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Mesenchymal stem/stromal cells (MSC) have displayed promising therapeutic potential. Nonetheless, no United States Food and Drug Administration (FDA)-approved MSC product exists due largely to the absence of a reliable potency assay based on the mechanisms of action to ensure consistent efficacy. MSCs are now thought to exert their effects primarily by releasing small extracellular vesicles (sEVs) of 50-200 nm. While non-living MSC-sEV drugs offer distinct advantages over larger, living MSC drugs, elucidating their mechanism of action to develop robust potency assays remains a challenge. A pivotal prelude to elucidating the mechanism of action for MSC-sEVs is how extracellular vesicles (EVs) engage their primary target cells. Given the inherent inefficiencies of processes such as endocytosis, endosomal escape and EV uncoating during cellular internalization, we propose an alternative EV-cell engagement: EMCEV (Extracellular Modulation of Cells by EV). This approach involves extracellular modulation by EV attributes to generate signaling/inhibitory molecules that have the potential to affect many cells within the vicinity, thereby eliciting a more widespread tissue response., Competing Interests: Conflict of Interest Statement The authors declare no conflicts of interest., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem/stromal cell-derived small extracellular vesicles (MSC-sEVs) are promising therapeutic agents. In this study, we investigated how the administration route of MSC-sEVs affects their therapeutic efficacy in a mouse model of bleomycin (BLM)-induced skin scleroderma (SSc). We evaluated the impact of topical (TOP), subcutaneous (SC), and intraperitoneal (IP) administration of MSC-sEVs on dermal fibrosis, collagen density, and thickness. All three routes of administration significantly reduced BLM-induced fibrosis in the skin, as determined by Masson's Trichrome staining. However, only TOP administration reduced BLM-induced dermal collagen density, with no effect on dermal thickness observed for all administration routes. Moreover, SC, but not TOP or IP administration, increased anti-inflammatory profibrotic CD163 + M2 macrophages. These findings indicate that the administration route influences the therapeutic efficacy of MSC-sEVs in alleviating dermal fibrosis, with TOP administration being the most effective, and this efficacy is not mediated by M2 macrophages. Since both TOP and SC administration target the skin, the difference in their efficacy likely stems from variations in MSC-sEV delivery in the skin. Fluorescence-labelled TOP, but not SC MSC-sEVs when applied to skin explant cultures, localized in the stratum corneum. Hence, the superior efficacy of TOP over SC MSC-sEVs could be attributed to this localization. A comparison of the proteomes of stratum corneum and MSC-sEVs revealed the presence of >100 common proteins. Most of these proteins, such as filaggrin, were known to be crucial for maintaining skin barrier function against irritants and toxins, thereby mitigating inflammation-induced fibrosis. Therefore, the superior efficacy of TOP MSC-sEVs over SC and IP MSC-sEVs against SSc is mediated by the delivery of proteins to the stratum corneum to reinforce the skin barrier.

Cell-based therapy using Mesenchymal Stromal Cell (MSC) has generally been efficacious in treating a myriad of diseases in animal models and clinical trials. The rationale for MSC therapy was predicated on the potential of MSC to differentiate and form new replacement cells in the diseased tissue. However, pre-clinical animal and clinical data were more consistent with a secretion- and not a differentiation-based rationale. Analysis of MSC secretion led to the identification of small extracellular vesicles (sEVs) as therapeutically active, secretory agents. MSC-sEVs are defined as bi-lipid membrane vesicles of 50-200 nm in diameter that are secreted by MSCs. They reportedly exert similar therapeutic efficacy as MSCs in many diseases including neurological diseases. MSC-sEVs being small and non-living are intrinsically safer than living MSCs. Manufacturing of MSC-sEVs may also be less complex. Nevertheless, realising the therapeutic potential of MSC-sEVs will require exacting scientific rigor and robustness, as well as compliance to regulatory oversight. This review summarises the scientific rationale for the transition of MSC therapy from a cell- to an EV-based therapy and discusses critical scientific issues in the development of MSC-sEVs therapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mesenchymal-stem/stromal-cell-derived small extracellular vesicles (MSC-sEV) have been shown to ameliorate many diseases in preclinical studies. However, translating MSC-sEV into clinical use requires the development of scalable manufacturing processes for highly reproducible preparations of safe and potent MSC-sEVs. A major source of variability in MSC-sEV preparations is EV producer cells. To circumvent variability in producer cells, clonal immortalized MSC lines as EV producer lines are increasingly being used for sEV production. The use of sEVs from immortalized producer cells inevitably raises safety concerns regarding the tumorigenicity or tumor promoting potential of the EV products. In this study, cells from E1-MYC line, a MSC cell line immortalized with the MYC gene, were injected subcutaneously into athymic nude mice. At 84 days post-injection, no tumor formation was observed at the injection site, lungs, or lymph nodes. E1-MYC cells pre-and post-sEV production did not exhibit anchorage-independent growth in soft agar. Daily intraperitoneal injections of 1 or 5 μg sEVs from E1-MYC into athymic nude mice with FaDu human head and neck cancer xenografts for 28 days did not promote or inhibit tumor growth relative to the xenograft treated with vehicle control. Therefore, MYC-immortalized MSCs are not tumorigenic and sEVs from these MSCs do not promote tumor growth. [ABSTRACT FROM AUTHOR]