This study outlines a method for differentiating mouse embryonic stem cells into cortical interneuron progenitors using small molecule inhibitors. The process involves the formation of embryoid bodies (EBs) and subsequent dissociation into single cells for culture.
Take mouse embryonic stem cells suspended in serum-free media containing small molecule inhibitors.
Seed the cells on a non-adherent culture plate, allowing them to proliferate and aggregate to form three-dimensional embryoid bodies or EBs.
Additionally, the inhibitors block non-neuronal signaling pathways and induce embryonic stem cell differentiation toward a cortical neural fate.
Transfer the EBs to a tube, centrifuge, and remove the supernatant.
Resuspend the EBs in a solution containing an enzyme cocktail and DNase.
The enzymes dissociate the EBs into single cells, while DNase degrades contaminating DNA.
Add serum-free media containing inhibitors to stop the enzymatic activity, then centrifuge and remove the supernatant containing enzymes.
Resuspend the cells in media containing small molecule inhibitors and transfer them onto culture plates coated with adhesion factors.
The cells attach to the coated plate surface.
The inhibitors prevent apoptotic cell death and induce cellular differentiation into cortical interneuron progenitors.
Start growing the cells as embryoid bodies by adding 75,000 cells per milliliter in KSRN2 media in the non-adherent tissue culture dishes and incubate them at 37 degrees Celsius. On DD1, prepare for cell landing by coating the tissue culture-treated dishes with poly-L-lysine for at least one hour or overnight at 37 degrees Celsius.
On DD2, aspirate the poly-L-lysine and coat the plates with laminin overnight at 37 degrees Celsius. On DD3, before beginning EB dissociation, aspirate laminin, and allow the plates to completely dry in a tissue culture hood.
Afterward, transfer the EBs with the media into a 15-milliliter tube and centrifuge for three to four minutes at 15 times g or until the EBs have pelleted. Aspirate the media and add 3 milliliters of cell detachment solution containing DNase. Then, incubate the sample at 37 degrees Celsius for 15 minutes.
Gently flick the tube every three minutes to aid in EB dissociation. Once the EBs are no longer visible or 15 minutes have elapsed, add 6 milliliters of KSRN2 containing DNase and centrifuge for five minutes at 200 times g. Then, plate the cells in KSRN2 containing LDN193189, XAV939, and the ROCK inhibitor Y27632 at 4.5 to 5 x 104 cells per centimeter squared.
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