Somatic mutations distinguish melanocyte subpopulations in human skin

To better understand the homeostatic mechanisms governing melanocytes, we performed deep phenotyping of single-cell expansions of melanocytes from human skin. In total, we interrogated the mutational landscapes, gene expression profiles, and morphological features of 297 melanocytes from 31 donors. To our surprise, a population of melanocytes with low mutation burden was maintained in sun damaged skin. These melanocytes were more stem-like, smaller, and less dendritic than their counterparts with high mutation burdens. We used single-cell spatial transcriptomics (10X Xenium) to reveal the spatial distribution of melanocytes inferred to have low and high mutation burdens (LowMut and HighMut cells), based on their gene expression profiles. LowMut melanocytes were found in hair follicles as well as in the interfollicular epidermis, whereas HighMut melanocytes resided almost exclusively in the interfollicular epidermis. We propose that melanocytes in the hair follicle occupy a privileged niche, protected from UV radiation, but periodically migrate out of the hair follicle to replenish the interfollicular epidermis after waves of photodamage. More broadly, our study illustrates the advantages of a cell atlas that includes mutational information, as cells can change their cellular states and positional coordinates over time, but mutations are like scars, providing a historical record of the homeostatic processes that were operative on each cell. We used the Xenium analyzer (10X Genomics) to interrogate the localization of melanocytes with high and low mutation burdens in human skin. The Xenium platform measures spatial expression patterns across hundreds of transcripts at subcellular resolution over a large field of view (up to 1x2cm). When choosing probes, we started with a generic skin panel, designed to mark common cell lineages in the skin, and supplemented it with custom targets, which included all genes upregulated in melanocytes with high and low mutation burdens (a total of 360 genes in the panel). We chose a formalin-fixed paraffin-embedded (FFPE) sample of normal skin from the back of a 63 year old male who had been diagnosed with melanoma. The skin came from the non-lesional portion (i.e. the normal, adjacent skin) of a wide excision biopsy of the patient’s melanoma.