Efficient derivation of sympathetic neurons from human pluripotent stem cells with a defined condition

Scientific Reports
Kosuke KirinoMegumu K Saito

Abstract

Sympathetic neurons (SNs) are an essential component of the autonomic nervous system. They control vital bodily functions and are responsible for various autonomic disorders. However, obtaining SNs from living humans for in vitro study has not been accomplished. Although human pluripotent stem cell (hPSC)-derived SNs could be useful for elucidating the pathophysiology of human autonomic neurons, the differentiation efficiency remains low and reporter-based cell sorting is usually required for the subsequent pathophysiological analysis. To improve the efficiency, we refined each differentiation stage using PHOX2B::eGFP reporter hPSC lines to establish a robust and efficient protocol to derive functional SNs via neuromesodermal progenitor-like cells and trunk neural crest cells. Sympathetic neuronal progenitors could be expanded and stocked during differentiation. Our protocol can selectively enrich sympathetic lineage-committed cells at high-purity (≈80%) from reporter-free hPSC lines. Our system provides a platform for diverse applications, such as developmental studies and the modeling of SN-associated diseases.

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Citations

Feb 1, 2019·Current Protocols in Stem Cell Biology·Kenyi Saito-DiazNadja Zeltner
Jan 12, 2019·Clinical Autonomic Research : Official Journal of the Clinical Autonomic Research Society·Kenyi Saito-Diaz, Nadja Zeltner
Sep 4, 2019·Current Protocols in Stem Cell Biology·Hsueh Fu Wu, Nadja Zeltner
Jun 19, 2020·Npj Regenerative Medicine·Suradip DasD Kacy Cullen
Aug 14, 2020·Physiology·Pien A GoldsteenReinoud Gosens
Feb 18, 2021·Development·Filip J WymeerschAnestis Tsakiridis
Feb 14, 2021·Npj Regenerative Medicine·Suradip DasD Kacy Cullen
Apr 3, 2020·Current Opinion in Pharmacology·Song Hui Chng, Vassilis Pachnis
May 14, 2021·Frontiers in Cellular Neuroscience·Zachary T Olmsted, Janet L Paluh
Jun 29, 2021·Frontiers in Physiology·Evanthia A Makrygianni, George P Chrousos
Jan 12, 2022·Biochemical Society Transactions·Fay Cooper, Anestis Tsakiridis

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Methods Mentioned

BETA
flow cytometry
FACS
PCR
transfection
fluorescence-activated cell sorting
electrophoresis

Software Mentioned

ImageJ
Excel

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