Generating Neurons and Glial Cells from Human Induced Pluripotent Stem Cell-Derived Embryoid Bodies

Take embryoid bodies, or EBs, derived from human induced pluripotent stem cells. These EBs are composed of three germ layers.

Transfer the EBs to a matrix-coated culture dish containing a suitable medium and incubate.

The matrix enables the EBs to attach to the culture dish.

The growth factors and nutrients in the medium facilitate the ectodermal cells of the EBs to form neuroepithelial aggregates arranged in a circular pattern termed a rosette.

Cut the rosette fragments and detach them from the dish.

Collect the fragments and spin them down.

Discard the supernatant and resuspend the fragments.

Using a pipette, move the fragments up and down to dissociate them.

Add the desired number of cells to a matrix-coated well plate containing a differentiation medium.

The components of the differentiation medium facilitate the neuroepithelial cells to transform into fully functional neuronal and glial cells, the primary cells of the nervous system.

On day two, aspirate standard matrix coating solution from a previously prepared 60-milliliter dish, and add 5 milliliters of complete neuroepithelial induction medium or NRI to the dish. Working under the stereoscopic microscope at 4x magnificati