Methylation levels in keratinocyte basal cells reflect donor age and associate with altered cellular proliferation pathways

To determine if epigenomic and mRNA associations with donor age are present in basal keratinocytes <i>in vitro</i>. Whole-genome methylation (EPIC array), RNAseq analysis, and in vitro cell growth assessments were performed on cultured keratinocytes (<i>n</i> = 9, 22–68 years), enriched for cells expressing the stem cell protein markers ITGB1 and EpCAM. Donor age associated with 1,244 differentially methylated positions (DMPs) (<i>p</i> &lt; 0.01, &gt;20% delta beta) and correlated with estimated ages from the Skin and Blood epigenetic clock (<i>r</i> = 0.83, <i>p</i> = 0.0018). The DMPs correlated with those in an <i>in vivo</i> epidermal dataset (<i>r</i> = 0.71, <i>p</i> &lt; 0.0001). Donor age associated (<i>p</i> &lt; 0.05) with 523 differentially expressed genes (DEGs), but the DEGs only weakly correlated with their changes in an <i>in vivo</i> epidermal dataset (<i>r</i> = 0.24, <i>p</i> &lt; 0.0001). The “cell growth and proliferation” ontology term was significantly enriched in the methylation and expression datasets despite only 13 overlapping annotated genes. Decreased keratinocyte proliferation, increased differentiation and a reduced re-epithelialization ability were observed for an older versus a younger cell strain. Basal keratinocytes maintain <i>in vivo</i> age-associated epigenomic changes <i>in vitro</i> making them a good model for studying the impact of age-associated epigenomic changes on cellular function and behavior <i>in vitro</i>.