Isolation, Cultivation And Phenotype Characterization Of Hair Follicle-derived Melonocytes

Background: Hair follicles(HF) is a complex epidermal appendage embedded deeply in the dermis,which is an important mechanism to keep homeostasis, to repair injury on the epidermis and to maintain skin’s and hair’s color.HF is an easily accessible rich source of autologous stem cells having been successfully used as cell source for treatment of skin loss and vitiligo. Hair pigmentation is provided by very active follicular melanocytes(MCs) located above the dermal papilla.Melanocytes are originally derived from the neural crest and become localized in hair follicles as well as in the epidermis where they deliver melanin to the hair and skin respectively. Mel SCs form a stem cell system within individual hair follicles and provide a ‘hair pigmentary unit’ for each cycle of hair pigmentation. During the transition from telogen to anagen, activation of Mel SC leads to the development of MC progenitors which, in turn, finally leads to differentiated MCs that produce melanin. Melanocytes in each hair follicle produce melanin pigments for the hair during each hair cycle. Melanocytes in the epidermis rarely proliferate and produce melanin pigments to protect the skin from ultraviolet light(UV), while melanocytes in hair follicles repeatedly proliferate and differentiate for hair pigmentation in every hair cycle.Lack in cell number or dysfunction will results in a series of pigment disorders. As a consequence the culture of melanocytes from hair follicles in vitro will supply autologous melanocytes for the treatment of this disease. It is of clinical importance to set up a technique for isolation and cultivation of melanocytes from hair follicle.Research Objectives: To set up a technique of isolation, purification and amplification of melanocytes from Hair Follicle.Methods: Full-thickness skin was harvested from dorsal of C57 mouse at anagen, cut into small pieces(0.2cm*0.2cm). The epidermis was removed from the skin of mice after digested with dispase II. The dermis was digested with collagenase I digestion for 1 hour. The hair follicles were released from the dermis and subsequently trypsinized. Cells from hair follicles were culture in the tissue culture dish in the culture medium M254 supplemented with HMGS. HE and toluidine blue staining were performed to inspect the location of HF-MCs in the skin. L-DOPA and immune fluorescence staining, RT-PCR and Western Blot were performed on HF-MCs.Results: HE and toluidine blue staining showed HF-MCs located in the out rout sheath and above the dermal papilla of hair follicles in anagen. Dopa staining showed HF-MCs displays bipolar or tripolar morphology, immune fluorescence assay showed positive staining against MITF, TYR and TRP1. RT-PCR and Western Blot showed HF-MCs expressed MITF, TYR, and TRP1 from m RNA and protein levels.All the results show that its melanocytes express the marker of the normal melanocytes and are functional which are what we expected.Conclusions:HF-MCs were isolated from hair follicles of mice. The HF-MCs expressed the specific markers of MCs from m RNA, protein levels by Dopa staining, immune fluorescence and RT-PCR as well as Western blot, which provides novel cell source for stem cell biology study and correction of melanocyte dysfunctions.