This article describes a method for differentiating human embryonic stem cells (hESCs) into dopaminergic progenitor neurons using a three-dimensional organoid culture system. The process involves neural induction, formation of neurospheres, and maturation into organoids that model aspects of the developing brain.
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.
Replace the stem cell medium used to maintain pluripotency features of hESCs, with a serum-free medium containing dual SMAD inhibition cocktail to start neural induction, and 10 micromolar ROCK inhibitor to increase the survival rate of cells during passage the next day. On day one, prepare the microwell plate with 2.5 milliliters per well of serum-free medium containing the same concentration of ROCK inhibitor and dual SMAD inhibition molecules.
To differentiate cells towards the neural tube ventral pattern, add SHH and FGF8 at 100 nanograms per milliliter and two micromolar smoothened agonist. After adding the medium, centrifuge the plate at 1,200 times g for five minutes to remove air bubbles from the microwells. Once the microwell plate is ready to use with half differentiating medium, remove the medium of hESCs, and quickly wash with calcium and magnesium chloride-free PBS.
Dissociate the colonies into a single-cell suspension by adding 7.5 mL of recombinant enzymatic solution into a T75 flask. Incubate the cells for two minutes at 37 degrees Celsius, and then add 7.5 of DMEM F-12. Subsequently, collect the cell suspension, and centrifuge at 300 times g for five minutes. Then, remove the supernatant, and count the cells in the same medium used to prepare the microwell plate.
Adjust the medium volume to obtain a cell suspension, allowing to form neurospheres containing 1,000 cells per microwell, by preparing 4.7 million cells in 2.5 milliliters of medium, and adding it to the previous 2.5 milliliters of medium already placed in the plate. In order to correctly distribute the cells in each microwell, gently shake the plate, and centrifuge it at 300 times g for five minutes. Next, incubate the plate at 37 degrees Celsius for 24 hours to generate spheres.
On day two, gently flush the microwells with medium, and transfer the spheres in a tissue-treated six well-plate. Place the spheres in rotation at 37 degrees Celsius, and change half the medium with fresh medium every two to three days. On day 3 to 13, to enhance neural induction and convert to neural progenitors with a midbrain identity, supplement the medium with three micromolar GSK-3-beta inhibitor. Split the sample into two new tissue-treated six well-plates to reduce the sphere density and avoid sphere aggregation.
On day eight, start the neural maturation by switching to new medium with growth factors, and change the medium every two to three days. On day 21, place one culture plate insert in one well of a new six-well plate, and add 1.2 milliliters of neural maturation medium used for neurosphere differentiation underneath the membrane insert. Then, depose the PTFE membrane on a culture plate insert.
Finally, to generate the neural organoid, seed around 100 neurospheres under air-liquid interface conditions on the PTFE membrane. Stop rotation from this step and change the medium every two to three days until the required differentiation time-point is achieved.
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