Abstract
Sympathetic neurons (SNs) capable of modulating the heart rate of murine cardiomyocytes (CMs) can be differentiated from human stem cells. The electrophysiological properties of human stem cell-derived SNs remain largely uncharacterized, and human neurocardiac cocultures remain to be established. Here, we have adapted previously published differentiation and coculture protocols to develop feeder-free SNs using human-induced pluripotent stem cells (hiPSCs). hiPSC-SNs were characterized in monoculture and coculture with hiPSC-CMs, using antibody labeling, enzyme-linked immunosorbent assay, and whole cell patch-clamp electrophysiology techniques. hiPSC-SNs stained positive for peripherin, tyrosine hydroxylase, and nicotinic acetylcholine receptors, the latter two colocalizing in somas and synaptic varicosities. hiPSC-SNs functionally matured in vitro and exhibited healthy resting membrane potentials (average = -61 ± 0.7 mV), secreted norepinephrine upon activation, and generated synaptic and action currents and inward and outward voltage-dependent currents. All hiPSC-SNs fired action potentials in response to current injection, local application of potassium, or spontaneously, followed by short-medium afterhyperpolarizations. hiPSC-SNs could successfully be maintained in coculture with hiPSC-CMs, and this induced further development of hiPSC-SN action potential kinetics. To test functional coupling between the neurons and cardiomyocytes, the hiPSC-CM beating response to nicotine-induced norepinephrine release was assessed. In neurocardiac cocultures, nicotine exposure significantly increased the hiPSC-CM spontaneous beating rate, but not in hiPSC-CM monocultures, supporting nicotinic neuronal hiPSC-SN stimulation directly influencing hiPSC-CM function. Our data show the development and characterization of electrophysiologically functional hiPSC-SNs capable of modulating the beating rate of hiPSC-CMs in vitro. These human cocultures provide a novel multicellular model to study neurocardiac modulation under physiological and pathological conditions.NEW & NOTEWORTHY We present data on a functional coculture between human-induced pluripotent stem cell-derived sympathetic neurons and cardiomyocytes. Moreover, this study adds significantly to the available data on the electrophysiological function of human-induced pluripotent stem cell-derived sympathetic neurons.