Lentiviral hTERT Overexpression Extends the Lifespan of Primary Human Epidermal Melanocytes and Reshapes Transcriptional Programs

Primary human epidermal melanocytes are valuable models for pigmentation research, but maintaining prolonged expansion together with stable melanocytic characteristics can be challenging. Here, primary melanocytes were transduced with an hTERT-overexpression lentiviral vector to generate a mixed population (MIX) and three single-cell-derived clones (SC1, SC3, and SC4). RNA sequencing was performed on passage-6 primary melanocytes, MIX cells, SC clones, and three melanoma cell lines (A375, WM-115, and SK-MEL-1). hTERT overexpression extended melanocyte culture lifespan by more than 10 passages. TERT expression increased in MIX cells and was highest in SC clones. Principal component and gene-expression analyses showed that MIX and SC cells remained globally closer to primary melanocytes than to melanoma cell lines, without an evident melanoma-like transcriptomic shift. However, lifespan extension and clonal selection were accompanied by marked morphological changes and broad transcriptional remodeling involving cell-cycle regulation, telomere-associated processes, antiviral responses, extracellular matrix organization, adhesion, dendrite-related programs, and pigmentation. Melanocyte differentiation and pigmentation programs were generally reduced, particularly in clonal cultures. These findings support hTERT-extended melanocytes as expandable in vitro models, while highlighting that hTERT expression and single-cell cloning can induce functional model drift.