Cellular, molecular and functional characteristics of induced pluripotent stem cell derived neurons from refractory chronic cough patients and healthy controls

The pathophysiology of refractory chronic cough (RCC) and the aetiology of associated cough hypersensitivity syndrome remains unclear, partly due to the absence of a suitable disease-specific sensory neuronal model. This study was designed to investigate whether induced pluripotent stem cell (iPSC)-derived sensory neurons (iPSC-Ns) from patients with RCC exhibit distinct phenotypic characteristics when compared to those from healthy controls (HC). Peripheral blood samples obtained from patients with RCC and HC were reprogrammed to iPSCs and subsequently differentiated into sensory neurons using defined culture conditions over 40 days. The neurogenic differentiation and functional characteristics of the iPSC-Ns were confirmed using molecular assays, live cell calcium mobilisation assays and patch clamp recordings. Transcriptomic differences between RCC and HC derived iPSC-Ns were assessed by RNA-sequencing. Successful differentiation of iPSC-Ns from iPSCs was observed as cells acquired a neuronal morphology, neuronal gene expression (peripherin), the neuronal marker β3-tubulin, and functional sodium channels. TRPV1 and P2X3 responses were heightened in RCC derived iPSC-Ns compared to HC with responses to capsaicin or αβmeATP blocked by the antagonists capsazepine or AF-353 respectively. Differential expression of 853 genes was noted between RCC- and HC-derived iPSC-Ns, with genes enriched in RCC-derived iPSC-Ns associated with sensory neuronal functions. Generation of an iPSC derived neuronal model from patients with RCC is feasible and offers insights into the pathophysiology of cough hypersensitivity syndrome. Heightened purinergic responses observed in iPSC-Ns from RCC compared to HC are consistent with the concept of cough hypersensitivity and support targeting P2X3 receptors as a therapeutic strategy.