Transcriptomic analysis and high throughput functional characterization of human induced pluripotent stem cell...

Peripheral sensory neurons are a primary effector in pain neurotransmission, and have become a useful cellular model for the study of pain. While rodent tissue has historically served as a source of these neurons, it has become increasingly clear that pain mechanisms in rodents and humans are substantially divergent. Sensory neurons harvested from cadaveric human tissue serve as a superior translational model for studying pain mechanisms, however their relative paucity limits their widespread utility. Theoretically, sensory neurons manufactured from human induced pluripotent stem cells (hiPSCs) could help bridge this translational gap given their relative abundance and potential similarity to primary human tissue. However, hiPSC-derived sensory neurons manufactured with the most common methodologies correlate poorly to human tissue both transcriptionally and functionally. In the present work, we compare a population of hiPSC-derived sensory neurons (hiSNs) generated using an accelerated directed differentiation method to previously published datasets and find this population to more closely resemble human primary dorsal root ganglia transcriptionally. Furthermore, we evaluate the heterogeneity of this novel population via single nucleus RNA sequencing and find it resembles specific nociceptor and mechanoreceptor subsets found in vivo. Finally, we assay the functionality of this population with high throughput automated patch clamp electrophysiology recordings of voltage-gated TTX-sensitive and TTX-resistant sodium (Na_v), potassium channels (K_v), ligand-gated ionotropic GABA and P2X receptors, and action potentials. Overall, we find this population of hiSNs demonstrates measurable expression and functional activity across multiple pain-relevant targets, making it suitable for interrogating diverse pain mechanisms on a fully humanized platform.