STEM CELLS 2007;25:402-410

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 (Kit1/CD452) cells from both embryonic and neonate skin by flow cytometry and cultured them on stromal cells. The Kit1/CD452 cells formed colonies containing neurons, glial cells, and smooth muscle cells, together with melanocytes. The Kit1/CD452 cells expressed Mitf-M, Sox10, and Trp-2, which are genes known to be expressed in melanoblasts. Even a single Kit1/CD452 cell formed colonies that contained neurons, glial cells, and melanocytes, confirming their multipotential cell fate. The colonies formed from Kit1/CD452 cells retained Kit1/ CD452 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 Kit1/ CD452 cells did not form colonies that contained multidifferentiated cells. These results indicate that melanoblasts isolated from skin have multipotency and self-renewal capabilities.

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. Developmental Dynamics 240:1681–1693, 2011. © 2011 Wiley‐Liss, Inc.

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.