In regenerative medicine, a robust protocol of differentiation of pluripotent stem cells toward a specific neural cell type is a valuable tool. In our case, this protocols were very useful to study both glioblastoma development and Parkinson's disease. So first, the generation of three-dimensional organoid has the advantage of closely mimicking physiological development, and also the production of size-calibrated neurospheres from pluripotent stem cells has high reproducibility.
The following procedure will be demonstrated by Manon Locatelli, a PhD student from our lab. 24 hours before starting the three-D culture, replace the hESC medium with serum-free medium supplemented with 10-micromolar ROCK inhibitor. The cells should be at 60%confluency.
The next day, detach hESC colonies as single cells by removing the medium and rinsing with calcium and magnesium-free PBS. Then, add five milliliters of enzymatic dissolution solution and incubate at 37 degrees Celsius for one to two minutes. Afterward, collect the cells in serum-free medium with 10-micromolar ROCK inhibitor and centrifuge the cells at 300 times g for five minutes.
After centrifugation, check the size of the cell pellet. Remove the supernatant and count the cells in 10 milliliters of serum-free medium supplemented with 10-micromolar ROCK inhibitor. In parallel, rinse the microwell plate with two milliliters of serum-free medium per well and centrifuge the plate at 1, 200 times g for five minutes to remove all bubbles in order to prevent neurosphere formation.
Next, prepare 28.2 million cells in 12.5 milliliters of serum-free medium supplemented with 10-micromolar ROCK inhibitor. Dispense 1, 000 cells per microwell. Centrifuge the cells at 300 times g for five minutes and check the homogenous 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 one to four, change the medium every two to three days before performing neural induction. From day four 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 one micromolar gamma-secretase inhibitor. On day 21, place one culture plate insert in one well of a new six-well plate.
Afterward, add one 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. On day zero, amplify hESCs in two-D culture up to 60%confluency.
Then, 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 micromolars 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 milliliters of recombinant enzymatic solution in a T75 flask.
Incubate the cells for two minutes at 37 degrees Celsius and then add 7.5 milliliters of DMEM F12. 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 three 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.
Here is a schematic of a standardized protocol for the generation of dopaminergic neural organoids. The immunofluorescence analysis of dopaminergic neural organoids shown in these images indicates TH immunoreactive cells coexpressing Nurr1, a midbrain-specific marker. These two graphs represent the kinetics of TH and Nurr1 gene expression evaluated by quantitative RT-PCR.
Here is an example of raw data recorded with the MEA platform. Each spike is displayed by a vertical line whereas the remaining trace is noise. And this picture represents a neurosphere deposited on the MEA.
These graphs show the superposition of typical spikes detected from the raw data. The black bold curve indicates the average of the corresponding red curves. This raster plot shows the time stamps associated with each spike detected.
The different colors highlight the different electrodes. This protocols allow researcher to answer question about cancer cell interaction with neural organoid as well as direct screening with dopaminergic organoid.
