Evaluation of Cultured Melanocytes in Terms of Pigmentation, Genetic Stability and Tumorigenicity in order to Vitiligo patients Transplantation

Abstract

Aims: Recently cultured melanocyte transplantation has been evolved as a promising modality against extensive depigmentation in vitiligo which are resistant to conventional treatments. Here, we aimed to find the best growth media for clinical purposes and to determine the tumorigenic activity of cultured melanocytes both in vitro and in vivo. Main methods: Human skin melanocytes were isolated from 8 patients, cultured under various growth media till passages 6. The best growth media based on doubling time (DT) and cell counts at each passages, was determined. Also, changes in BRAF, NRAS, and HRAS genes expressions in cultured cells were assessed by real-time PCR and gene sequencing of BRAF V600E and NRAS in exons 1 and 2 as compared to melanoma cell lines. Karyotyping of the passage 6 melanocytes was done to assess the genetic stability. Cells of passages 5, 6 were subcutaneously injected into BALB/c nude mice to evaluate the risk of tumorigenesis after transplantation. Key findings: A mixture of MGM-M2 supplemented with a combination of melanocyte growth factors two times plus 4% fetal bovine serum (FBS), had the best results in terms of PDT and melanocyte count during passages 0 to 6. Karyotypes of melanocytes at passages 5 and 6 in all patients displayed no structural alterations in the 15 metaphase plate spreads. HRAS and BRAF gene expressions of melanocytes at passage 6 showed <2-fold increases, while HRAS and BRAF gene expressions in melanoma cell lines were up-regulated to more than 3 and 8 folds, respectively. Furthermore, NRAS gene expression in melanocytes at passage 6, showed 5-fold change which was lower than NRAS gene expression in melanoma cell line. Gene sequencing of BRAF V600E and NRAS in exons 1 and 2 displayed no mutation. Melanocytes injected into BALB/c nude mice showed no risk of tumor formation. Also, gene sequencing of BRAF and NRAS in injected melanocytes displayed no mutation 16 weeks after transplantation.Significance: Our results revealed that culturing human melanocytes using standard growth media protocols, may increase the expression of specific proto-oncogenes, however will not lead to tumorigenic mutations, in vitro and in vivo.