Functional coculture of sympathetic neurons and cardiomyocytes derived from human-induced pluripotent stem cells

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 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.

From Mice to Mainframes: Experimental Models for Investigation of the Intracardiac Nervous System

“…Pluripotent stem cells have emerged as an important source of human cells in which cellular development and function can be studied in vitro under physiological and pathophysiological conditions [114]. Using human-iPSC (hiPSC), hiPSC-derived sympathetic neurons have been characterised in monoculture and in co-culture with hiPSC-derived CM [114].…”

“…Pluripotent stem cells have emerged as an important source of human cells in which cellular development and function can be studied in vitro under physiological and pathophysiological conditions [114]. Using human-iPSC (hiPSC), hiPSC-derived sympathetic neurons have been characterised in monoculture and in co-culture with hiPSC-derived CM [114]. It has been shown that the differentiation of hiPSC into norepinephrine-secreting cells produces sympathetic neurons that are able to modulate the beating rate of co-cultured CM [114].…”

“…Using human-iPSC (hiPSC), hiPSC-derived sympathetic neurons have been characterised in monoculture and in co-culture with hiPSC-derived CM [114]. It has been shown that the differentiation of hiPSC into norepinephrine-secreting cells produces sympathetic neurons that are able to modulate the beating rate of co-cultured CM [114]. It was found that nicotine exposure significantly increased CM beating rate in sympathetic neuron-CM co-cultures, but not in CM monocultures, suggesting a direct influence of nicotinic sympathetic neuron stimulation [114].…”

“…It has been shown that the differentiation of hiPSC into norepinephrine-secreting cells produces sympathetic neurons that are able to modulate the beating rate of co-cultured CM [114]. It was found that nicotine exposure significantly increased CM beating rate in sympathetic neuron-CM co-cultures, but not in CM monocultures, suggesting a direct influence of nicotinic sympathetic neuron stimulation [114]. Fluorescence labelling revealed post-synaptic nicotinic acetylcholine receptors co-localised to TH-positive somas and synaptic varicosities in sympathetic neurons, which mediate the effect of nicotine.…”

The Intrinsic Cardiac Nervous System and Its Role in Cardiac Pacemaking and Conduction

“…Apart from the effects on pacemaker cells, sympathetic neurons also modulate the contractility of rodent cardiomyocytes [ 46 , 157 , 158 ] and induced pluripotent stem cells (iPSC)-derived cardiomyocytes [ 159 , 160 ]. Sympathetic innervation in the murine heart is more abundant in the epicardial layer and it diminishes towards the endocardium [ 161 , 162 , 163 ].…”