Generating Postganglionic Sympathetic Neurons from Human Pluripotent Stem Cells

Take an extracellular matrix-coated microplate and add human pluripotent stem cells suspended in a differentiation medium.

The medium contains signaling molecules that influence cellular signaling pathways, directing differentiation into neural progenitor cells.

Introduce another differentiation medium containing signaling molecules that induce differentiation into neural crest cells, or NCCs, precursors to neuronal and non-neuronal cells.

Add enzymes to disrupt the matrix, thereby dissociating the cells.

Wash to neutralize the enzymes, then centrifuge and discard the supernatant. Resuspend the NCCs in a spheroid medium and transfer them to a microplate.

The medium's growth factors and small molecules induce spheroid formation.

Supplement with retinoic acid, which promotes differentiation into sympathetic progenitor neurons.

Transfer the spheroid onto an adhesion protein-coated microplate in a sympathetic neuron maturation medium, causing spheroid attachment.

Neural growth factors and retinoic acid in the medium promote maturation into postganglionic sympathetic neurons, which are part of the peripheral nervous system and regulate involuntary processes.