Pathways regulating the excitability of sympathetic neurons derived from human induced pluripotent stem cells

Postganglionic sympathetic neurons not only regulate target organs but also their own intrinsic activity and can be further modulated by parasympathetic neurons. This crosstalk and automodulatory signalling have been implicated in cardiovascular disorders, with the majority of earlier work employing rodent models. Here, we have used human sympathetic neurons derived from induced pluripotent stem cells (hiPSCs), as a scalable human in vitro system with the aim to investigate these pathways. Efficient differentiation of hiPSCs into sympathetic neurons was confirmed using molecular characterisation for the expression of PHOX2B , DBH , TH , PRPH . We employed Ca ²⁺ imaging and whole-cell patch-clamp electrophysiology, to examine the neuronal functional properties and found that hiPSC-derived sympathetic neurons recapitulate key physiological features of the rodent native counterparts. Most cells responded to nicotine and expressed functional α2 adrenergic and muscarinic receptors, involved in sympathetic autoregulation and parasympathetic crosstalk. We further demonstrated that α2 adrenergic and muscarinic receptors inhibit membrane excitability (increased rheobase, hyperpolarisation, reduced input resistance) and that both types of receptors converge on inwardly rectifying K ⁺ channels (GIRK) as effectors. The GIRK blocker Tertiapin-Q significantly reduced the α2 adrenergic and muscarinic responses, while the activator ML297 mimicked their action. Analysis of mouse stellate scRNA-seq confirmed that the receptors and GIRK subtypes studied here are prominently expressed in native sympathetic neurons. Overall, our data show that GIRK channels play an important role in the regulation of sympathetic neurons excitability and that hiPSC-derived neurons provide an attractive in vitro tool for drug discovery to study sympathetic autoregulation and parasympathetic-sympathetic crosstalk.