A More Rapid Method for Culturing LUHMES-Derived Neurons Provides Greater Cell Numbers and Facilitates Studies of Multiple Viruses

The ability to study mature neuronal cells ex vivo is complicated by their nature as non-dividing and difficulty in obtaining large numbers of primary cells from organisms. Thus, numerous transformed progenitor models have been developed that can be routinely cultured then scaled and differentiated to mature neurons. Here, we present a new method for differentiating one such model, the LUHMES dopaminergic neurons. This method is two days faster than some established protocols, results in nearly five times greater numbers of mature neurons, and involves fewer handling steps that could introduce technical variability. Moreover, it overcomes the problem of cell aggregate formation that commonly impedes high-resolution imaging, cell dissociation and downstream analysis. While recently established for herpes simplex virus type 1, we demonstrate that LUHMES neurons can facilitate studies of other herpesviruses, as well as RNA viruses associated with childhood encephalitis and hemorrhagic fever. This protocol provides an improvement in the generation of large-scale neuronal cultures, which may be readily applicable to other neuronal 2D cell culture models and provides a system for studying neurotrophic viruses. We name this method the Streamlined Protocol for Enhanced Expansion and Differentiation Yield, or SPEEDY method. Overall design: rRNA depleted total RNA-seq and SLAM-seq of undifferentiated and differentiated LUHMES cells, with and without HSV-1 infection at given timepoints