An In Vitro Cell Model for Toxicity Studies and Pigmentation Regulation Utilizing hTERT-immortalized Neonatal Dermal Melanocytes

Skin pigmentation is a complex process; melanocytes produce melanin and package it into melanosomes that are in turn exocytosed, then endocytosed by neighboring keratinocytes. Numerous genes play roles in controlling pigmentation at various levels of melanin production. Mutations in these genes are characteristic of multiple skin disorders, including hyperpigmentation, hypopigmentation, and mixed hyper-/hypopigmentation. Additionally, extrinsic factors secreted by the surrounding resident cell types also regulate the melanin expression in melanocytes. Human primary cells can be a useful model for elucidating melanocyte biology and their toxicity to dermal agents. However, primary cells have their limitations such as donor variability and limited lifespan. Consequently, a need exists for a more robust human cell model system for the study of skin pigmentation and agents which damage melanocytes.

In this study, we immortalized primary epidermal neonatal melanocytes by expressing human telomerase reverse transcriptase (hTERT) in cells that were isolated from a neonatal donor. The immortalized primary melanocytes were cultured continuously for more than 35 population doublings without any signs of replicative senescence, yet retained melanin production. The immortalized primary melanocytes maintained a consistent expression of the melanocyte-specific marker TRP-1, and lacked expression of the fibroblast-specific marker TE7. In addition, we demonstrate the capability of these immortalized primary neonatal melanocytes to transfer melanosomes to keratinocytes utilizing a 3-D human dermal organotypic culture, and the ability to modulate melanogenesis with depigmentation agents and these agents’ effects on cell viability. Taken together, the hTERT immortalized primary neonatal melanocytes described here provide a versatile in vitro cell model for the study of toxicity and melanogenesis regulation.