Generating Cortical Interneuron Precursors From Mouse Embryonic Stem Cells
Transkript
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.
