A human-derived 3D brain organoid model to study JC virus infection

Abstract

Progressive multifocal leukoencephalopathy (PML) is a frequent neurological complication in immunosuppressed patients. PML is caused by the JC virus (JCV), a neurotropic DNA polyomavirus that infects oligodendrocytes and astrocytes, causing inflammation and demyelination which lead to neurological dysfunction. The pathogenesis of PML is poorly understood due to the lack of in vitro or animal models to study mechanisms of disease as the virus most efficiently infects only human cells. We developed a human-derived brain organotypic system (also called brain organoid) to model JCV infection. The model was developed by using human-induced pluripotent stem cells (iPSC) and culturing them in 3D to generate an organotypic model containing neurons, astrocytes, and oligodendrocytes which recapitulates aspects of the environment of the human brain. We infected the brain organoids with the JCV MAD4 strain or cerebrospinal fluid of a patient with PML. The organoids were assessed for evidence of infection by qPCR, immunofluorescence, and electron microscopy at 1, 2, and 3 weeks post-exposure. JCV infection in both JCV MAD4 strain and PML CSF-exposed brain organoids was confirmed by immunocytochemical studies demonstrating viral antigens and electron microscopy showing virion particles in the nuclear compartment of oligodendrocytes and astrocytes. No evidence of neuronal infection was visualized. Infection was also demonstrated by JCV qPCR in the virus-exposed organoids and their media. In conclusion, the brain organoid model of JCV infection establishes a human model suitable for studying the mechanisms of JCV infection and pathogenesis of PML and may facilitate the exploration of therapeutic approaches.

Keywords: Human cells; In vitro model; JC virus; JCV; Microphysiological system; Organoid.

Fig. 1

Experimental design and cell populations in the brain organoid model. A Timeline of the brain organoid differentiation and experimental stages. B Mature brain organoid cell populations at 5 weeks of in vitro differentiation contain neurons (MAP2⁺), astrocytes (GFAP⁺), and oligodendrocytes (O1⁺ cells). Scale bar: 20 microns

Fig. 2

Immunocytochemical confocal imaging micropathology of the JCV-infected brain organoids. JCV MAD4 virus-exposed brain organoids at 2 weeks post-infection revealed nuclear VP-1 capsid protein (PAB-597 antibody) positive inclusions in GFAP⁺ astrocytes (A) as well as perinuclear (B) and nuclear (C) inclusions expressing T-antigen (SV-40 antibody) in O1⁺ oligodendrocytes. Brain organoids exposed to JCV⁺CSF also revealed VP-1⁺ nuclear inclusions in oligodendroglia (D). Scale bar: 20 microns

Fig. 3

Electron microscopy imaging of JCV-infected brain organoids. (A) At 2 weeks post-infection, JCV-MAD4-exposed brain organoids exhibited nuclear inclusions with 45-nm icosahedral viral particles, (B) and (C) showing a close-up of the area in the yellow rectangle. Inclusion clusters were also seen in the cytoplasm of some cells (D), close-up (E), (F) and necrotic cells (G), close-up (H) and (I). Yellow arrows point to viral particles

Fig. 4

JCV qPCR results. (A) qPCR results of the brain organoid cell pellets expressed as the mean number of JCV copies/10 μL across the 3 experiments for time points 0,1, 2, and 3 weeks post-infection. (B) JC viral load in the brain organoid media over time expressed as the number of JCV copies/mL at days 2, 5, 7, 9, 12, and 14 post-infection