Emergence of nociceptive functionality and opioid signaling in human iPSC-derived sensory neurons.

Induced pluripotent stem cells (iPSC) have enabled the generation of various difficult to access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdN) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdN, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored.Here we used an immunocytochemical high content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this time period the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude and membrane capacitance increased. After 70 days hiPSCdNs exhibited robust physiological responses induced by GABA, pH-shift, ATP and capsaicin. Direct activation of protein kinase A type II (PKA-II) via adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time-points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation.Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable hiPSCdN-based drug discovery.