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

Hsueh-Fu Wu; Jennifer Art; Tripti Saini; Nadja Zeltner

doi:10.1016/j.xpro.2024.102970

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

STAR Protoc. 2024 Mar 21;5(2):102970. doi: 10.1016/j.xpro.2024.102970. Online ahead of print.

Authors

Hsueh-Fu Wu¹, Jennifer Art², Tripti Saini¹, Nadja Zeltner³

Affiliations

¹ Center for Molecular Medicine, University of Georgia, Athens, GA 30602, USA; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
² Center for Molecular Medicine, University of Georgia, Athens, GA 30602, USA; Biomedical and Translational Sciences Institute, Neuroscience Program, University of Georgia, Athens, GA 30602, USA.
³ Center for Molecular Medicine, University of Georgia, Athens, GA 30602, USA; Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA; Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA. Electronic address: nadja.zeltner@uga.edu.

Abstract

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.

Keywords: Biotechnology and bioengineering; Cell Biology; Developmental biology; Neuroscience; Stem Cells.