The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell–derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

Screening against a disease-relevant phenotype to identify compounds that change the outcome of biological pathways, rather than just the activity of specific targets, offers an alternative approach to find modulators of disease characteristics. However, in pain research, use of in vitro phenotypic screens has been impeded by the challenge of sourcing relevant neuronal cell types in sufficient quantity and developing functional end-point measurements with a direct disease link. To overcome these hurdles, we have generated human induced pluripotent stem cell (hiPSC)-derived sensory neurons at a robust production scale using the concept of cryopreserved "near-assay-ready" cells to decouple complex cell production from assay development and screening. hiPSC sensory neurons have then been used for development of a 384-well veratridine-evoked calcium flux assay. This functional assay of neuronal excitability was validated for phenotypic relevance to pain and other hyperexcitability disorders through screening a small targeted validation compound subset. A 2700-compound chemogenomics screen was then conducted to profile the range of target-based mechanisms able to inhibit veratridine-evoked excitability. This report presents the assay development, validation, and screening data. We conclude that high-throughput-compatible pain-relevant phenotypic screening with hiPSC sensory neurons is feasible and ready for application for the identification of new targets, pathways, mechanisms of action, and compounds for modulating neuronal excitability.