Modeling Poliovirus Infection Using Human Engineered Neural Tissue Enriched With Motor Neuron Derived From Embryonic Stem Cells

Figures

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  Figure 1. MN ENT generation and characterization. (A) Schematic representation of hESC differentiation toward lnENTs and MN ENT. (B) RT-PCR analysis of the expression of pluripotency (OCT4 and NANOG), neuroprogenitor cells NPCs (NES and PAX6), motor neuron progenitor cells (MN NPCs) (NGN, PAX6, and OLIG2), motor neuron enriched ENTs (CHAT and HB9), and markers for neuronal, astroglial and oligodendroglial cells (MAP2, TUBB3, GFAP, and OLIG2). Human lymphocytes B were used as negative controls. (C) Immunofluorescence showed NeuN, CHAT, and HB-9-immunoreactive cells present in the whole MN ENT. Scale bar = 50 μm. (D) RT-PCR analysis of the expression of pluripotency (OCT4 and NANOG), neuroprogenitor cells NPCs (NES and PAX6), and non-directed late neural ENTs (MAP2, TUBB3, GFAP, and OLIG2). Human lymphocytes B were used as negative controls. (E) Immunofluorescence showed βIII-Tubulin, NeuN, and HB-9-immunoreactive cells present, or not, in the lnENT. Scale bar = 100 μm.

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  Figure 2. MN ENT shows a greater permissiveness for PV-3 compare to lnENT. (A) Infection of motor neurons (MN), neural progenitors (NPC), and human embryonic stem cells (hESCs) by PV-3. Immunofluorescence showed PV-3 (green), HB-9, Nestin, and Oct4 (all in red) in MN, NPC, and PSC, respectively. Scale bar = 50 μm. (B) Schematic representation of PV-3 and EV-71 infection of ENTs. (C) Immunohistochemistry showing PV-3-immunoreactive cells within MN ENTs (left panel) and lnENT (right panel).

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  Figure 3. PV-3 preferentially infects motor neurons in MN ENTs. (A) Schematic representation of PV-3 infection of MN ENTs. (B) Quantitative RT-PCR of mRNA of PV-3 in PV-3-infected MN ENTs at day 1, 3, 5, and 7. Data are normalized to housekeeping genes; mean ± SEM with ***p < 0.01 for n = 3 independent experiments. (C) MN ENTs were fixed at 3, 5, and 7 days after PV-3 infection, sectioned, stained by immunohistochemistry with antibodies against PV-3. Scale bar = 50 μm. (D) MN ENTs were fixed at 3, and 5 days after PV-3 infection, sectioned, and stained with antobodies against PV-3 (in green), CHAT (in red) and with DAPI (in blue). Scale bar = 50 μm.

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  Figure 4. PV-3 infection also affects neural progenitors and induces an innate antiviral response. (A) RBFOX3, NES, and TUBB3 mRNA expression was determined by qPCR in PV-3-infected MN ENT at day 1, 3, and 5. (B) Immunofluorescence showing βIII-Tubulin, and NeuN -immunoreactive cells within the MN ENTs at day 3, 5, and 7. (C) Quantitative RT-PCR analysis of interferon-related or interferon stimulated mRNA expression in by qPCR in PV-3-infected MN ENTs at day 1, 3, and 5. Data are represented as mean (n = 3) ± SEM (*p < 0.05).

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  Figure 5. PV-3 infection induces the expression of genes involved in EGR-EP300 complex. (A) Volcano plot representing the differentially expressed genes upon PV-3 infection at day 5. (B) Immunohistochemistry showed GALR3-immunoreactive cells present in uninfected MN ENT (top panel) and infected MN ENT at day 3, 5, and 7. (C) String analysis shows known and predicted interactions between identified genes expressed upon PV-3 infection.