Melanocytic Differentiation Of Hair Follicle-derived Neural Crest Stem Cells And Age-related Impairment Of Stemness

BackgroundVitiligo is a disorder that causes depigmentation of patches of skin. It occurs when melanocytes, the cells responsible for skin pigmentation, lost or are unable to function. Although the cause of vitiligo is unknown, but research suggests that it may arise from autoimmune, genetic, oxidative stress, neural, or viral causes. The incidence of vitiligo in China is about 0.1-2.9%. There are a number of treatments that improve the hypopigmentation, but there is no cure for vitiligo. Our project focused on a stem cell-based technology and tried to figure out the regulation mechanism of skin melanocytes development.ObjectiveStem 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, (â…±) migrate into the skin during embryogenesis and form skin melanocytes, neurons, Schwann cells and mesenchymal cells, (â…²) 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.ResultsPartâ… . Isolation of hair follicle NCSCsHair 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, which are distinctively different from known epithelial stem cells. These bulge precursor cells, grown as three-dimensional spheroid structures, are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They do not express squamous markers but express immature neural crest cell markers p75, nestin and Sox10 as well as the embryonic stem cell transcription factors Sox2, Klf4 and cMyc. They exhibit clonal multipotency that can give rise to myogenic and neuronal cell lineages after in vitro clonal single cell culture. In addition, these SCs show differentiation potential toward mesenchymal lineages, and they can be differentiated into adipocyte, chondrocyte, and osteocyte lineages.Partâ…¡. Melanocytic differentiation of hair follicle NCSCsEpidermal melanocytes play an important role in protecting skin from ultraviolet (UV) rays, and they are implicated in a variety of pigmentation skin disorders. Next step, we developed an efficient method for differentiating hair NCSCs into melanocytes. After 2-3 weeks induction in a cocktail medium containing several growth factors, 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. This technology provides a novel in vitro system for studying the developmental biology and diseases of melanocytes. It also offers new opportunities for the use of these cells in regenerative medicine.Partâ…¢. Age-related sternness impairment of hair follicle NCSCsAging is an age-related decline in physical and mental function. Its powerful and irremovable effect makes researchers very frustrated. Moreover a major unanswered question in autologous cell therapy is the appropriate timing for cell isolation. Many of the putative target diseases arise with old age and previous evidence, mainly from animal models, suggests that the stem/progenitor cell pool decreases steadily with age. Studies with human cells have been generally hampered to date by poor sample availability. Although a large number of studies deal with their differentiation ability and potential applications in tissue damage repair, few studies have concentrated on the regulation of NCSC sternness. Here we show an analysis of hair follicle NCSCs abundance and in vitro differentiation potential, by using bulges isolated from mice with different age. Hair NCSC abundance and differentiation potential decrease sharply with age, being extremely difficult to isolate, expand, differentiate and migrate when obtained from the elderly. Our data suggest preserving bulge NCSCs banks early in life would be desirable for use in clinical protocols in the aging population.