Selective Induction of Human Autonomic Neurons Enables Precise Control of Cardiomyocyte Beating

Abstract

The autonomic nervous system (ANS) regulates tissue homeostasis and remodelling through antagonistic effects of noradrenergic sympathetic and cholinergic parasympathetic signalling. Despite numerous reports on the induction of sympathetic neurons from human pluripotent stem cells (hPSCs), no induction methods have effectively derived cholinergic parasympathetic neurons from hPSCs. Considering the antagonistic effects of noradrenergic and cholinergic inputs on target organs, both sympathetic and parasympathetic neurons are expected to be induced. This study aimed to develop a stepwise chemical induction method to induce sympathetic-like and parasympathetic-like ANS neurons. Autonomic specification was achieved through restricting signals inducing sensory or enteric neurogenesis and activating bone morphogenetic protein (BMP) signals. Global mRNA expression analyses after stepwise induction, including single-cell RNA-seq analysis of induced neurons and functional assays revealed that each induced sympathetic-like or parasympathetic-like neuron acquired pharmacological and electrophysiological functional properties with distinct marker expression. Further, we identified selective induction methods using appropriate seeding cell densities and neurotrophic factor concentrations. Neurons were individually induced, facilitating the regulation of the beating rates of hiPSC-derived cardiomyocytes in an antagonistic manner. The induction methods yield specific neuron types, and their influence on various tissues can be studied by co-cultured assays.

Human Neurons Form Axon-Mediated Functional Connections with Human Cardiomyocytes in Compartmentalized Microfluidic Chip

“…3D projections of cocultures confirmed that axons and the clustered synaptic protein SynI were located very close to CMs, indicating that neurons and CMs can interact via axon-mediated synaptic connections. In previous neuron–CM coculture studies, the presence of synaptic proteins near CMs has not been evaluated except for by Takayama et al [ 13 ], although later, they showed that hiPSC-neurons expressed synaptic proteins in free cocultures with CMs [ 15 ].…”

“…Previously, the functionality of neuron–CM interactions has been reported in models utilizing rat primary neurons and CMs [ 10 , 11 ] and rat primary neurons and hiPSC-CMs [ 12 ] with integrated MEAs. Furthermore, MEAs have been utilized in cardiac innervation models where CMs have been seeded on top of neurons [ 15 , 35 ]. In these studies, neuronal stimulation evoked a response in the CMs.…”

“…Moreover, a coculture of hiPSC-derived PNS neurons with hiPSC-CMs in a compartmentalized chip has been reported; however, only evidence of physical neuromuscular connections was shown, without further evaluation of electrophysiological functionality [ 13 ]. hiPSC-neurons and hiPSC-CMs have also been cocultured without restrictions of cell-to-cell contacts, showing the formation of functional interactions between the cell types [ 14 , 15 ]. Previously reported neuron–CM coculture models have not shown functional connections between human neurons and CMs via axon-mediated interactions alone.…”

Angiotensin II and the Cardiac Parasympathetic Nervous System in Hypertension

“…The advancement of neuron-specific mouse models; viral gene transfer; and new technologies, including optogenetic and chemogenomic stimulation, has allowed for more specific study within these pathways [140]. Another limitation to studying the role of Ang II in parasympathetic neurotransmission is the lack of reliable and reproducible 'parasympathetic' cell lines, although recent progress may change this in the future [141]. A stepwise neurotropic chemical induction method of human pluripotent stem cells developed by Takayma et al can be used to induce both sympathetic-like and parasympathetic-like autonomic nervous system neurons [141].…”

“…Another limitation to studying the role of Ang II in parasympathetic neurotransmission is the lack of reliable and reproducible 'parasympathetic' cell lines, although recent progress may change this in the future [141]. A stepwise neurotropic chemical induction method of human pluripotent stem cells developed by Takayma et al can be used to induce both sympathetic-like and parasympathetic-like autonomic nervous system neurons [141]. Work has been published on modifying this technique to study sympathetic neurons; however, its use for differentiation of parasympathetic cells is still untested [142].…”