Modeling HSV-1 infections in human CNS neuronal cells using two-dimensional and three- dimensional cultures derived from induced pluripotent stem cells.

HSV-1 establishes latency in both peripheral nerve ganglia and the central nervous system (CNS). The outcome of acute and latent infections in these different anatomic sites appear to be distinct. It is becoming clear that many of the existing culture models using animal primary neurons to investigate HSV-1 infection of the CNS are limited and not ideal, and most do not recapitulate features of CNS neurons. Human induced human pluripotent stem cells (hiPSCs) and neurons derived from them as tools to study aspects of neuropathogenesis are documented but few have focused on modeling infections of the (CNS). Here, we characterize functional two-dimensional (2D) CNS-like neuron cultures and three-dimensional (3D) brain organoids made from hiPSCs to model HSV-1-human-CNS interactions. Our results show that: i) hiPSC-derived CNS neurons are permissive for HSV-1 infection; ii) a quiescent state exhibiting key landmarks of HSV-1 latency described in animal models can be established in hiPSC-derived CNS neurons; iii) the complex laminar structure of the organoids can be efficiently infected with HSV, with virus being transported from the periphery to the central layers of the organoid; iv) the organoids support reactivation of HSV, albeit less efficiently than 2D cultures. Collectively, our results indicate that hiPSC-derived neuronal platforms, especially 3D organoids, offer an extraordinary opportunity for modeling the interaction of HSV-1 with the complex cellular and architectural structure of the human CNS. IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model acute and latent HSV-1 infections in two-dimensional (2D) and three-dimensional (3D) CNS neuronal cultures. We successfully established acute HSV-1 infections and infections showing features of latency. HSV-1 infection of the 3D organoids was able to spread from the outer surface of the organoid and was transported to the interior lamina providing a model to study HSV-1 trafficking trough complex neuronal tissue structures. HSV-1 could be reactivated in both culture systems tough in contrast to 2D cultures, it appears to be more difficult to reactivate HSV-1 in 3D cultures, potentially paralleling the low efficiency of HSV-1 reactivation in the CNS of animal models. The reactivation events were accompanied by dramatic neuronal morphological changes and cell-cell fusion. Together our results provide substantive evidence of the suitability of hiPSC-based neuronal platforms to model HSV-1 CNS interactions in a human context.