Regenerative therapy based on the use of mesenchymal stem cells (MSC) is a promising approach for the treatment of stroke. The beneficial effects of MSC appears to be mainly related to the secretion of cellular factors and/or extracellular vesicles (EV). Heparan sulfates present on the surface of producing and recipient cells as well as on EV could play a critical role in the EV-mediated communication. The MAESTROVE project will explore, through the use of innovative approaches and models developed by 4 partners, the ability of combining human MSC-derived EV with a HS mimetic (HSm) agent, i.e. OTR4132, to enhance EV-mediated tissue regeneration and functional recovery following stroke, thus opening a new and rapid perspective for a development more easily industrialized for the treatment of stroke. The research hypothesis of the project lies on the fact that OTR4132 will create/restore a favourable tissue environment in which MSC-derived EV will be satisfactorily trapped and thus exert their beneficial effects in an optimal manner in the damaged brain tissue after stroke. Moreover, in vitro MSC priming with OTR4132 could improve EV biogenesis, cargo composition and regenerative properties. Combining HSm-based matrix therapy and MSC-derived EV therapy has never been studied. Another originality of this project is to test a priming strategy of MSC with this HSm-based matrix therapy to improve EV cargo constitution. This project will also evaluate for the first time a high-yield and scalable turbulence EV production approach for post-ischemic stroke treatment. This therapeutic strategy will be tested in relevant animal models of stroke towards the clinic, including the integration of the main comorbidity factor, i.e. the pre-existence of chronic arterial hypertension and an original non-human primate model. Overall, this project aims to provide an improved EV-based therapy that could represent a new clinically cell-free feasible paradigm for stroke. MAESTROVE gathers 4 partners with complementary expertise, most of them collaborating for a long time together with publications, patents and joint funding including 3 academic laboratories (Partner 1: ISTCT unit, Partner 3: Gly-CRRET unit and Partner 4: MSC unit) and 1 SME (Partner 2: OTR3). This project is subdivided into 5 Work Packages with WP1 for project management; WP2 for EV production by turbulence and characterization; WP3 for in vitro potency studies of EV (derived from MSC primed or not with OTR4132) in association or not with OTR4132 on neuronal, glial and endothelial cells survival submitted to an ischemic-like stress; WP4 for effects of combined therapies (OTR4132/EV) in different stroke models in the rat and marmoset; WP5 for effects of combined therapies (OTR4132/EV) on endogenous GAG modifications induced by stroke and blood markers identification of treatment efficacy. The MAESTROVE project proposes to develop a new therapy concept with the combination of OTR4132 and EV produced by human MSC. The success of the project is strongly supported by the proven effectiveness of OTR4132 and combined OTR4132/MSC in stroke tissues. Partner 2 has exclusive and worldwide rights for the engineering of matrix agents (RGTA®, ReGeneraTing Agents including OTR4132) and its applications in nervous system pathologies. Besides, the development of GMP EV production by turbulence from human MSC is on ingoing for a future clinical via a bioproduction start-up (EverZom) exploiting the patent from Partner 4.