Molecular heterogeneity of quiescent melanocyte stem cells revealed by single-cell RNA-sequencing.

Melanocyte stem cells (McSCs) of the hair follicle are a rare cell population within the skin and are notably underrepresented in whole-skin, single-cell RNA sequencing (scRNA-seq) datasets. Using a cell enrichment strategy to isolate KIT+/CD45- cells from the telogen skin of adult female C57BL/6J mice, we evaluated the transcriptional landscape of quiescent McSCs (qMcSCs) at high resolution. Through this evaluation, we confirmed existing molecular signatures for qMcCS subpopulations (e.g., Kit+, Cd34+/-, Plp1+, Cd274+/-, Thy1+, Cdh3+/-) and identified novel qMcSC subpopulations, including two that differentially regulate their immune privilege status. Within qMcSC subpopulations, we also predicted melanocyte differentiation potential, neural crest potential, and quiescence depth. Taken together, the results demonstrate that the qMcSC population is heterogenous and future studies focused on investigating changes in qMcSCs should consider changes in subpopulation composition. Follicular melanocyte stem cells reside in the cellular state of quiescence for the majority of their lifespan and are essential for the regeneration of hair pigmentation during hair cycling. Functional, anatomical and molecular evidence indicates that quiescent melanocyte stem cells are heterogenous and this heterogeneity is a characteristic that defines the stem and lineage potential of these cells. To further interrogate the transcriptional programs that drive this heterogeneity we isolated quiescent melanocyte stem cells from the dermis of female, 8-week-old, C57BL/6J mice (n=3) using KIT+/CD45- enrichment using flow cytometry. Isolated quiescent melanocyte stem cells were processed for single cell RNA-seq (10X genomics) to generate a high resolution map of these cells (5545 total cells analyzed after quality control and filtering). We also performed single cell RNA-seq on dissociated dermis without enrichment as a comparison.