Neural crest cells retain their capability for multipotential differentiation even after lineage-restricted stages.

Multipotency of neural crest cells (NC cells) is thought to be a transient phase at the early stage of their generation; after NC cells emerge from the neural tube, they are specified into the lineage-restricted precursors. We analyzed the differentiation of early-stage NC-like cells derived from Sox10-IRES-Venus ES cells, where the expression of Sox10 can be visualized with a fluorescent protein. Unexpectedly, both the Sox10+/Kit- cells and the Sox10+/Kit+ cells, which were restricted in vivo to the neuron (N)-glial cell (G) lineage and melanocyte (M) lineage, respectively, generated N, G, and M, showing that they retain multipotency. We generated mice from the Sox10-IRES-Venus ES cells and analyzed the differentiation of their NC cells. Both the Sox10+/Kit- cells and Sox10+/Kit+ cells isolated from these mice formed colonies containing N, G, and M, showing that they are also multipotent. These findings suggest that NC cells retain multipotency even after the initial lineage-restricted stages.

Unexpected multipotency of melanoblasts isolated from murine skin.

Melanoblasts, precursor of melanocytes, are generated from the neural crest and differentiate into melanocytes during their migration throughout the entire body. The melanoblasts are thought to be progenitor cells that differentiate only into melanocyte. Here, we show that melanoblasts, even after they have already migrated throughout the skin, are multipotent, being able to generate neurons, glial cells, and smooth muscle cells in addition to melanocytes. We isolated Kit-positive and CD45-negative (Kit+/CD45-) cells from both embryonic and neonate skin by flow cytometry and cultured them on stromal cells. The Kit+/CD45- cells formed colonies containing neurons, glial cells, and smooth muscle cells, together with melanocytes. The Kit+/CD45- cells expressed Mitf-M, Sox10, and Trp-2, which are genes known to be expressed in melanoblasts. Even a single Kit+/CD45- cell formed colonies that contained neurons, glial cells, and melanocytes, confirming their multipotential cell fate. The colonies formed from Kit+/CD45- cells retained Kit+/CD45- cells even after 21 days in culture and these retained cells also differentiated into neurons, glial cells, and melanocytes, confirming their self-renewal capability. When the Kit signal was inhibited by the antagonist ACK2, the Kit+/CD45- cells did not form colonies that contained multidifferentiated cells. These results indicate that melanoblasts isolated from skin have multipotency and self-renewal capabilities.

Isolation and characterization of Kit-independent melanocyte precursors induced in the skin of Steel factor transgenic mice.

Steel factor (SLF, also called KIT-ligand, mast cell growth factor, or stem cell factor) acting through the tyrosine kinase receptor KIT is thought to be indispensable for the early phase of melanocyte development both in vivo and in vitro. In the present study, Kit-independent precursor cells were generated in mice expressing exogenous SLF in their skin keratinocytes and were detected as pigmented spots after administration of Kit function-blocking antibody. We successfully purified these precursor or stem cells as Kit+CD45- cells by flow cytometry. The purified cells showed normal but delayed differentiation into mature melanocytes, indicating the immature nature of Kit-independent precursors. The Kit-independent interfollicular population generated in SLF transgenic mice was suggested to be the counterpart of the follicular melanocyte stem cell based on the Kit-independent nature for their survival.

Multipotent cell fate of neural crest-like cells derived from embryonic stem cells.

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.