• Culture iPSCs on Matrigel in six-well plates in mTesR medium before differentiation. Start the differentiation process when iPSCs colonies reach 80-90% confluence.
Differentiation of human iPSCs to Purkinje progenitors
• Day 0: Treat iPSCs with 1mg/ml Collagenase IV at 37℃ for 15-20 min. Discard collagenase IV and replace with DMEM/F12 when the edges of iPSC colonies peel off from the surface of the dishes. Scrape the iPSC colonies with the tip of a 2 ml pipette through drawing cross lines to cut large cell colonies into small clusters. Collect the cell clusters into 15ml centrifuge tube. Centrifuge for 5 min at 300g. Resuspend the cells with gfCDM medium without bFGF. The cell clusters should re-aggregate and form embryonic bodies (EBs) in gfCDM medium after overnight incubation.
Caution: Insulin is a very important factor for EB formation. If EBs cannot be derived from iPSCs, please check the concentration and quality of insulin.
• Day 1: Pipet the cells up and down with 5ml pipette to get rid of the dead cells hanging on the surface of the EB balls. Transfer the cell suspension to a 50ml centrifuge tube. Centrifuge at 300g for 5 min. Discard the supernatant and resuspend with gfCDM medium containing 20ng/ml bFGF.
• Day 2-7: Change the medium to gfCDM with 20ng/ml bFGF every other day.
• Day 7-10: Change the medium to gfCDM medium with 20ng/ml bFGF and 10 μg/ml cyclopamine.
• Day 10: Transfer gfCDM EBs to a 50 ml centrifuge tube. Pipet up and down to get rid of the dead cells hanging on the surface of the EB balls. Discard the supernatant and suspend the EB balls with gfCDM containing 20 ng/ml bFGF (without cyclopamine). Transfer EBs to poly-D-lysin/laminin-coated 6-well-plates to allow attachment and formation of rosette-like structures.
TIP: Whether EBs can attach relies on EB quality. EBs of good quality should assume spherical morphology and smooth edges. EBs with irregular shapes or ragged surfaces imply insufficient quality, and may lead to the failure to attach and rosette formation. If EBs of heterogeneous qualities are formed during differentiation, those of good quality can be enriched by selecting under microscope.
• Day 10-20: Change medium every other day with gfCDM with 20ng/ml bFGF.
Co-culture of human Purkinje progenitors with cerebellum slices
• Sterilize the surgical instruments and disinfect preparation room by ultraviolet light.
• Adjust the vernier calipers on McIlwain tissue chopper to 350 μm in thickness. Modify the angle of the chopper's arm with a blade to a proper position to make sure the blade can touch the platform horizontally. Set up the speed and strength to cut the tissue continuously.
TIP: The platform needs to be sterilized with 75% ethanol beforehand, and make sure that the platform is completely dry before slicing.
• Place Millicell insert into the well of six-well plate. Add 1 ml slice medium on the membrane of the insert. Transfer the plates with inserts to 37℃, 5% CO2 incubator.
• Prepare HBSS with 5mg/ml D-Glucose, and cool the solution in ice.
• Deeply anesthetize the SD (postnatal day 10) rat on ice, clean with 75% ethanol, and remove the brain rapidly. Separate the brain as soon as possible and transfer it into cold HBSS with 50 mg/ml D-glucose. Dissect out the rat's cerebellum and place the tissue in a sagittal direction on the platform of McIlwain tissue chopper.
TIP: To avoid movement of tissues during slicing, suck off the liquid surrounding the tissue with a pipet.
• Start the dissection procedure.
• Add some HBSS to merge the chopped slices on the platform. Carefully transfer slices into the dishes containing cold HBSS supplemented with 5 mg/ml D-glucose.
TIP: To prevent tissue damage during the transfer process, add some HBSS to the slices and use a tweezer to slide them off into culture dishes containing HBSS.
• Separate slices under a dissecting microscope using micro tweezers, and carefully transfer slices into the slice medium on the prepared Millicell membrane inserts using a flat shovel. Put three or four slices on one insert. Carefully unfold the slices in medium to make sure that slice surfaces can touch the membrane. Carefully transfer the medium (1ml) on the insert into the same well underneath the insert.
TIP: The insert membrane is semipermeable. One ml medium is just enough to touch the membrane and keep it wet. The slices should be cultured on the gas-liquid interface.
• Day 1-7: Change medium every day.
• From Day 8 onward: Transplant sorted neph3+ cells onto the surface of cerebellum slices. Change Purkinje cell differentiation medium every other day.
Caution: The cerebellar slices from newborn rats are small and the added cells may flow out of the slice surface. To avoid cell loss, add 1μl cell suspension each time and add multiple times.
Human fetal cerebellum slice culture
• All experiments involving electively aborted fetus should strictly follow the institutional ethical guidelines and regulations. The slicing and culture of fetal cerebella are similar to those of rat ones.
Sorting of Neph3+ cells
• Cell preparation: Treat differentiated cells with cell dissociation buffer at 37℃ for 20 min. Discard dissociation buffer and add 2ml DPBS into each well. Pipet the treated cells with 1ml tips to separate the rosette-like clusters to single cell suspension. Transfer the cell suspension to 50ml centrifuge tubes. Centrifuge at 300g for 5 min. Discard the supernatant and resuspend cells with 30ml DPBS to wash the cells.
• Antibody preparation: Reconstitute the lyophilized antibodies with Tris-buffered saline (TBS, PH=7.3) to a final concentration of 500ug/ml. Label the secondary antibody with Lightning-Link® PE/Cy5 DIY Antibody Labeling Kit according to the procedures provided by the manufacture.
• Antibody labeling: Resuspend the cell pellet with PBS containing 0.1% BSA. Adjust the volume to get a density of 107/ml. Transfer 100μl cell suspension to another two centrifuge tubes as blank and negative control, respectively. Add Neph3 primary antibody to a final concentration of 10 μg/ml and incubate at 4℃ for 30min.Wash the cells two times with DPBS. Resuspend the cells with DPBS and adjust the DPBS volume to reach 2×106/ml density.
• Cell sorting: Sort Neph3+ cells using BD FACSAria II. Re-suspend sorted neph3+ cell with gfCDM media. Adjust the concentration to 1×107 cells/ml for co-culture with slices. Resuspend the cells with HBSS containing 5mg/ml D-glucose and 20 ng/ml bFGF. Adjust the concentration of cells to 2×107 cells/ml for microinjection.
Microinjection of human Purkinje progenitors into cerebellum of SCID mice
• Deeply anesthetize the newborn SCID/Beige on ice.
• Inject 1μl of cell suspension (2×104 cells) into the cerebellum region using a glass micropipette connected to a microinjector.
• Four weeks after injection, sacrifice the mice and perfuse with 4% PFA. Dissect the cerebellum and section at 40 μm thickness for further analysis.