Differentiation and Maturation of Human Embryonic Stem Cells into Neural Organoids

Begin with human embryonic stem cells in a culture medium.

Incubate for cell proliferation and sphere formation.

Transfer these spheres to a new well with a medium containing differentiation factors.

Incubate with gentle shaking. The differentiation factors drive the stem cells towards the neural lineage.

Regularly replace half of the medium.

Replace the medium with a neural induction medium, then incubate to obtain neural progenitors.

Switch to a growth-factor-rich medium and incubate to promote cell proliferation and neural rosette formation.

Then, culture in a medium containing inhibitors to block undesired cell differentiation.

Replace this medium with a medium containing growth factors and inhibitors. Incubate to induce differentiation into glial cells and primary neurons.

Later, place the neural rosettes on a hydrophobic membrane in an insert-carrying well containing an inhibitor-rich maturation medium.

Incubate without shaking to allow neural and glial cell maturation at an air-liquid interface, forming neural organoids.