In vitro expansion of immature melanoblasts and their ability to repopulate melanocyte stem cells in the hair follicle.

Elucidation of the molecular mechanisms underlying stem cell regulation is of great importance both for basic biology and for clinical applications. Melanocyte stem cells (MSCs) are an excellent model in which to study the molecular basis of stem cell regulation, as the genetic alterations involved in the maintenance of the stem cells are readily identifiable by a premature hair graying phenotype. Research on MSCs has been hampered by the lack of a reliable system to assay their function. Here, by co-culturing highly purified melanoblasts (MBs) with XB2 keratinocytes, we describe an efficient culture method that allows the expansion of immature MBs in vitro. These MBs are also capable of undergoing terminal differentiation into mature melanocytes (MCs) when differentiation is induced. Furthermore, by performing a hair-follicle reconstitution assay in which expanded MBs in a mixture of epidermal and dermal cells were grafted to reconstitute a hair follicle, we demonstrate that the expanded MBs retain their capacity to become incorporated into newly developed hair follicles and repopulate the MC stem cell population there. Thus, by integrating genetic manipulations in cultured MBs in vitro, this method provides a powerful tool with which to study the molecular basis of stem cell regulation.

Molecular characterization of melanocyte stem cells in their niche.

Emerging evidence from stem cell (SC) research has strengthened the idea that SC fate is determined by a specialized environment, known as the SC niche. However, because of the difficulty of identifying individual stem cells and their surrounding components in situ, the exact mechanisms underlying SC regulation by the niche remain elusive. To overcome this difficulty, we employed melanocyte stem cells (MSCs), which allow the identification of individual SCs in the niche, the lower permanent portion of the hair follicle (HF). Here, we present molecular makers that can distinguish MSCs from other melanocyte (MC) subsets in the HF. We also describe a simple and robust method that allows gene expression profiling in individual SCs. After isolating individual MSCs from transgenic mice in which the MCs are marked by green fluorescence protein (GFP), we performed single-cell transcript analysis to obtain the molecular signature of individual MSCs in the niche. The data suggest the existence of a mechanism that induces the downregulation of various key molecules for MC proliferation or differentiation in MSCs located in the niche. By integrating these data, we propose that the niche is an environment that insulates SCs from various activating stimuli and maintains them in a quiescent state.