Project Summary Neurological damage resulting from stroke is a leading cause of long-term disability in the United States with more than 795,000 people suffering a stroke annually, of which approximately 17% result in death. Unfortunately, only one drug, tissue plasminogen activator, is clinically effective with a narrow 4.5-h therapeutic window that benefits only 5% of patients. Thus, it is evident that an effective therapy is needed to minimize short- and long- term damages, and human mesenchymal stem cells (hMSC) have been shown to exhibit therapeutic effects on tissue recovery following neurological damage. This study evaluates the therapeutic effects of intra-arterially administered hMSC-derived therapy in the clinically relevant treatment window (4-6 h) targeting acute neurodegeneration using an ischemic stroke rat model. Tissue recovery, notably cerebral sodium fluctuations and corresponding metabolite changes resulting from induced transient ischemia, will be mapped temporally utilizing innovative magnetic resonance imaging and spectroscopy (MRI/S) acquisition techniques at ultra-high field. MR assessment of recovery will be correlated to behavioral and immunohistochemical analysis. Specifically, this work proposes investigation into the effects of hMSC preconditioning under 2D culture with hypoxia or dissociated cells from 3D aggregates to establish the role of 3D pre-conditioning during cellular expansion on therapeutic efficacy. Preconditioning methods aim to enhance cell robustness and secretory capacity to improve the efficacy of hMSC-based treatment for cerebral ischemia. Such preconditioning can reap benefits for transplanted cells as well as cell-derived exosomes, providing two cellular-based approaches to ischemic therapy that will help to identify and optimize the primary mechanism of hMSC impact—whether by direct cell interaction or paracrine effect. As a natural extension, exosomes derived either from hMSC or neural progenitor cells (NPC) will be evaluated for their role in secretory therapeutic effects on tissue recovery. To evaluate homing, cell-based therapies will be labeled intracellularly with an iron oxide MR contrast agent. Additionally, an innovative and novel method for tracking exosomes is being proposed utilizing a 19F labeling contrast agent composed of multifunctional fluorinated polymers to visualize and quantify exosome delivery to the ischemic infarct. Our goal is to determine if the presence of hMSC in an ischemic region is required or if delivery of cell secretions alone improves outcomes, locally or by recruiting endogenous regenerative action.