Embryonic Tongue Mesenchyme Isolation

The E11.5 tongue swellings were dissected from the mandible and the mesenchyme was separated from the epithelium, as previously described (Liu et al., 2008 (link)). At this stage, the mesenchyme is primarily populated by NC-derived cells (Han et al., 2012 (link)) and very few non-NC-derived myoprogenitors are present in the tongue mesenchyme (Han et al., 2012 (link)). Separated mesenchyme was then transferred to a culture dish, cut into small pieces and cultured in a humidified CO₂ incubator at 37°C in a serum-free medium, i.e. a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's nutrient F12 (11320033, DMEM/F12, Gibco) containing 50 μg/ml gentamicin sulfate (15750060, Gibco). After 1 day in culture, the medium was replaced with fresh medium and continued to culture for 2 days. Culture medium was collected as conditioned medium. To inhibit the p-Smad1/5/8 activity, dorsomorphin (3093, R&D Systems) was added at 30 µg/ml to the cultures of mesenchyme cells from wild-type mouse tongue.