Molecular and cellular neurocardiology in heart disease.

This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study 'disease in a dish'. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia.

A modular platform to generate functional sympathetic neuron-innervated heart assembloids.

The technology of human pluripotent stem cell (hPSC)-based 3D organoid/assembloid cultures has become a powerful tool for the study of human embryonic development, disease modeling and drug discovery in recent years. The autonomic sympathetic nervous system innervates and regulates almost all organs in the body, including the heart. Yet, most reported organoids to date are not innervated, thus lacking proper neural regulation, and hindering reciprocal tissue maturation. Here, we developed a simple and versatile sympathetic neuron (symN)-innervated cardiac assembloid without the need for bioengineering. Our human sympathetic cardiac assembloids (hSCAs) showed mature muscle structures, atrial to ventricular patterning, and spontaneous beating. hSCA-innervating symNs displayed neurotransmitter synthesis and functional regulation of the cardiac beating rate, which could be manipulated pharmacologically or optogenetically. We modeled symN-mediated cardiac development and myocardial infarction. This hSCAs provides a tool for future neurocardiotoxicity screening approaches and is highly versatile and modular, where the types of neuron (symN or parasympathetic or sensory neuron) and organoid (heart, lung, kidney) to be innervated may be interchanged.

Parasympathetic neurons derived from human pluripotent stem cells model human diseases and development.

Autonomic parasympathetic neurons (parasymNs) control unconscious body responses, including "rest-and-digest." ParasymN innervation is important for organ development, and parasymN dysfunction is a hallmark of autonomic neuropathy. However, parasymN function and dysfunction in humans are vastly understudied due to the lack of a model system. Human pluripotent stem cell (hPSC)-derived neurons can fill this void as a versatile platform. Here, we developed a differentiation paradigm detailing the derivation of functional human parasymNs from Schwann cell progenitors. We employ these neurons (1) to assess human autonomic nervous system (ANS) development, (2) to model neuropathy in the genetic disorder familial dysautonomia (FD), (3) to show parasymN dysfunction during SARS-CoV-2 infection, (4) to model the autoimmune disease Sjögren's syndrome (SS), and (5) to show that parasymNs innervate white adipocytes (WATs) during development and promote WAT maturation. Our model system could become instrumental for future disease modeling and drug discovery studies, as well as for human developmental studies.

Protocol for generating postganglionic sympathetic neurons using human pluripotent stem cells for electrophysiological and functional assessments.

Assessing the development and function of the sympathetic nervous system in diseases on a large scale is challenging. Here, we present a protocol to generate human pluripotent stem cell (hPSC)-derived postganglionic sympathetic neurons (symNs) differentiated via neural crest cells (NCCs), which can be cryopreserved. We describe steps for hPSC replating, NCC replating and cryobanking, and symN differentiation. We then demonstrate the functionality of the hPSC-derived symNs, focusing on electrophysiological activity, calcium flux, and norepinephrine dynamics. For complete details on the use and execution of this protocol, please refer to Wu et al.1,2.