Dynamics of plasma membrane surface related to the release of extracellular vesicles by mesenchymal stem cells in culture

Abstract

Extracellular vesicles (exosomes and shedding vesicles) released by mesenchymal stem cells (MSCs) are regarded as a storable, cell-free alternative with comparable therapeutic potential to their parent cells. Shedding vesicles originate as bulges on the cell surface but little is known about their turnover or how their formation can be stimulated. We have used atomic force microscopy (AFM) to follow the formation dynamics of bulges in living adipose tissue-derived MSCs. AFM images showed that, in general, MSCs present hundreds of nanosized protrusions on their surface with life spans of 10–20 min. Scanning electron microscopy confirmed those images and showed that bulges are also formed on filamentous processes. Extracellular vesicles deposited on the culture surface have comparable sizes to those of bulges showing up on the cell surface. The amount of protrusions on cells treated with progesterone or PDGF-BB, two treatments that stimulate the secretion of extracellular vesicles in MSCs, was evaluated by AFM. Measurements of the cross-area at 50 nm over the cell surface provided estimates of the amount of protrusions and showed that these values increased with the stimulating treatments. Our study suggests that shedding vesicles constitute a large population of the extracellular vesicle pool.

A shared pathway of exosome biogenesis operates at plasma and endosome membranes

“…The model of exosome biogenesis posited here is also consistent with prior observations of exosome biogenesis via the plasma membrane (Anderson, 1969;Anderson et al, 2005;Bianchi et al, 2014;Booth et al, 2006;Cantaluppi et al, 2012;Casado et al, 2017;Fang et al, 2007;Shen et al, 2011a;Shen et al, 2011b) as well as prior observations of exosome biogenesis via endosome membranes (Colombo et al, 2014;Crenshaw et al, 2018;Desrochers et al, 2016;Harding et al, 1983;Harding et al, 1984;Mathew et al, 1995;Mathieu et al, 2019;Pan and Johnstone, 1983;Pegtel and Gould, 2019;van Niel et al, 2018). Furthermore, it adheres to the principle of maximum parsimony by offering a simpler explanation for a wider array of data than is possible under the 'endosome-only' hypothesis of exosome biogenesis.…”

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

“…Specific EV proteins are commonly detectable by immunoblotting for EV-associated proteins (e.g., ALIX, TSG101 and specific tetraspanins, such as CD63, CD9, and CD81), frequently used to confirm the presence of EVs in fluids [84,132]. The most direct method to determine EV size and morphology is electron microscopy (EM), including both transmission EM (TEM) [133,134] and scanning EM (SEM) [39,135,136]. To avoid sample dehydration, cryogenic EM techniques have been developed, of which cryogenic TEM (cryo-TEM) is the most appropriate [137] (Figure 1).…”

Immunoregulatory Effects of Stem Cell-Derived Extracellular Vesicles on Immune Cells

“…An increasing number of studies report the process of secretion of EVs by various cells (15,18). A large number of protuberances and pits on the membrane surface of viable SCs are observed to be dynamic; the protuberances may shed as microvesicles (MVs), and the pits may form when multivesicular bodies (MVBs) fuse with the plasma membrane to release exosomes (18).…”