Complementary In Vivo Approaches Reveal Quiescent Melanocytes in Anagen Outside the Hair Follicle Bulge

Melanocyte stem cells (McSCs) are key components of the hair follicle (HF) stem cell system that are derived from neural crest during embryogenesis and are responsible for regeneration of differentiated melanocytes during successive HF cycles. Our previous research has shown presence of two subsets of phenotypically and functionally distinct McSCs exist in murine telogen HFs, CD34+ McSCs in the bulge/lower permanent portion (LPP) and CD34- McSCs in the secondary hair germ (SHG). Whether these subsets are maintained independently or exist in a developmental hierarchy is not yet known. Using Dct-H2BGFP mice, we analyzed the quiescent and proliferative properties of McSCs and melanocytes in anagen and telogen. We found unexpectedly that Kit+Nestin- quiescent melanocytes are maintained outside of the bulge/LPP region throughout anagen in addition to the Kit+Nestin+ quiescent melanocytes of the bulge/LPP. Both subpopulations express lower levels of melanocyte differentiation markers Mitf, Pax3, Dct, Tyrp1 and Tyr compared to differentiated melanocytes of the HF bulb/matrix. These results suggest that quiescent melanocytes localized in the outer root sheath, both in and below the bulge/LPP) retain the stem cell phenotype observed in quiescent McSCs during telogen. This finding has implications for maintenance of distinct subsets of McSCs throughout successive HF cycles.