Research On Purification, Stress And Reprogramming Of Epidermal Melanocytes

Objective:(1)To optimize the protocols for the purification and growth of human epidermal melanocytes by using various coated tissue culture plates and cultural conditions,and look for a convenient and efficient approach to purify melanocyte cells(MCs).(2)To isolate melanocytes from human epidermis and prepare melanocyte spheroids(MS)by repetitive long-term trypsinization.To characterize these MS with immunofluorescent staining,immunohistochemical examination,transmission electron microscopy,and tumorigenicity analysis.(3)To prepare human induced pluripotent stem cells(hiPSCs)derived from MCs transfected with a combination of 3 or 4 transcription factors.The hiPSCs will be characterized with specific markers and their pluripotency will be investigated by ectoderm differentiation.Methods:(1)The tissue specimens and single cells were obtained from human foreskin,cultured in the plates coated with Matrigel and laminin,respectively.The primary cells at 80–90% confluence were treated with 0.05% trypsin-EDTA for 4 minutes,and then resuspended in M254 medium complemented with G418 and 5-BrdU respectively.Whereafter,the purity of melanocytes was estimated by immunofluorescence staining of specific antibody.(2)The primary melanocytes at 70–80% confluence were incubated with 0.25% trypsin-EDTA solution for 2 h,and then resuspended in M254 medium and reseeded in 24-well plates.The cells reached 70–80% confluence within approximate one week,trypsinized for 4 h in the same way as the above-mentioned.Following the same procedure,the cells were cultured and trypsinized for 6 h.(3)Combination of three factors(Oct4,klf-4 and c-Myc)or four factors(Sox2,Oct4,klf-4 and c-Myc)were respectively used for MC transfection.The generated clones were subsequently characterized by immunofluorescent staining with specific antibodies of pluripotent stem cells and induced differentiation into the typic cells of the ectoderm,such as nerve cells.Results:(1)The observation of cell culture indicated that the proliferation of cell suspension was faster than those of skin explants and epidermal specimens.Moreover,the cells migrated from the epidermis earlier and proliferated quickly than from the skin explants.The melanocytes in the plates coated with laminin grew better than those in the plates coated with Matrigel.The unwanted keratinocytes and fibroblasts were eliminated by using differential trypsinization and by adding G418 and 5-BrdU respectively.(2)The MS were isolated from human epidermis by repetitive long-term trypsinization(LTT),and maintained an aggregated themorphology for a short period in certain conditions.The MS were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and re-inoculated in 24-well plates.(3)Transfection of MCs by using various combination of factors was successful.The cells transfected by combination of four factors generated clones than those by three factors.These obtained clonal cells were positive for staining with factors Sox2 and Oct4 respectively.Under certain induced conditions,the differentiated neural cells(represent an ectodermal layer)were less than other cell types,suggesting the induced condition will be improved.Conclusions:(1)The purified melanocytes could be obtained by culturing single cells from foreskin in the plates coated with laminin,and by differential trypsinization and additing small doses of G418 or 5-BrdU into medium.(2)Immunohistochemical analysis of MS revealed that they were positive for HMB45,a melanosome-specific marker.No melanomas occurred when MS were transplanted into mice.This study provides a promising approach for treating vitiligo by transplantation of MS.(3)MCs could be reprogrammed and generated iPSCs via being transfected with three or four factors.The hiPSCs expressed specific stem cell markers and could differentiated to neuron-like cells in suitable conditions.