A Study Of The Direct Reprogramming Of Fibroblasts Into Melanocytes

Objective: The loss of melanocyte function leads to the occurrence of vitiligo,which impacts patient physically and psychologically.At present,there is a lack of a small trauma,long-lasting and effective treatment method for vitiligo,and the use of regenerative medicine technology to directly reprogram melanocytes seems to provide new ideas for the treatment of vitiligo.If the original non-pigmented skin cells can be directly reprogrammed into functional melanocytes,it is bound to help improve the plight of vitiligo patients.Although the current direct reprogramming method is very popular,the method for direct reprogramming into melanocytes is inconclusive,and the selection of transcription factors is numerous and dazzling.We need to construct a simple and high-efficiency method to directly reprogram melanocytes,screen transcription factors and directly reprogram melanocytes with optimal transcription factor combinations to increase the efficiency and safety of reprogramming,and verify the functionality of induced melanocytes(iMels)obtained by direct reprogramming in vitro & in vivo to prove the possibility of its clinical application.Methods: Firstly,a simple and repeatable concentrated lentivirus packaging system was established to deliver the corresponding transcription factors.The virus titer was detected by flow cytometry.The best infection method,infection time and multiplicity of infection(MOI)were determined by observing the infected fluorescence rate,flow cytometry and quantitative real-time fluorescence polymerase chain reaction analysis(q RT-PCR),respectively;Then,six transcription factors(Sox10,Mitf,Pax3,Sox2,Sox9 and Snai2)were introduced into mouse embryonic fibroblasts(MEFs)for direct reprogramming into melanocytes.The characteristics of multi-factor direct reprogramming iMels were preliminarily verified by watching the changes in cell morphology and detecting the changes in expression levels of transcription factor genes and melanocytic genes by q RT-PCR;The direct reprogramming system was upgraded and optimized in the following ways: 1)Increase cell proliferation: adding small molecules of insulin,corticosteroids and adenosine in permutation and combination,detecting cell proliferation by cell counting kit(CCK8),and detecting the gene expression of transcription factors and melanocytic markers by q RT-PCR;2)Improve the purity of infected cells: puromycin was added to screen cells,and the positive rate of green fluorescent protein(GFP+)was detected by flow cytometry;3)Improve cell attachment matrix: comparing the results of using different matrix gels Gelatin and Fibronectin,and detecting the expression levels of transcription factor genes and melanocytic genes by q RT-PCR;4)Activate melanogenesis pathway: adding Wnt pathway activator Chir99021,and detecting the expression levels of transcription factor genes and melanocytic genes by q RTPCR;For the screening of transcription factors,five transcription factors were tranfected into MEFs(one of the six transcription factors was removed at random),and the melanocytic genes in each group were detected by q RT-PCR to find the important transcription factors.Then the important transcription factors were used to infect the MEFs in different permutations,and the melanocytic genes in each group were detected to find the most effective transcription factor combination and the reprogramming efficiency was detected by flow cytometry;Finally,the iMels generated by the optimized direct reprogramming system and the best combination of transcription factors were identified.Identification of the phenotype and functionality in vitro was performed by morphological observation,β-galactosidase staining to detect cellular senescence,q RT-PCR to detect transcription factor genes and melanocytic genes,immunofluorescence to detect melanocytic proteins,Masson-Fontana staining and L-DOPA staining to detect melanin secretion;In the in vivo characterization,follicle reconstruction was performed by intradermal injection method and small chamber method to transplant iMels,and the in vivo functionality of iMels was demonstrated by immunofluorescence for melanocytic proteins,Masson-Fontana staining and LDOPA staining for melanin secretion.Results:(1)Direct reprogramming into melanocytes using transcription factors Sox10,Mitf,Pax3,Sox2,Sox9 and Snai2 resulted in iMels with morphology similar to melanocytes,but the expression of melanocytic genes was upregulated for the first 7 days of direct reprogramming and then downregulated;(2)In the optimized direct reprogramming system,CCK8 assay revealed the addition of hydrocortisone,insulin and adenine significantly increased the proliferation of direct reprogramming cells,and significantly increased the expression of melanocytic genes(Tyrp1 and S100);The purity of transformed cells after puromycin screening was over 95%;The expression of melanocytic genes was higher in the Gelatin group than in the Fibronectin group;Wnt pathway activator(small molecule Chir99021)upregulated the expression of the transcription factors Mitf and Sox10 during direct reprogramming into melanocytes and upregulated the expression of melanocytic genes in iMels;(3)In the screening of transcription factors,after removing Sox10,Mitf and Pax3,the expression of melanocytic genes in transformed cells was basically silenced.iMels obtained using the two factors Sox10 and Mitf(2F iMels)had the highest melanocytic gene expression and reprogramming efficiency of about 43%;(4)In the identification of 2F iMels,cell senescence β-galactosidase staining demonstrated mature ageing of cells cultured beyond 20 days,and the expression of melanocytic genes was close to that of real melanocytes.The expression of melanocytic markers TYRP1 and DCT was positive,and the melanin specific Masson-Fontana staining and L-DOPA staining were also positive;(5)The site of 2F iMels transplantation was positive for melanin characteristic markers TYRP1 and DCT immunostaining,melanin specific Masson-Fontana staining and L-DOPA staining.Conclusions: iMels directly reprogrammed with transcription factors Sox10,Mitf,Pax3,Sox2,Sox9 and Snai2 are poorly proliferative and the expression of melanocytic genes differs considerably from that of real melanocytes,and the previous direct reprogramming system to melanocytes needs to be optimized;In the optimized direct reprogramming system: 1)The optimal induction medium supplemented with hydrocortisone,insulin and adenine can improve the proliferation of direct reprogrammed cells and promote the expression of melanocytic genes;2)In terms of matrix gels,Gelatin is more suitable for melanocyte transdifferentiation than Fibronectin;3)Wnt pathway activator can promote direct reprogramming into melanocytes;In the screening of transcription factors,direct reprogramming into melanocytes can be performed with Sox10 and Mitf,successfully obtaining 2F iMels;In functional characterization,2F iMels undergo mature aging without tumorigenicity and 2F iMels are functional both in vitro and in vivo.