The proposed study is designed to determine how distinct subsets of melanocyte stem cells (MSCs) we have identified are regulated and maintained in the stem cell state and contribute to pigment regeneration. The rationale for the study is based upon our discovery that MSCs not only populate a region of the murine hair follicle (HF) termed the bulge, also the site of keratinocyte stem cells (KcSCs) of the HF, but also the secondary hair germ (SHG), a transient structure at the base of the telogen, or resting, HF adjacent to the dermal papilla. Our laboratory has developed methods to separate and study these two cell subsets in the viable state using a combination of a unique transgenic mouse system and fluorescence-activated cell sorting (FACS).The objectives of our studies are to determine which of these MSC subsets has greater regenerative potential, and to describe the interrelationship between and maintenance determinants of these distinct subsets. To realize this objective, we plan to test in vitro and in vivo the capacity of these cell subsets to self-renw, maintain quiescence, and also regenerate pigmentation in previously unpigmented living HFs. These studies will be accomplished a unique, bitransgenic mouse line we have developed, Dct-H2BGFPki, in combination with the FACS facility and other core resources associated with my research laboratory located in the Department of Biochemistry and Molecular Biology at the University of Maryland School of Medicine. The results of these studies should have a positive impact on the health care of the general public. By determining which murine MSC subsets are most effective at regenerating pigmentation, we will be able to apply that knowledge to human melanocytes, isolating or generating human cells with potent regenerative properties. These cells can be developed to treat patients with the depigmenting disease vitiligo or for regenerating pigmentation in healing wounds or scars. In addition, stem cells are related to cancer because genes repressed epigenetically in stem cells are more likely to be suppressed in the corresponding cancer cell type. By determining the epigenetic state of key melanocyte and growth genes in MSCs, we may highlight molecular mechanisms leading to irreversible gene repression in melanoma developing, thereby revealing how particular cells of origin contribute to the development of this particular malignancy.