Mesenchymal stem cells (MSCs) are the most widely used type of cells in stem cell therapy, with over 1,000 registered clinical trials primarily focused on immunosuppression of inflammatory disease. “MSCs” are often termed “medicinal stem cells”, as they secrete multiple beneficial factors in the form of extracellular vesicles (EVs) that have trophic homing properties for sites of inflammation. However, the use of MSCs is limited by complex regulatory issues and logistics, low cell viability, and high costs. The field of cell therapy has now been shifting to the production of EVs as a “cell-free” alternative. Similar to tracking whole cells, it will be imperative to be able to track the biodistribution of these therapeutic EVs in vivo in order to interpret and improve therapeutic outcome. We propose to develop two types of labeled MSC-derived EV formulations and will compare their in vivo imaging (MRI and MPI) and therapeutic properties to unmodified, naïve EVs, that we hypothesize may still be trackable with CEST MRI by virtue of their mannose-rich membrane composition. Based upon the existing clinical trials using MSCs in patients with multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), we will evaluate our MSC-EV formulations in vivo using an experimental autoimmune encephalomyelitis (EAE) MS mouse model and a superoxide dismutase-decificient transgenic ALS mouse model. To this end, we propose the following specific aims: AIM 1: To develop magnetically labeled MSC-EVs as a bimodal negative MRI contrast agent and magnetic particle imaging (MPI) tracer. We will use electroporation to load EVs with superparamagnetic iron oxides (SPIOs). Unlike with MRI, the number of MSC- SPIO-EVs can be quantified with MPI. AIM 2: To develop gadolinium-labeled MSC-EVs as positive MRI contrast agent. We will use lipophilic gadolinium chelates and lipid insertion techniques to render EVs with paramagnetic membranes. AIM 3: To develop unlabeled MSC-EVs as diaCEST MRI contrast agent. We will take advantage of the natural expression of high-mannose type N glycans on EV membranes which provide contrast on mannose-weighted CEST MRI, without the need of further vesicle manipulation. AIM 4: To track the in vivo distribution and therapeutic outcome of labeled and unlabeled EVs in a mouse models of EAE and ALS. This will serve as an internal JHU validation study. We will investigate various administration routes and use standard behavioral scoring tests to correlate the diagnostic and therapeutic outcome measures of our nanotheranostic approach. We will compare and validate all three MSC-EV imaging strategies in terms of sensitivity, specificity, and EV functionality. Upon proper synthesis, characterization, and in vivo validation, we will disseminate our SPIO-MSC-EVs, Gd-MSC-EVs, and unlabeled EVs to the CPS, SPs, and beyond.