Functional characterization of human induced pluripotent stem cell-derived sympathetic neurons

The peripheral nervous system (PNS) influences the function of nearly all tissues in the body, regulating various physiological processes. When compared to the central nervous system (CNS) type of neurons, an equivalent understanding of the functional properties of human PNS neurons is lacking in vitro while there is a need for better characterization of electrophysiologically mature human PNS neurons. Generating subtypes of human PNS neurons opens possibilities for physiologically more relevant innervation and OoC models both in health and disease.Here, we modified existing differentiation protocol of human induced pluripotent stem cell (hiPSC)- derived sympathetic neurons. Functional characterization was done with multielectrode array (MEA) measurements combined with pharmacological treatments. The development of neuronal functionality was followed over time and characterized with various functional parameters. The expression of specific neuronal markers was studied with immunocytochemistry while PCR was performed for gene-level analyses. Moreover, calcium dynamics of mature cultures were analyzed.HiPSC-derived sympathetic neurons expressed robust spontaneous functionality on MEA. When compared to hiPSC-derived cortical neurons they possessed distinct electrophysiological features during the functional maturation and high peak activity. Neurons responded to excitatory and inhibitory pharmacological agents specific for peripheral neurons. Expression of neuronal and synaptic markers and genes increased during and after the differentiation. Also, the upregulation of cholinergic acetylcholine receptors on similar timepoints with pharmacology further supported the maturation of the neurons.This study provides novel knowledge on the functional properties of human sympathetic neurons paving the way for more accurate in vitro PNS and innervation models.