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.

Begin embryoid body formation by harvesting embryonic stem cells with 0.25% trypsin-EDTA for 2 to 5 minutes at 37 degrees Celsius, 5% carbon dioxide. Add fresh Iscove's Modified Dulbecco's medium or IMDM with 15% FBS to the detaching cells, then transfer the cell suspension to a 15-milliliter tube.

Centrifuge at 160 x g for 5 minutes at room temperature. Then, count the cells using a hemocytometer and prepare a 5 x 10⁵ cells per milliliter suspension in IMDM with 0.5 micromolar retinoic acid. Next, use an 8-channel pipette and 200-microliter tips to plate 120-microliter drops per 100-milliliter Petri dish. Invert the dish and fill the inverted lid with PBS to prevent hanging drops from drying. Culture at 37 degrees Celsius, 5% carbon dioxide for 3 to 4 days.