To induce neural crest formation, replace the maintenance medium of the human pluripotent stem cell culture with 200 microliters of medium A per square centimeter of the well-surface. Incubate the culture under 5%carbon dioxide supplementation at 37 degrees Celsius for two days. Gently aspirate the medium from the well-containing 100%confluence cells then pipette about 200 microliters of medium B per square centimeter of the well-surface area.
On day four, feed the cells with 200 microliters of medium B per square centimeter of the well-surface. Incubate the cells for two more days, then replace the medium B with 400 microliters per square centimeter of medium C.
