Induction of neural crest cells from mouse embryonic stem cells in a serum-free monolayer culture

The neural crest (NC) is a group of cells located in the neural folds at the boundarybetween the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells,including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early developmentremain poorly understood. We previously established a defined serum-free culture condition formouse embryonic stem (mES) cells without feeders. Here, using this defined condition, we havedeveloped a protocol to promote mES cell differentiation into NC cells in an adherent monolayerculture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblastgrowth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we haveestablished a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocolexpressed EGFP. This protocol could be used to help clarify the mechanisms by which cellsdifferentiate into the NC lineage and to assist the development of applications for clinical therapy.