Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Hsueh  Fu Wu; Nadja Zeltner

doi:10.3791/60843

Method Article

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

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DOI:

10.3791/60843

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May 24th, 2020

Hsueh Fu Wu¹^,² , Nadja Zeltner¹^,²^,³

¹Center for Molecular Medicine, University of Georgia, ²Department of Biochemistry and Molecular Biology, Franklin College of Arts and Sciences, University of Georgia, ³Department of Cellular Biology, Franklin College of Arts and Sciences, University of Georgia

Corresponding Authors: Nadja Zeltner <Nadja.Zeltner@uga.edu>

Summary

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Abstract

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

Introduction

Postganglionic sympathetic neurons (symNs) belong to the autonomic nervous system (ANS) and have multiple important roles in responding and regulating homeostasis of the body independent of consciousness. For example, stress stimulates symNs and evokes the fight-or-flight response that leads to an increase in heart rate, blood pressure, and sweating. SymNs are affected in multiple human disorders due to genetics, toxicity/injury, or as companions to other diseases. An example of a genetic neuropathy is the childhood disorder Familial Dysautonomia (FD), where a severe dysregulation of symNs causes dysautonomic crisis, evident by sweating, blotching of the skin, vomitin....