Speaker
Description
"Introduction. Extracellular vesicles (EVs) are involved in a plethora of physiological and pathophysiological contexts, and their potential regenerative applications have attracted special interest. Ease of autologous isolation, low immunogenicity and lack of reproductive potential are only some of the enticing characteristics that turn the spotlight increasingly towards EV-based therapy. However, too many unknowns regarding the biology of EVs remain. Within the scope of our research, we focus on peripheral nerve regeneration, where we challenge the gold standard of autologous reconstruction with alternative therapeutic approaches, including EVs. Schwann cells (SCs) have been ascribed an essential role in nerve repair. Their response to nerve damage includes debris clearance, attraction of macrophages, and providing structural and trophic support for the regrowing axon. In this study, adipose tissue derived stem cells (ASCs) serve as a source for EVs. Here, we follow the journey of ASC-EVs to the recipient SCs and decipher how they are able to transmit their multifaceted signals and actuate downstream processes, including proliferation.
Methods. EVs were isolated by differential ultracentrifugation and characterized according to the MISEV guidelines. Imaging flow cytometry (IFC) allowed immunophenotyping with a single-vesicle resolution, while nanoparticle tracking analysis (NTA) was used to determine concentration and size distribution. The entire SC – ASC-EV interaction was observed with live-cell imaging (LCI), from initial contact to subsequent internalization and perinuclear translocalization, which was confirmed with 3D image reconstructions of high-resolution confocal micrographs. Scanning electron microscopy enabled us to elucidate the initial contact in detail, while transmission electron microscopy granted us a closer look at the vesicle transit through the cellular membrane. We further broke down the membrane transit on a molecular level by pairing well-established pharmacological inhibitors of major endocytotic mechanisms with state-of-the-art IFC. The cellular response to ASC-EV treatment was quantified via EdU incorporation during DNA synthesis.
Results. Upon initial contact with SCs, ASC-EVs were moved along the membrane until they were internalized and subsequently transported towards the cell’s nucleus, where they were accumulated. The inhibition of specific endocytosis pathways revealed that in SCs, the internalization of ASC-EVs is mainly mediated by clathrin, though alternative modes of membrane transit are likely involved, as no complete block of ASC-EV-internalization could be achieved. Upon internalization of ASC-EVs, we observed an increase in SC proliferation in a time- and dose-dependent manner, up to 2.5-fold compared to untreated SCs within 72h.
Conclusions. We established that ASC-EVs can enhance proliferation in SCs, crucial for peripheral nerve regeneration. This response is activated upon internalization of ASC-EVs. We identified the major mode of internalization, however, alternative modes of internalization likely involved. The potential therapeutic application of EVs necessitates understanding the underlying processes, especially the interaction with target cells. Our investigations provide a deeper understanding of the cellular signal transduction during peripheral nerve regeneration upon stimulation with ASC-EVs and adds to the knowledge needed to harness the full potential of EVs for therapeutic purposes."
41883636477
