Synergy of Human Platelet-Derived Extracellular Vesicles with Secretome Proteins Promotes Regenerative Functions

Abstract

Platelet-rich plasma is a promising regenerative therapeutic with controversial efficacy. We and others have previously demonstrated regenerative functions of human platelet lysate (HPL) as an alternative platelet-derived product. Here we separated extracellular vesicles (EVs) from soluble factors of HPL to understand the mode of action during skin-organoid formation and immune modulation as model systems for tissue regeneration. HPL-EVs were isolated by tangential-flow filtration (TFF) and further purified by size-exclusion chromatography (SEC) separating EVs from (lipo)protein-enriched soluble fractions. We characterized samples by tunable resistive pulse sensing, western blot, tandem mass-tag proteomics and super-resolution microscopy. We evaluated EV function during angiogenesis, wound healing, organoid formation and immune modulation. We characterized EV enrichment by TFF and SEC according to MISEV2018 guidelines. Proteomics showed three major clusters of protein composition separating TSEC-EVs from HPL clustering with TFF soluble fractions and TFF-EVs clustering with TSEC soluble fractions, respectively. HPL-derived TFF-EVs promoted skin-organoid formation and inhibited T-cell proliferation more efficiently than TSEC-EVs or TSEC-soluble fractions. Recombining TSEC-EVs with TSEC soluble fractions re-capitulated TFF-EV effects. Zeta potential and super-resolution imaging further evidenced protein corona formation on TFF-EVs. Corona depletion on SEC-EVs could be artificially reconstituted by TSEC late fraction add-back. In contrast to synthetic nanoparticles, which commonly experience reduced function after corona formation, the corona-bearing EVs displayed improved functionality. We conclude that permissive isolation technology, such as TFF, and better understanding of the mechanism of EV corona function are required to realize the complete potential of platelet-based regenerative therapies.

A functional corona around extracellular vesicles enhances angiogenesis, skin regeneration and immunomodulation

“…As a proof of concept, we visualised the corona formation by super‐resolution microscopy using fluorescent albumin and fluorescent VEGF. During the review process of this study, we also demonstrated in a separate publication (Gomes et al., 2022) that loss of function of EVs after SEC can be fully reverted by adding the SEC late fraction. Corona formation was indicated by means of fluorescent albumin as a model protein (Gomes et al., 2022).…”

“…For statistical analysis, three fields of view were acquired for three independent donors. To visualise the protein corona around EVs, EV TUCF coated with a fluorescent albumin‐AF488 or VEGF‐CF488A corona were stained with tetraspanin CD9, (R&D Systems, USA) CD63, (Becton Dickinson, USA) and CD81 (R&D Systems, USA), all conjugated to Alexa flour 647 (tetramix) as published (Gomes et al., 2022). Quantification of colocalizations was performed as reported with a limit of 150 nm radius and five localizations for quantifying an event as colocalization.…”

IL-10 partly mediates the ability of MSC-derived extracellular vesicles to attenuate myocardial damage in experimental metabolic renovascular hypertension

“…The immunomodulatory effects of EVs with or without IL-10 were evaluated in stimulated T-cells ( 21 – 23 ). Fresh human CD3+ Pan T-cells (4W-350, Lonza, Walkersville, MD, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (10-040-CM, Corning Life Sciences, Corning, NY, USA) supplemented with 10% FBS for 6hrs after been thawed.…”

Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles

“…The application of size exclusion chromatography is prevalent across laboratories worldwide (Royo et al, 2020) due to its gentle nature and efficiency for eliminating contaminating materials (Baranyai et al, 2015;Guan et al, 2020;Lobb et al, 2015;Stam et al, 2021). Based on the growth in the use of isolation techniques that preserve EV integrity and prevent EV aggregation (Royo et al, 2020), as well as recent reports of the application of tangential flow filtration (Andrade et al, 2021;Binder et al, 2021;Busatto et al, 2018;El Baradie et al, 2020;Haraszti et al, 2018;Heinemann et al, 2014) coupled with size exclusion chromatography (Coenen-Stass et al, 2019;Corso et al, 2017;Gomes et al, 2022;Mcnamara et al, 2018;Nordin et al, 2019;Wolf et al, 2022), it can be anticipated that this combinatorial method may become standard for many laboratories in the near future.…”