The Effects Of NB-UVB/308Nm Excimer Laser On The Hair Follicle-derived Neural Crest Stem Cells Differentiating Into Melanocyte Lineage In Vitro

BackgroundMelanocytes are specialized melanin producing cells, and are responsible for skin, hair, and eye pigmentation in vertebrate organisms. Melanocytes in vertebrates are derived from the neural crest of ectoderm, which migrate intensively to the epidemis and hair follicle where they differentiate into melanocytes during embryogenesis. Our project focused on a stem cell-based technology and tried to figure out the regulation mechanism of skin melanocytes development.Stem cells are primitive, undifferentiated cells with the capacity for self-renewal and the ability to differentiate into mature cell lineages. Skin neural crest stem cells (NCSCs) derive from embryonic neural crest, migrate into the skin during embryogenesis and form skin melanocytes, neurons, Schwann cells and mesenchymal cells, and persist within specific niches such as bulge, dermal papilliar and dermis. They display multi-lineage differentiation potential, producing both neural and mesodermal progeny in vitro, and are an apparently novel precursor cell type that is distinct from other known precursors within the skin. Furthermore NCSCs play a key role in the skin melanogenesis, hair growth and wound healing and are considered as a potential therapeutic approach in skin pigment diseases.Narrow-band UVB (NB-UVB) and308nm excimer laser are two effective therapeutic option in the treatment of vitiligo. Despite the apparent clinical efficacy, the underlying mechanism of how topical NB-UVB/308nm excimer laser induces repigmentation in vitiligo has not been clearly elucidated. ObjectivesTo investigate the effects of NB-UVB/308nm excimer laser on the maturation of melanocyte lineage differentiated from hair follicle-derived neural crest stem cells (HF-NCSCs) in vitro.MethodsHF-NCSCs were isolated from mouse whisker follicles. The isolated cells were multipotent and expressed embryonic NCSC biomarkers. The effects of NB-UVB on development and differentiation of HF-NCSCs were evaluated. We assessed cell viability, melanogenesis and migration of melanocytes derived from HF-NCSCs after NB-UVB radiation. Tyrosinase, Tyrpl, Dct, Kit, Mc1R, Fzd4, NT3R, Ednra, EPI, TGFfiR, Sox10, Mitf, Lefl and Pax3gene expression was measured by quantitative RT-PCR, while Tyrosinase, Sox10and Mitf protein expression were measured by Western blot analysis. Cell migration was measured by Boyden chamber transwell assay.ResultsPart I. Isolation of HF-NCSCs and melanocytic differentiationHair follicles are known to contain a well-characterized niche for adult stem cells:the bulge, which contains epithelial-and neural crest stem cells (NCSCs). Using a serum-free culture condition, we isolated a population of adult stem cells with NCSC characteristics from the bulge area of mouse whisker follicles. These bulge precursor cells, grown as three-dimensional spheroid structures. They do not express squamous markers but express immature neural crest cell markers p75, nestin, Sox10, Snail-1, Twist-1as well as the embryonic stem cell transcription factors Myo10, Msx2, Klf4, Wnt1. They exhibit clonal multipotency that can give rise to melanocyte, myogenic and neuronal cell lineages after in vitro clonal single cell culture. Next step, we used M254for differentiating hair NCSCs into melanocytes. After1-2weeks induction, the NCSCs-derived melanocytes demonstrated a characteristic bipolar-or tripolar morphology and expressed melanocyte specific markers MITF and Tyrosinase. Furthermore NCSCs-derived cells exhibited positive to DOPA reaction and possessed a capacity of melanin pigment production. Part Ⅱ. The effects of NB-UVB on the hair follicle-derived neural crest stem cells differentiating into melanocyte lineage in vitroNB-UVB increased the expression of tyrosinase during melanocytic differentiation from mouse HF-NCSCs, however, NB-UVB inhibited proliferation of melanocytes derived from HF-NCSCs. Furthermore, the migration of the HF-NCSCs-derived melanocytes was downregulated as NB-UVB doses increased. However, the migration of HF-NCSCs was upregulated under0.4J NB-UVB radiation.Part Ⅲ. The Mechanism of NB-UVB on the hair follicle-derived neural crest stem cells differentiating into melanocyte lineage in vitroMechanistically, increased melanocyte maturation after NB-UVB treatment was resulted from increased expression of several key melanogenic factors, including Sox10, Kit and Mc1R, which play a critical role to promote tyrosinase expression.Part Ⅳ. Comparison of308nm excimer laser and NB-UVB on the maturation of melanocytes differentiated from hair follicle-derived neural crest stem cells in vitroCompare to NB-UVB,308nm excimer laser could increase the expression of tyrosinase, Tyrp1, Tyrp2during melanocytic differentiation from mouse HF-NCSCs more effectively at the same dose and time.ConclusionsI. HF-NCSCs is an ideal cell model to investigate the differentiation and development of the melanocyte precursors.II. NB-UVB increase the maturation of melanocyte lineage differentiated from HF-NCSCs in vitro.III. Mechanistically, several key melanogenic factors, including Sox10, Kit and Mc1R, which play a critical role to promote melanocyte maturation after NB-UVB treatment on HF-NCSCs.Ⅳ. Compare to NB-UVB,308nm excimer laser could increase the maturation of melanocytic differentiation from mouse HF-NCSCs more effectively at the same dose and time. Those data provide in vitro evidence demonstrating some direct effects of NB-UVB/308nm excimer laser on pigmentation of melanocyte lineage differentiated from HF-NCSCs, and may provide a possible mechanism for the effect of NB-UVB/308nm excimer laser in vitiligo.