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