Generation of Dopaminergic Neuronal Organoids from Human Embryonic Stem Cells

Take human embryonic stem cells, or hESCs, in an extracellular matrix-coated flask.

Replace the medium with a neural induction medium and incubate, differentiating hESCs into neural progenitor cell, or NPC, colonies.

Add enzymatic solution to dissociate the colonies.

Centrifuge, remove the supernatant, and resuspend cells in an induction medium with growth factors to support NPC differentiation.

Add the cell suspension onto inverse pyramidal-shaped microwells to form three-dimensional neurospheres.

Transfer neurospheres to an extracellular matrix-coated plate containing a differentiation medium with growth factors and small molecules. Incubate under agitation to promote differentiation into dopaminergic progenitor neurons.

Replace the medium with neural maturation medium and place neurospheres on a membrane disc in a culture-well insert, allowing diffusion of air and nutrients.

Incubate under static conditions to form three-dimensional organoids consisting of mature dopaminergic neurons.

These organoids, mimicking a developing brain, are useful for studying dopaminergic neuron degeneration, a characteristic of Parkinson's disease.