Despite the strong evidence for the immunomodulatory activity of mesenchymal stromal cells (MSCs), clinical trials have so far failed to clearly show benefit, likely reflecting methodological shortcomings and lack of standardization. MSC-mediated tissue repair is commonly believed to occur in a paracrine manner, and it has been stated that extracellular vesicles (EVs) secreted by MSCs (EVMSC) are able to recapitulate the immunosuppressive properties of parental cells. As a next step, clinical trials to corroborate preclinical studies should be performed. However, effective dose in large mammals, including humans, is quite high and EVs industrial production is hindered by the proliferative senescence that affects MSCs during massive cell expansion. We generated a genetically modified MSC cell line overexpressing hypoxia-inducible factor 1-alpha and telomerase to increase the therapeutic potency of EVMSC and facilitate their large-scale production. We also developed a cytokine-based preconditioning culture medium to prime the immunomodulatory response of secreted EVs (EVMSC-T-HIFc). We tested the efficacy of this system in vitro and in a delayed-type hypersensitivity mouse model. MSC-T with an HIF-1α-GFP lentiviral vector (MSC-T-HIF) can be effectively expanded to obtain large amounts of EVs without major changes in cell phenotype and EVs composition. EVMSC-T-HIFc suppressed the proliferation of activated T-cells more effectively than did EVs from unmodified MSC in vitro, and significantly blunted the ear-swelling response in vivo by inhibiting cell infiltration and improving tissue integrity. We have developed a long-lived EV source that secretes high quantities of immunosuppressive EVs, facilitating a more standard and cost-effective therapeutic product.

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Bacterial eVs have been related to inter-kingdom communication between probiotic/pathogenic bacteria and their hosts. our aim was to investigate the transcytosis process of B. subtilis eVs using an in vitro intestinal epithelial cell model. In this study, using Confocal Laser Scanning Microscopy, we report that uptake and internalization of cfSe-labeled B. subtilis EVs (115 nm ± 27 nm) by Caco-2 cells are time-dependent. to study the transcytosis process we used a transwell system and eVs were quantified in the lower chamber by Fluorescence and Nanoparticle Tracking Analysis measurements. Intact EVs are transported across a polarized cell monolayer at 60-120 min and increased after 240 min with an estimated average uptake efficiency of 30% and this process is dose-dependent. EVs movement into intestinal epithelial cells was mainly through Z axis and scarcely on X and Y axis. this work demonstrates that eVs could be transported across the gastrointestinal epithelium. We speculate this mechanism could be the first step allowing EVs to reach the bloodstream for further delivery up to extraintestinal tissues and organs. the expression and further encapsulation of bioactive molecules into natural nanoparticles produced by probiotic bacteria could have practical implications in food, nutraceuticals and clinical therapies.

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