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

Am J Physiol Heart Circ Physiol. 2020 Nov 1;319(5):H927-H937. doi: 10.1152/ajpheart.00546.2020. Epub 2020 Aug 21.


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.

Keywords: human-induced pluripotent stem cells; neurocardiac coculture; sympathetic modulation of heart rate; sympathetic neurons.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials
  • Adult
  • Cells, Cultured
  • Cellular Reprogramming
  • Cellular Reprogramming Techniques / methods*
  • Coculture Techniques / methods*
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Male
  • Myocardial Contraction
  • Myocytes, Cardiac / cytology*
  • Myocytes, Cardiac / metabolism
  • Myocytes, Cardiac / physiology
  • Neurons / cytology*
  • Neurons / metabolism
  • Neurons / physiology
  • Norepinephrine / metabolism
  • Peripherins / genetics
  • Peripherins / metabolism
  • Receptors, Nicotinic / genetics
  • Receptors, Nicotinic / metabolism
  • Tyrosine 3-Monooxygenase / genetics
  • Tyrosine 3-Monooxygenase / metabolism


  • Peripherins
  • Receptors, Nicotinic
  • Tyrosine 3-Monooxygenase
  • Norepinephrine