Isolation and culture of EMSCs from dental germs

The first molars of the upper and lower jaws were dissected from seven to ten embryos of E18.5d rats. The minced tissue was mixed with 1% trypsin/1 mM of the EDTA solution (Sigma, St. Louis, MO, United States) at 37°C for 10 min and neutralized with Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12) (Sigma, St. Louis, MO, United States) containing 10% of FBS (Gibco Waltham, MA United States). Next, the suspension was centrifuged at 800 rpm for 5 min. The cell pellet was resuspended in DMEM/F12 supplemented with 10% FBS and antibiotics (100 μg/ml of penicillin and 100 μg/ml of streptomycin) at a density of 2 * 10⁵/ml and then cultured at 37°C in a 5% CO2 humidified incubator. The culture medium was changed every 2 days and cells were passaged when the cell density was fused to 70 %–80%. E18.5 d rat EMSCs at passage three were used in the following experiment.