Generation of human sensory neurons derived from embryonic stem cells as an in-vitro model for varicella zoster virus infection.

Primary varicella-zoster virus (VZV) infection causes chickenpox and establishes a lifelong latent infection in peripheral neurons. Reactivation of VZV is frequently complicated by acute pain that can remains for months or years. Little is known about VZV latency and reactivation, due to lack of a suitable in vitro model. We have previously published a model system in which human embryonic stem cells (hESC) are differentiated into neurons via aggregates. These hESC-derived neurons can host a latent infection that can be reactivated experimentally, but at low efficiency. We hypothesized that a monolayer culture system might improve access to the virus and yield more latently-infected neurons. Here we present two approaches to generate monolayer cultures of neurons. In one approach we disassociated hESC-derived neuronal aggregates into single cells. In order to eliminate non-neuronal cells and precursors, we added mitotic inhibitors. A second approach was to overexpress a neuronal “master gene” in hESC that directs differentiation to neuronal precursors. CRISPR-Cas9 was used to introduce a Neurogenin2 (NGN2) cassette under the control of the tetracycline promoter into hESC at the AAVS1 safe-haven locus, allowing temporally limited expression of the transgene. NGN2-trangenic hESC treated for 4 days with doxycycline and cultured with neurotrophic factors yielded monolayers of cells with extensive processes and expressing multiple neuronal markers within 14 days. Both approaches show promise as improved models for investigating human neurotropic viruses in general, and should prove invaluable specifically in the study of latency and reactivation of VZV.