Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells

Abstract

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.

Figure 1. Neuronal differentiation from iPSCs generated from fibroblasts.

(a) Typical morphology of an iPS colony cultured for 7 days on matrigel with mTeSR1 medium. (b) Spherical cluster of cells containing neural stem/progenitor cells. (c) neural rosettes. (d) Neurosphere-like structures formed 1 day after culturing in suspension dissected spherical cluster and neural rosettes. (e) neural progenitor cells (NPCs). (f) neuron differentiating from neurosphere-like structures. (g–h) Staining of neurons with ß-tubulin III (Tuj1) (g) and MAP2 (h). Glutamate administration (10 µM) in NPCs cultures (i) did not evoked a significant calcium influx (Ca²⁺ basal levels: 0.26 ± 0.02; after glutamate: 0.27 ± 0.03; n = 128; not significant), whereas higher calcium basal levels were recorded in NPCs-neurons (j) (0.30 ± 0.02; n = 136; p<0.005 compared to NPCs calcium basal levels) and glutamate administration evoked a quicker and stronger response in term of Calcium influx (0.42 ± 0.03; n = 136; p<0.005 compared to calcium basal levels). The data are representative of four different experiments. The significant increase of glutamate-mediated Ca2+ influx suggests that the iPS-derived neurons are functional. Scale bar is 50 µm.

Figure 2. Microphotographs of HCMV-infected (a) and mock infected iPS cells.

Scale bar is 50 µm.

Figure 3. Effect of HCMV on differentiation of neural rosettes.

Top panel. Identification of neurons differentiating in mock infected (a) and infected (b) cultures by Tuj 1 immnostaining (red). The presence of HCMV antigens in infected cells displaying CPE (c, e) was determined by staining for pp65 (d) and blend (f, see material and methods). (g–j): Analysis of neurons differentiating from infected neural rosettes. Co-immunostaining for Tuj1 (red) and pp65 (green) showed that cells expressing the virus antigen (h) strongly stain for Tuj1 (g). Most of the cells with neuronal morphology stain for Tuj1 (g, within the box, left) but not for pp65 (g, within the box, left). Co-expression of ß-tubulin III (i) and pp65 (j) was observed only in a small fraction of neurons (indicated by arrowheads). Scale bar is 50 µm. Bottom panel. FACS analysis of neural stem cells viability after HCMV infection (day 15 p.i.).

Figure 4. HCMV infection of NPCs.

Left panel. (a) microphotograph of NPCs before infection. The percentage of progenitors in the culture approach 90% as showed by staining with nestin (b). Cytopathic effect developed in the infected cultures (d, f). Microphotographs of mock infected NPCs are depicted in (c) and (e). Scale bar is 50 µm. Right panel. FACS analysis of NPCs viability after HCMV infection gated on the nestin-positive cells.

Figure 5. Mature neurons exposed to HCMV.

Neuron-enriched cultures were infected with HCMV an MOI of 3. Expression of HCMV proteins was detected only in a small fraction of neurons (indicated by arrowhead) at day 3 post infection. (a) Bright-field. (b) Staining for early immediate, immediate and late HCMV antigens. (c) Nuclei counterstained with Hoechst. (d) Overlay of HCMV antigens and bright-field. (e–f) Staining for early immediate, immediate, and late HCMV antigens shows the presence of vial proteins (green) along neural processes at day 5 post infection. (f) Overlay of viral antigens and bright-field. Scale bar is 50 µm.

Figure 6. Effect of HCMV on neural viability and function.

HCMV reduces neuronal viability and down-regulates NR1 subunit of NMDA receptor. At day 7 post infection most neurons showed degeneration as highlighted by immunostaining for Tuj1 (b), whilst mock infected neurons maintain their integrity (a). (c) Immunoblot showing activation of caspase 3 in infected neuron cultures. (d) Normalized cleaved caspase 3 expression from data in (c). (e–f): Measurements of calcium influx in mock infected (e) and infected (f) neurons induced by administration of 10 µM glutamate [Glu]. (g) Immunoblot showing reduced level of NR1 subunit in HCMV infected neuron cultures. (h) Normalized NR1 expression from data in (g). Scale bar is 50 µm. The data represent an average of three independent experiments.