SUMMARY The loss of tissue homeostasis and regenerative capacity with age underlies some of the most challenging health issues in the elderly. A major contributor to age-related declines in the structure and function of many tissues is the loss of stem cell function that occurs during the aging process. To slow, arrest, or even reverse those age-related declines in stem cell function would represent a major therapeutic success based upon advances in both stem cell biology and the biology of aging. The primary focus of this Program is to understand the molecular basis of age-related changes in stem cell function. An underlying premise is that such an understanding will reveal ways to enhance aged stem cell function and tissue repair. Based on many studies, including innovative work from the laboratories of this Program, there is increasing evidence of dysfunctional genetic, epigenomic, transcriptional, and metabolic changes in stem cells with age. A central concept that is woven throughout this Program is that systemic factors may mediate some of these changes, and that, conversely, cell-extrinsic influences may restore youthful properties to aged stem cells. Another unifying concept is how changing genetic and epigenetic diversity in stem cell populations combine with age-associated alterations in their niches to create an ever changing adaptive landscape, one that may select for subsets of stem cells that are actually suboptimal for tissue homeostasis and repair. To examine those concepts experimentally, this Program includes Projects that focus on stem cell populations from three tissues – muscle (Project 1), brain (Project 2), and blood (Project 3). In the current proposal, we extend our previous studies along several themes that are shared among the Projects. These themes include changes in stem cell heterogeneity with age, the role of metabolic changes as determinants of the aged epigenomic state, and the impact of rejuvenating interventions on stem cells and their niches. The Projects are supported by three essential Cores – an Administrative Core; a Single Cell and Spatial Proteomics Core, which includes cutting-edge spatial proteomic technologies; and a Bioinformatics Core both to serve as the critical hub for storing, processing, visualizing, analyzing, and sharing data from the three Projects, and also to develop pioneering spatial transcriptomics. Overall, the investigators who are Project Leaders and Core Directors are internationally recognized experts in their respective fields. They bring to the Program the full breadth of expertise, creativity, and records of accomplishment to assure an integrated, innovative, and highly successful Program to explore the molecular mechanisms of stem cell aging and rejuvenation. The successful completion of the studies of this proposal has the potential to advance therapeutics to enhance tissue homeostasis and repair in elderly individuals.