A Technique to Generate a 2D Monolayer of Cerebellar Cells from Induced Pluripotent Stem Cells

Begin with human induced pluripotent stem cell colonies cultured on an adherent basement membrane matrix.

Add a cerebellar differentiation medium containing a specific hormone, growth factor, and small molecule inhibitors.

Lift the colonies using a glass pipette, and transfer them to an ultra-low attachment plate.

The low-attachment surface maintains the cells in suspension, facilitating their aggregation into three-dimensional embryoid bodies, or EBs.

The hormone, growth factor, and inhibitors bind to their respective target sites, promoting stem cell differentiation into neuronal progenitors.

Transfer the EBs to culture plate wells coated with a synthetic polymer and an adhesion protein to facilitate EB attachment.

Replace the medium with an inhibitor-free differentiation medium.

The cells begin to migrate outward from the EBs, forming a 2D monolayer.

Remove the medium and add a cerebellar maturation medium.

Growth factors in the medium induce the maturation of neuronal progenitors into distinct cerebellar neuronal precursors.

On day zero, add 1 milliliter of cerebellar differentiation medium supplemented with 10 micromolar Y27632 and SB431542 to each well of a six-well ultra-low attachment or ULA plate, and place it in the incubator until the lifted colonies are ready to be added to the wells. Clean the differentiated cells using a pulled glass pipette. Aspirate the medium, and add 1 milliliter of iPSC passaging solution for each 35-millimeter dish.

After three minutes of incubation at 37 degrees Celsius, aspirate the medium and add 2 milliliters of cerebellar differentiation medium supplemented with 10 micromolar Y27632 and SB431542. Under 4X magnification of a transmitted light-inverted microscope, lift the colonies using the bent edge of the pulled glass pipette. Once every colony is lifted, gently transfer all the colonies to one well of the 6-well ULA plate using a 10-milliliter serological pipette. Repeat this process for each iPSC plate, and incubate the cells at 37 degrees Celsius with 5% carbon dioxide.

On day 2, add FGF2 to each well to adjust a final concentration of 50 nanograms per milliliter. On day 7, change one-third of the medium by aspirating 1 milliliter of spent medium, and replacing it with 1 milliliter of fresh cerebellar differentiation medium. Incubate the embryoid bodies for seven days.

On day 14, swirl the plate to gather all the embryoid bodies in the center of the plate. Then, tilt the plate, and aspirate all the spent medium using a 1000-microliter pipettor from the edge. As the medium amount decreases, slowly lay the plate flat and continue to aspirate, leaving enough medium to avoid drying the embryoid bodies out.

Next, add 3 milliliters of fresh cerebellar differentiation medium supplemented with 10 micromolar. Y27632. Then, transfer the embryoid bodies using a 10-milliliter serological pipette to a poly-L-Ornithine laminin-coated dish. On day 15, aspirate the medium and replace it with fresh cerebellar differentiation medium.