Extracellular vesicle heterogeneity and its impact for regenerative medicine applications

Extracellular vesicles (EVs) are cell-derived membrane-enclosed particles that are involved in physiologic and pathologic processes. EVs are increasingly being studied for therapeutic applications in the field of regenerative medicine. Therapeutic application of stem cell-derived EVs has shown great potential to stimulate tissue repair. However, the exact mechanisms through which they induce this effect have not been fully clarified. This may to a large extent be attributed to a lack of knowledge on EV heterogeneity. Recent studies suggest that EVs represent a heterogeneous population of vesicles with distinct functions. The heterogeneity of EVs can be attributed to differences in their biogenesis, and as such, they can be classified into distinct populations that can then be further subcategorized into various subpopulations. A better understanding of EV heterogeneity is crucial for elucidating their mechanisms of action in tissue regeneration. This review provides an overview of the latest insights on EV heterogeneity related to tissue repair, including the different characteristics that contribute to such heterogeneity and the functional differences among EV subtypes. It also sheds light on the challenges that hinder clinical translation of EVs. Additionally, innovative EV isolation techniques for studying EV heterogeneity are discussed. Improved knowledge of active EV subtypes would promote the development of tailored EV therapies and aid researchers in the translation of EV-based therapeutics to the clinic

Cardiac progenitor cell-derived extracellular vesicles promote angiogenesis through both associated- and co-isolated proteins

Extracellular vesicles (EVs) are cell-derived lipid bilayer-enclosed particles that play a role in intercellular communication. Cardiac progenitor cell (CPC)-derived EVs have been shown to protect the myocardium against ischemia-reperfusion injury via pro-angiogenic effects. However, the mechanisms underlying CPC-EV-induced angiogenesis remain elusive. Here, we discovered that the ability of CPC-EVs to induce in vitro angiogenesis and to stimulate pro-survival pathways was lost upon EV donor cell exposure to calcium ionophore. Proteomic comparison of active and non-active EV preparations together with phosphoproteomic analysis of activated endothelial cells identified the contribution of candidate protein PAPP-A and the IGF-R signaling pathway in EV-mediated cell activation, which was further validated using in vitro angiogenesis assays. Upon further purification using iodixanol gradient ultracentrifugation, EVs partly lost their activity, suggesting a co-stimulatory role of co-isolated proteins in recipient cell activation. Our increased understanding of the mechanisms of CPC-EV-mediated cell activation will pave the way to more efficient EV-based therapeutics

Cardiac progenitor cell-derived extracellular vesicles promote angiogenesis through both associated- and co-isolated proteins

Extracellular vesicles (EVs) are cell-derived lipid bilayer-enclosed particles that play a role in intercellular communication. Cardiac progenitor cell (CPC)-derived EVs have been shown to protect the myocardium against ischemia-reperfusion injury via pro-angiogenic effects. However, the mechanisms underlying CPC-EV-induced angiogenesis remain elusive. Here, we discovered that the ability of CPC-EVs to induce in vitro angiogenesis and to stimulate pro-survival pathways was lost upon EV donor cell exposure to calcium ionophore. Proteomic comparison of active and non-active EV preparations together with phosphoproteomic analysis of activated endothelial cells identified the contribution of candidate protein PAPP-A and the IGF-R signaling pathway in EV-mediated cell activation, which was further validated using in vitro angiogenesis assays. Upon further purification using iodixanol gradient ultracentrifugation, EVs partly lost their activity, suggesting a co-stimulatory role of co-isolated proteins in recipient cell activation. Our increased understanding of the mechanisms of CPC-EV-mediated cell activation will pave the way to more efficient EV-based therapeutics

Size matters: Functional differences of small extracellular vesicle subpopulations in cardiac repair responses

Cardiac progenitor cell (CPC)-derived small extracellular vesicles (sEVs) exhibit great potential to stimulate cardiac repair. However, the multifaceted nature of sEV heterogeneity presents a challenge in understanding the distinct mechanisms underlying their regenerative abilities. Here, a dual-step multimodal flowthrough and size-exclusion chromatography method was applied to isolate and separate CPC-derived sEV subpopulations to study the functional differences related to cardiac repair responses. Three distinct sEV subpopulations were identified with unique protein profiles. Functional cell assays for cardiac repair-related processes demonstrated that the middle-sized and smallest-sized sEV subpopulations exhibited the highest pro-angiogenic and anti-fibrotic activities. Proteasome activity was uniquely seen in the smallest-sized subpopulation. The largest-sized subpopulation showed no effect in any of the functional assays. This research uncovers the existence of sEV subpopulations, each characterized by a distinct composition and biological function. Enhancing our understanding of sEV heterogeneity will provide valuable insights into sEV mechanisms of action, ultimately accelerating the translation of sEV therapeutics

Large-scale production of extracellular vesicles: Report on the “massivEVs” ISEV workshop

Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor-made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field

Extracellular Vesicle Heterogeneity and its Translational Implications for Cardiac Repair

Extracellular vesicles (EVs) are small vesicles naturally produced by cells to transfer bioactive content to other cells. This thesis addresses key challenges in translating EV therapeutics in the context of cardiac repair. This work encompasses multiple projects with a focus on enhancing their functional delivery, purification, preservation, and understanding of their functional heterogeneity. The study begins by demonstrating the potential of EV-liposome hybrid nanoparticles for delivering therapeutic cargo, showcasing their promise as future carriers for siRNA. The development of novel EV isolation methods is crucial for large-scale applications; a multimodal flow-through chromatography (MFC) method is introduced as a scalable alternative to conventional methods. Furthermore, preserving EV stability during concentration and storage is explored, resulting in optimized conditions that significantly boost EV recovery rates while maintaining functionality. The thesis also delves into the complexity of EV heterogeneity and its impact on their therapeutic potential. By purifying EV subpopulations using the combination of MFC and size-exclusion chromatography, differences in properties, proteomic composition, and functionality among subpopulations were unveiled, particularly in the context of cardiac regeneration. Asymmetric flow field flow fractionation is introduced as an alternative method for EV separation, offering higher yields and better preservation of EV function compared to MFC, with the added capability of subpopulation separation. Collectively, these findings provide valuable insights for advancing EV-based therapeutics. By addressing critical challenges in delivery, isolation, preservation, and understanding heterogeneity, this work contributes to the potential clinical translation of EVs. The study underscores the promise of EVs in regenerative medicine and drug delivery, offering tailored approaches to optimize their therapeutic potential. Advances in these areas will enhance the efficiency and reproducibility of EV treatments and ultimately accelerate the translation of EV-based therapeutics towards clinical application