Abstract

Neural crest cells migrate throughout the embryo and differentiate into diverse derivatives: the peripheral neurons, cranial mesenchymal cells, and melanocytes. Because the neural crest cells have critical roles in organogenesis, detailed elucidation of neural crest cell differentiation is important in developmental biology. We recently reported that melanocytes could be induced from mouse ESCs. Here, we improved the culture system and showed the existence of neural crest-like precursors. The addition of retinoic acid to the culture medium reduced the hematopoiesis and promoted the expression of the neural crest marker genes. The colonies formed contained neural crest cell derivatives: neurons and glial cells, together with melanocytes. This suggested that neural crest-like cells assuming multiple cell fates had been generated in these present cultures. To isolate the neural crest-like cells, we analyzed the expression of c-Kit, a cell-surface protein expressed in the early stage of neural crest cells in vivo. The c-Kit-positive (c-Kit(+)) cells appeared as early as day 9 of the culture period and expressed the transcriptional factors Sox10 and Snail, which are expressed in neural crest cells. When the c-Kit(+) cells were separated from the cultures and recultured, they frequently formed colonies containing neurons, glial cells, and melanocytes. Even a single c-Kit(+) cell formed colonies that contained these three cell types, confirming their multipotential cell fate. The c-Kit(+) cells were also capable of migrating along neural crest migratory pathways in vivo. These results indicate that the c-Kit(+) cells isolated from melanocyte-differentiating cultures of ESCs are closely related to neural crest cells.