 3 (left) and normalized to VP1 signal (right) across multiple Z-stacks and individual organoids (N ≥ 3). (E and F) 3-DiSC hSCOs at 14 days post-differentiation were infected with 105 PFU/mL of either US/KY/14-18953 or E11. hSCOs were fixed for immunofluorescence at 24 hpi and TUNEL assay was performed. Whole organoids were stained with anti-BrdU (green) to demonstrate DNA breaks and nuclei were stained with propidium iodide (PI). (E) MFI was quantified for TUNEL for each condition across multiple Z-stacks and individual organoids (N ≥ 3). (*P < 0.05, ****P < 0.0001). Scale bar, 100 µm.