Differential Gene Expression and Tumorigenicity Analysis of CulturedMelanocyte Comparing Melanoma

This study aimed to identify the optimal growth media for culturing human skin melanocytes for
clinical applications and to assess their tumorigenic potential both in vitro and in vivo. Various growth
media were tested to determine the most effective and safest for melanocyte culture, avoiding harmful
growth factors such as TPA and colorant toxins. The study evaluated changes in RAF and NRAS gene
expression through real-time PCR and gene sequencing of BRAF V600E and NRAS in exons 1 and 2,
comparing these with melanoma. Melanocytes were subcutaneously injected into BALB/c nude mice
to assess tumorigenic risk. Results indicated that a mixture of MGM-M2 supplemented with
melanocyte growth factors provided the best outcomes in terms of cell proliferation and melanocyte
count. Gene expression analysis revealed that HRAS and BRAF expressions in melanocytes at passage
6 showed less than 2-fold increases, whereas these genes were up-regulated by more than 3 and 8 folds,
respectively, in melanoma cell lines. NRAS expression in melanocytes at passage 6 increased by 5-fold
but remained lower than in melanoma cell lines. Gene sequencing of BRAF V600E and NRAS in exons
1 and 2 showed no mutations, and melanocytes injected into BALB/c nude mice exhibited no tumor
formation risk. Furthermore, gene sequencing of BRAF and NRAS in the injected melanocytes 16
weeks’ post-transplantation revealed no mutations. These findings suggest that while standard growth
media protocols may elevate specific proto-oncogene expressions, they do not induce tumorigenic
mutations in melanocytes, both in vitro and in vivo.