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.

Dispense 1,000 thousand cells per microwell. Centrifuge the cells at 300 times g for 5 minutes, and check the homogeneous repartition of cells in the microwell plate under a microscope. Place the plate in the incubator at 37 degrees Celsius overnight.

The next day, examine the microwell plate to check the size of formed spheres in each microwell. Transfer the spheres to a six-well plate using the medium, supplemented with dual SMAD inhibition cocktail to promote fast neural induction. From this step forward, the spheres are cultured in rotation at 60 RPM to prevent them from sticking together or to the plate. On day 1 to 4, change the medium every two to three days before performing neural induction.

From day 4 to 11, promote proliferation of hESC-derived neural rosettes by adding EGF and bFGF at 10 nanograms per milliliter to the B27 medium, supplemented with dual SMAD cocktail. From day 11 to 13, culture the spheres in B27 medium, supplemented with 0.5 micromolar BMP inhibitor. From day 13 to 21, culture the spheres in B27 medium supplemented with GDNF and BDNF at 10 nanograms per milliliter and 1 micromolar gamma-secretase inhibitor.

On day 21, place one culture plate insert in one well of a new six-well plate. Afterward, add 1 milliliter of B27 medium, supplemented with growth factors and inhibitors to each well underneath the membrane insert. Subsequently, plate the spheres on a hydrophilic PTFE membrane deposited on a culture plate insert and change the medium every two to three days for the following three weeks of differentiation. Stop rotation from this step.

The presence of rosettes indicates the initiation of neural differentiation. From day 21 to 25, cultivate human neural organoids in the same neural maturation medium. Then, from day 25 to 28, only complement B27 medium with one micromolar gamma-secretase inhibitor. From day 28 to 39, stop adding the gamma-secretase inhibitor and continue human neural organoid culture in B27 medium only. After three weeks, neural organoids are ready to use for GIC implantation.