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

Yuzo Takayama,Yutaro Kumagai,Hiroko Kushige,Yasuyuki S Kida,Yuka Akagi,Yutaka Suzuki

doi:10.1038/s41598-020-66303-3

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.

Highlights

Devices often include microfluidic structures for mimicking inter-organ communication in blood flow, including physiological nutrition and biomolecular gradients[3]
We hypothesized that a stepwise induction strategy, including an neural crest (NC) induction step and subsequent autonomous specification step with restricted WNT signalling inhibition, sonic hedgehog (SHH) signalling inhibition, and bone morphogenetic protein (BMP) signalling activation can lead to the specification of human pluripotent stem cells (hPSCs) into autonomic nervous system (ANS) lineages, inducing noradrenergic sympathetic neurons and cholinergic parasympathetic neurons (Supplementary Fig. 1a)
In the autonomic neural crest (aNC) method, WNT activation via CHIR99021 (CHIR), BMP inhibition via dorsomorphin (DM), and tumour growth factor (TGF)-β inhibition via SB431542 (SB) for NC induction were carried out for 7 days. This was followed by treatment with a combination of WNT signalling inhibitor (IWR1), SHH signalling inhibitor (SANT1), and recombinant human bone morphogenetic protein 4 (BMP4) for 6 days to promote the specification of cells into the ANS lineage, in contrast with treatment for 11 days with CHIR and SB in the conventional NC (cNC) method

Summary

Introduction

Devices often include microfluidic structures for mimicking inter-organ communication in blood flow, including physiological nutrition and biomolecular gradients[3]. These studies have induced trunk NC cells or sympathoadrenal cells to efficiently generate sympathetic neurons on the basis of the regulation of the dorso-ventral or anterior-posterior axis[10,11,12] in hPSC differentiation pathways. We hypothesized that a stepwise induction strategy, including an NC induction step and subsequent autonomous specification step with restricted WNT signalling inhibition, SHH signalling inhibition, and BMP signalling activation can lead to the specification of hPSCs into ANS lineages, inducing noradrenergic sympathetic neurons and cholinergic parasympathetic neurons (Supplementary Fig. 1a). Through stepwise induction and selective culture conditions, we confirmed that hPSC-derived sympathetic-like and parasympathetic-like neurons regulated the beating rates of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in vitro

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