This method can help in studying early human cerebellar development and how it might be altered in neuropsychiatric disorders. The key advantage of this method is generating developmentally early human cerebellar cells in a 2D layer structure without performing complicated steps. It is also cost efficient.
Making pull pipettes can be tricky at the beginning, but with practice and time, it gets easier to pull the pipettes in a way that will be easier to work with. Begin the experimental preparation by pulling a 22.9 centimeter Pasteur pipette approximately two centimeters below the neck, above the bunsen burner to create two glass pipettes. The thinner side will be approximately four centimeters shorter than the other.
Bend the tips of the pulled side on the flame to create a smooth R, then place the pulled pipettes in autoclave sleeves and sterilize them in an autoclave. On day zero, add one milliliter of cerebellar differentiation medium supplemented with 10 micromolar Y-27632 and SB-431542 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 one millimeter of iPSC passaging solution for each 35 millimeter dish. After three minutes of incubation at 37 degrees Celsius, aspirate the medium and add two millimeters of cerebellar differentiation medium supplemented with 10 micromolar Y-27632 and SB-431542 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 six-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 two, add FGF2 to each well to adjust a final concentration of 50 nanograms per milliliter. On day seven, change one third of the medium by aspirating one milliliter of spent medium and replacing it with one 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 pipetter from the edge.
As the medium amount decreases, slowly lay the plate flat and continue to aspirate, leaving enough medium to avoid drawing the embryoid bodies out. Next, add three milliliters of fresh cerebellar differentiation medium supplemented with 10 micromolar Y-27632. 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. On day zero, iPSC colonies were cleaned and lifted into cerebellar differentiation medium containing SB-431542 and Y-27632 and were placed into ultra-low attachment plates for generating embryoid bodies. The addition of FGF2 on day two and a change of 1/3 medium on day seven resulted in embryoid bodies showing enlargement on the 14th day.
Cells started migrating outward from the embryoid bodies along the coated surface on the 15th day. The complete change of the medium to cerebellar maturation medium from the 21st day resulted in a monolayer of cells with neuronal-like morphology and complexity by the 35th day. The gene expression analysis at day 35 revealed that the cells express early cerebellar progenitor markers such as glutamatergic progenitors, ATOH1, GABAergic progenitors, PTF1 alpha, and the Purkinje progenitor cell markers KIRREL2 and SKOR2.
In addition, the expression of rhombic lip marker OTX2 and mostly anterior neuron specific SIX3 were also observed. The immunofluorescent labeling of the cells harvested on the 35th day showed positive staining for the cerebellum markers EN2 and PTF1 alpha, the neuronal marker beta tubulin III, and the proliferation marker KI67. Nuclear staining with 4'6-diamidino-2-phenylindole, or DAPI, showed cell nuclei.
For a successful differentiation, it is crucial to start with healthy and undifferentiated iPSC colonies and use reagents that are as freshly reconstituted as possible.
