Single-cell RNA-seq of nevus-derived melanocytes and matching perilesional skin melanocytes

Melanocytic nevi are considered to be benign melanocytic proliferations which are driven primarily via BRAF V600E MAPK activation. Using scRNA-seq we explored the signalling pathways in nevus derived melanocytes compared to perilesional skin. Overall design: Each nevus was bisected; one half prepared for standard histopathological diagnosis, while the other half was dissected to separate nevus from adjacent non-lesional skin (hereafter referred to as perilesional skin). In nevi that were deemed to have sufficient size to attempt cell culturing, the bisected portion that was not used for diagnostic purposes, was bisected again, with one half prepared for cell culturing. Briefly, the epidermis was separated from the dermis by a 3-hour incubation at 37°C with Dispase II solution (Roche, Rotkreutz, Switzerland) followed by vortexing for 15 seconds and stirring with a pipette tip until the skin layers separated. Samples were then mixed with 2mL melanoblast culture media (MCBD 153 medium [Sigma-Aldrich, St Louis, USA], 10% chelated fetal bovine serum (8), 2% fetal bovine serum, 2mM glutamine, 1.66µg/L cholera toxin, 100nM endothelin-3 [ENZO Life Sciences, Farmingdale, USA], 10ng/mL stem cell factor [Sigma-Aldrich, St Louis, USA], 2.5ng/mL basic fibroblast growth factor 2) and transferred to a culture plate coated with collagen matrix (Thermo Fisher Scientific Inc, Waltham, USA) to be incubated for 72 hours at 37°C. The melanocytes were grown using media designed to promote the transition of melanocytes back to the melanoblast cell state to allow the expansion of cells in cell culture flasks. From prior experience, melanocytes derived from nevi do not commonly grow very efficiently which may be in part due to the phenomenon of oncogene-induced growth arrest. To overcome growth arrest induced by the presence of the BRAF V600E mutation, once the culture was approximately 50% confluent, cells were treated with a lentiviral CDK4 R24C vector. This vector has previously been shown to allow oncogene driven melanocytes (NRAS Q61K) to enter into the senescence bypass by rendering CDK4 to be insensitive to inhibition by p16 and p15. Stably transduced cell lines were generated by transducing cells in 12- or 6-well plates at approximate 50:1 ratio of viral particles to cells. Wells containing confirmed >99% GFP- or RFP-positive cells were expanded for stock freezing and/or assays.