Abstract
Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioactivity is most often attributed to their protein and nucleic acid components, while the function of exosomal lipids remains comparatively unexplored. This work specifically assesses the involvement of lipids and the transmembrane enzyme CD73 in the exosomes’ biological activity in stimulating the cutaneous wound healing process. Since exosome preparation processes are not harmonized yet, certain production and purification parameters are first systematically investigated, enabling the optimization of a standardized protocol delivering high exosome integrity, yield, and purity. An in situ enzymatic assay is introduced to specifically assess the vesicle functionality, and quantitative proteomics is employed to establish the cell culture conditions yielding a stable exosome protein profile. Using a combination of in vitro approaches, CD73 and constitutional lipids of HPV‐16 E6/E7 transformed human bone marrow stromal cell‐derived exosomes are identified as key bioactive components promoting the exosome‐driven acceleration of processes required for wound repair. A pilot wound healing study in mice indeed suggests a role of lipids in the exosomes’ biological activity. Strikingly, the extent of the bioactivity of these exosomal components is found to be dependent on the target cell type.
Highlights
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Exosomes were produced by HS-5 cells for 24 h under serum-free conditions in order to obviate a possible contamination with serumderived components such as extracellular vesicles (EV), lipids, and RNA.[1,23]
As measured by nanoparticle tracking analysis (NTA), the UC-isolated vesicles had a modal diameter of 139 ± 6 nm, while those obtained by UF-size exclusion chromatography (SEC) were smaller, measuring 121 ± 6 nm (Figure 1A,B)
Summary
The purity and functionality of exosomal formulations is highly dependent on the exosome isolation method.[20,21,22] In order to obtain a high yield of enzymatically active exosomes, which can be reproducibly used for in vitro and in vivo wound healing studies, the HPV-16 E6/E7 transformed human bone marrow mesenchymal stromal cell line HS-5 was selected for a detailed investigation of two common purification strategies. The vesicles harvested by either isolation method exhibited a distinct cup-shaped morphology, which has been previously reported as an artefact of the preparatory fixation process required for TEM.[23]. Immunoblotting revealed that both isolates were enriched in the exosome marker proteins TSG101, CD9, CD63, and CD73 (Figure 1C). The CD73 enzymatic activity following UC isolation was tenfold higher than that after UF-SEC (4.4 ± 0.7 U mg−1 for UC vs 0.4 ± 0.1 U mg−1 for UF-SEC, whereby 1 unit (U) is defined as 1 μmol min−1) This could indicate a partial activity loss upon the UF-SEC procedure or, in line with Western blot analysis, a lower purity of the UF-SEC sample (i.e., less CD73 per isolated μg of protein). In contrast to UF-SEC, UC was able to purify enzymatically active HS-5 exosomes in a more reproducible fashion, and was retained as purification strategy in subsequent experiments
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