Functional Coculture of Sympathetic Neurons and Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

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 uncharacterised, 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 mono- and coculture with hiPSC-CMs, using antibody labelling, 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 colocalising in somas and synaptic varicosities. hiPSC-SNs functionally matured in vitro and exhibited healthy resting membrane potentials (average -61±0.7 mV), secreted noradrenaline 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 afterhyperpolarisations. 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, hiPSC-CM beating response to nicotine-induced-noradrenaline-release was assessed. In neurocardiac coculture, nicotine exposure significantly increased hiPSC-CM spontaneous beating rate, but not in hiPSC-CM monoculture, supporting nicotinic neuronal hiPSC-SN stimulation directly influencing hiPSC-CM function. Our data show the development and characterisation of electrophysiologically functional hiPSC-SNs capable of modulating the beating rate of hiPSC-CMs in vitro. These human cocultures provide a novel multi-cellular model to study neurocardiac modulation under physiological and pathological conditions.