This article outlines a protocol for differentiating human pluripotent stem cells (hPSCs) into neuroectodermal spheroids. The process involves the use of specific inhibitors and growth factors to guide the maturation and self-organization of stem cells.
Begin with colonies of human pluripotent stem cells or hPSCs attached to a suitable substrate in a culture medium.
Remove the medium. Add a neuronal medium containing specific inhibitors and incubate.
These inhibitors direct the maturation of hPSCs toward a neuronal lineage, forming neuroectodermal colonies. These are precursors to small, spherical structures called spheroids.
Remove the medium and wash with a salt buffer.
Incubate with a digestive enzyme to detach the colonies from the plate.
Add the neuronal medium to stop the enzymatic activity.
Collect the colonies and transfer them to a tube.
Allow them to settle, and then discard the supernatant.
Resuspend the colonies in a neuronal medium containing a growth factor.
Transfer the colonies to a multi-well plate and incubate.
This growth factor facilitates the self-organization and multiplication of neuroectodermal cells, leading them to form a spheroid.
To begin, plate the hPSC colonies on an hESC-qualified basement membrane matrix from one well of a 6-well plate at 60% confluency into three wells to achieve 20% to 30% density, and then, proceed with induction of 2D neuroectodermal colonies.
Add fresh N2 media supplemented with dual SMAD inhibitors daily to each well for the next 3 days. For generating 3D neuroectodermal spheroids from induced 2D neuroectodermal colonies, remove 2 milliliters of N2 medium from the 6-well plate, and wash one time with HBSS to ensure that all of the N2 medium is removed.
Add 1 milliliter of dispase to each colony-containing well. Incubate the plate for 20 to 25 minutes at 37 degrees Celsius, and check for colony detachment regularly. At the end of the incubation, to stop the activity of the dispase enzyme, add 1 milliliter of N2 medium to the well. Using a wide-bore P1000 pipette tip or a modified P1000 pipette tip cup with sterile scissors, transfer the colonies into a 15-milliliter tube, and allow the colony clumps to sink to the bottom of the tube with gravity.
Then, with a standard P1000 pipette tip, carefully remove the supernatant. Replace it with 1 milliliter of fresh N2 medium, and repeat washing thrice to ensure complete removal of dispase. After resuspending the cell clumps and N2 medium, transfer the cell suspension to one well of a 6-well plate, and add 40 nanograms per milliliter of bFGF.
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