Scott Schachtele1 Nicola Secomandi2, Matilde Bergamini2, Federico Olivero2, Megan Pritchard2, Edward B. Stevens2, Rebecca Fiene1, Coby Carlson1, Anthony M. Rush2.
1FUJIFILM Cellular Dynamics, Madison, WI, USA
2Metrion Biosciences, Granta Park, Cambridge, UK
Humanized models of pain are critical for confidently translating the effectiveness of therapeutic compounds from rodents to man. Human induced pluripotent stem cell (iPSC)-derived sensory neurons are emerging as a key in vitro human model of nociception that can bridge preclinical discovery data from cultured rodent dorsal root ganglion neurons or in vivo rodent models of pain to a human-relevant system. Despite initial success in generation of iPSC-derived sensory neurons within academia/pharma, lengthy differentiation protocols, batch-to-batch variability or lack of clear sensory neuron phenotype has limited use of the model to support pain drug discovery. In this study, we evaluated the phenotype and robustness of a commercial source of human iPSC-derived sensory neurons (iCell Sensory Neurons) using gene expression and electrophysiology. These neurons, differentiated from a female donor (21527), exhibit high sensorineural purity (>80% BRN3A+/UCHL1+).
Bulk RNASeq analysis in sensory neurons cultured for 21 days in vitro shows the expression of sensory neuron ion channels, including sodium channels (SCN9A, SCN10A) and sensory receptors (TRPV1, P2RX3, PIEZO2). Specifically, robust expression of NaV1.7 (SCN9A) was observed in the population. This was confirmed using single cell RNAseq analysis (21 days in vitro) where approximately 60% of neurons population expressed SCN9A. Electrophysiological properties of sensory neurons were recorded between 28 and 56 days in vitro using whole-cell patch clamp technique. In terms of passive membrane properties, there was a gradual hyperpolarization in resting membrane potential and increase in rheobase during time in culture. Evoked action potentials from the iPSC-derived sensory neurons displayed a waveform typical of human/rat DRGs, with a broad action potential and ‘hump’ in the repolarizing phase. Current injections evoked multiple firing, where the proportion of cells displaying multiple firing increased between 28 and 56 days in culture. Repetitive firing evoked by a current injection ramp protocol was inhibited by the selective NaV1.7 inhibitor, PF05089771 (100 nM), confirming the functional expression of NaV1.7.
The presence of sensory neuron markers, excitability properties consistent with sensory neurons and evidence of nociceptor ion channels (using both RNASeq and electrophysiology) provides strong evidence that iCell Sensory Neurons have a robust sensory neuron phenotype suitable for supporting pain discovery programs.