Project Summary/Abstract The appearance of fatty and fibrotic infiltration in aged skeletal muscle causes a decline in muscle function and an increase in metabolic disorders. However, a means to mitigate this detrimental process remains absent, as the cellular and molecular mechanisms that lead to the intramuscular accumulation of these adverse atypical tissues are not well understood. Recently identified mesenchymal stem cells (MSCs) that occupy the interstitial space of skeletal muscle (interstitial MSCs, iMSCs) have been postulated as the foremost cellular mediators of adiposis and fibrosis. In young tissue iMSCs do not covert to these fates, but instead, remain as progenitors and are assumed to support the processes of tissue homeostasis and regeneration. We hypothesize that the fate of iMSCs determine their impact on skeletal muscle and with age, iMSCs become the adipocytes and fibrocytes that negatively impact the organism as a whole. In the studies of this proposal, we will define the role of iMSCs in intramuscular fatty and fibrotic infiltration by genetically targeting the cell in vivo and examining its developmental potential in models of aged skeletal muscle (Aim 1). We will also determine the necessity of iMSCs in skeletal muscle homeostasis and regeneration in young and aged milieus by specifically depleting iMSCs in vivo and then examining these activities (Aim 2). Finally, we will define the molecular pathways of iMSC fate progression by modeling this process in vitro and using advanced RNA sequencing technologies to reveal non-coding and coding RNA species that can functionally modulate cell fate determination (Aim 3). Our overarching goal is to elucidate the impact iMSC fate has on skeletal muscle and to leverage these findings to develop therapeutic targets to mitigate age-related skeletal muscle decline. The candidate, Dr. Michael Wosczyna, has extensive expertise in experimental progenitor cell biology and has spent more than a decade studying stem cell-related disorders. During the mentoring phase of this award, Dr. Wosczyna aims to advance his knowledge in age-related stem cell decline and in massively parallel technologies used for uncovering genetic pathways of fate determination. Dr. Wosczyna's mentor and co- mentor, Dr. Thomas Rando and Dr. Judith Campisi, respectively, have comprehensive expertise in these areas. They will provide an enriching environment for Dr. Wosczyna to develop as a prominent independent investigator in aging research.