Protocol for generating postganglionic sympathetic neurons using human pluripotent stem cells for...

Area of Science:

• Neuroscience
• Stem Cell Biology
• Developmental Biology

Background:

• Assessing sympathetic nervous system development and function in disease is difficult at scale.
• Human pluripotent stem cells (hPSCs) offer a potential model for studying neuronal development.
• Neural crest cells (NCCs) are precursors to sympathetic neurons.

Purpose of the Study:

• To develop a scalable protocol for generating functional human pluripotent stem cell (hPSC)-derived sympathetic neurons (symNs).
• To establish a method for cryopreservation of these derived sympathetic neurons for future use.
• To validate the functionality of the differentiated sympathetic neurons.

Main Methods:

• Protocol development for hPSC replating and neural crest cell (NCC) differentiation.
• NCC replating and cryobanking procedures.
• SymN differentiation from NCCs and subsequent functional assays.

Main Results:

• Successful differentiation of hPSC-derived NCCs into sympathetic neurons.
• Demonstrated cryopreservation viability and recovery of sympathetic neurons.
• Verified functionality through electrophysiological recordings, calcium flux measurements, and norepinephrine dynamics.

Conclusions:

• A robust protocol for generating functional, cryopreservable sympathetic neurons from hPSCs is established.
• This method facilitates large-scale studies of sympathetic nervous system development and disease.
• The hPSC-derived sympathetic neurons serve as a valuable tool for disease modeling and drug discovery.