SUMMARY Human skin is the largest and one of the most complex organs in the human body. It performs diverse functions, ranging from protection, sensation, heat regulation, absorption of gases, excretion of sweat, and water resistance. Skin structure and functions gradually deteriorate with age. While there is a substantial effort towards understanding cellular mechanisms that cause cellular aging and influence the lifespan of model organisms, the molecular mechanisms of age-related alterations in human skin in vivo are barely explored. The limited number of studies on this subject conducted by bulk sequencing or metabolomics analyses of whole tissue lysates failed to provide evidence on robust transcriptional, epigenetic or metabolic alterations that drive skin aging or generate any reliable theory on the molecular determinates of these processes. We hypothesize that aging in the human epidermis affects only the discrete populations of cells with stem and progenitor features and results in the deterioration of their ability to drive self-renewal. Our preliminary data show that cycling stem cells maintain epidermal homeostasis by undergoing a profound metabolic reprograming, which determines their ability to differentiate and build the cornified barrier. We also found that the aptitude to initiate such metabolic adaptations is progressively impaired in aging stem cells, which correlates with decreased expression of certain metabolic enzymes. Consequently, we propose that exhaustion of metabolic reprogramming capacity is caused by aging of the epidermal stem cells and is highly relevant for age-related alterations in the skin. We plan in aim 1 to utilize our unique repository of primary human epidermal stem cells derived from donors of different ages and further expose the metabolic failure of elderly progenitors. We will define the transcriptional alterations underlying the defects in reprogramming from cytosolic glycolysis to mitochondrial oxidative phosphorylation. Aim two will employ human artificial skin equivalents and a recently developed total thickness human skin regeneration system as models of epidermal homeostasis and wound healing to address the functional significance of altered metabolism-associated genes identified in Aim 1. Based on these findings, we will further explore the function of the gene candidates in vivo in the mouse epidermis (in aim 3) using a highly efficient in utero gene-editing system.