Melanocytic Differentiation Of Human Hair Follicle-derived Neural Crest Stem Cells

BackgroundVitiligo is one of the most common acquired pigmentary disorders.Its etiology is complex,and the pathogenesis is not yet clear,may be related to heredity,immunity,neuropsychiatric factors,self-destruction of black cells etc.,resulting in the destruction of epidermal melanocytes,causeing skin,mucous membrane depigmentation and even hair pigment loss.Although vitiligo lesions is asymptomatic,the damage of self-image has a great impact on the patient's psychology and communication,patients with visible lesions often have several psychological diffi culties,such as shame,depression,and anxiety.So far,there is no cure for vitiligo,current treatment results vary between individuals.Successful repigmentation of the lesions of patients with vitiligo mainly presents in a perifollicular pattern.Certain anatomic sites that lack hair follicles are difficult to treat,including the lips,genitals,hands(particularly the knuckles and fingertips),feet and other bony prominences.In addition,lesions in which the hair is depigmented have a poor prognosis.Therefore,it is of great scientific,economic and social significance to explore the mechanism of hair follicle repigmentation,further study the pathogenesis of vitiligo,and find new effective treatment methods.As a dynamic micro organ,hair follicle(HF)plays an important role in maintaining biological function.It not only regulates hair growth and shedding,but also has important sensory and immune functions.Recent studies suggest that the presence of neural stem cells(NCSCs)in the hair follicle outer root sheath bulge region.NCSCs has the unique characteristics of migration and differentiation,under certain conditions can differentiate into sensory neurons,bone cells,chondrocytes and melanocytes,Schwann cells,etc.Based on this,we conceive that the human hair follicle neural crest stem cells can differentiate into melanocytes and produce melanin,which induce the skin lesions of vitiligo repigmentation.In the previous study,the neural crest stem cells were isolated from the hair follicles and were initially identified as neural crest stem cells.This project intends to separate neural crest stem cells from hair follicles,culture in vitro and induce differentiation into melanocytes.Thus,providing a basis for the development of hEPI-NCSCs as the target for the treatment of vitiligo.ObjectiveThis experiment is intended to demonstrate the differentiation potential of human hair follicle neural crest stem cells cultured in vitro,inducing differentiation of neural crest stem cells into melanocytes and preliminary identification of melanocytes.Methods1.The specimens were derived from the scalp tissue containing a certain number of hair follicles obtained from the first affiliated hospital of Zhengzhou University,such as dermatology,orthopedic surgery,and neurosurgery.There were no organic lesions,no pigmentary diseases and no history of topical medication during nearly one month in the skin tissue.2.In the sterile environment,flush the scalp tissue with DPBS buffer,cut loose adipous and dermal tissue around the follicle with the straight scissors,cut into small pieces,and the single hair follicle was microscopically isolated,then the hair follicle bulge region was cut off.3.The hair follicles were cultured by neural stem cell culture medium in vitro,observe the time,morphology,and quantity of cells migrated from the hair follicle bulge region,and immunofluorescence was used to identifiy the expression of Sox10 and Nestin in cells.4.Replace the culture medium to melanocyte differentiation medium,and the morphological and quantitative changes of the cells were observed.5.Identification of differentiated cells: Dopa reaction;and the expression of Tyrosinase,Mitf in cells was detected by immunofluorescence.Results1.When hair follicles were cultured in vitro,a small amount of cells migrated from the bulge region on the third day.On the sixth day,a large number of efferent cells were seen around the hair follicles.The cells were elliptical,spindle-shaped,full of nuclei,and immunofluorescence results.It was shown that Sox10 and Nestin were positively expressed in cells released from the hair follicle bulge region.2.With different separation methods,hair follicles without enzymatic digestion are more likely to dissociate cells.3.After 48 hours of changing the differentiation medium of melanocyte,the morphology of neural crest cells changed significantly,showing typical dendritic structure;differentiated cells were positive for dopa,cytoplasm showing grayish black;Immunofluorescence staining showed that the differentiated cells were positive for Tyrosinase,Mitf.ConclusionsIn vitro culture,human hair follicles can move out of cells,and the hair folliclederived cells can be directed to differentiate into melanocytes when cultured in induction differentiation medium culture,...