Differentiating Embryoid Body Cells into Neural Progenitors Using Retinoic Acid

Begin with embryonic stem cells cultured over a layer of fibroblast feeder cells, which maintain the stem cells in their undifferentiated state.

Add trypsin enzymes and EDTA to disrupt cell-to-cell adhesion and detach the cells.

Later, add a serum-enriched culture medium to stop the enzyme activity, and transfer the cell suspension into a tube.

Centrifuge to collect the cells, remove the supernatant, and then resuspend the cells in a medium containing retinoic acid.

Place droplets of this cell suspension onto the base of a non-adhesive culture dish.

Invert the base to form hanging drops and position it over the culture dish lid filled with a buffer to prevent the droplets from drying.

In hanging drops, gravity causes the cells to settle at the bottom, eventually aggregating into three-dimensional structures termed embryoid bodies.

Further, the cells absorb retinoic acid, which modulates various intracellular signaling pathways. 

This modulation stimulates the differentiation of stem cells into neural progenitor cells with the neuron-producing ability.