Hacking Extracellular Vesicles: Using Vesicle-Related Tags to Engineer Mesenchymal Stromal Cell-Derived Extracellular Vesicles

Abstract

Background/Objectives: Extracellular Vesicles (EVs) have shown great promise as diagnostic and therapeutic tools, as well as pharmacological nanocarriers. Various strategies are being explored to develop EVs for monitoring, imaging, loading with pharmacological agents, and surface decoration with tissue-specific ligands. EVs derived from Mesenchymal Stromal Cells (MSC-EVs) are of particular interest both as therapeutics per se and as natural nanocarriers for the targeted delivery of biotherapeutics. Methods: In this study, we investigated the ability of different tags to deliver a reporter protein into canine MSC-EVs with the aim of identifying the most effective endogenous loading mechanism. To this aim, canine MSCs were engineered to express the Green Fluorescent Protein (GFP) fused to CD63, Syntenin-1, TSG101, and the palmitoylation signal of Lck, which were expected to promote GFP incorporation into EVs. Overexpression of tagged GFP in canine MSCs was confirmed by Western blotting and examined by confocal microscopy and transmission electron microscopy to map intracellular localization. Results: All tags were able to deliver GFP into EVs. Syntenin-1 showed relatively high loading efficiency and secretion index but exhibited a diffuse localization pattern in the transfected cells. The palmitoylation signal showed low loading efficiency and localization specificity. TSG101 displayed a morphological pattern consistent with specific localization in endosomal structures, but its low expression level prevented further evaluations. Finally, CD63 showed the highest expression efficiency, as GFP-CD63 levels were approximately 5-fold higher than untagged GFP. Conclusions: In conclusion, CD63 emerged as the most suitable tag for canine MSC-EV engineering. Indeed, even if the secretion index favours Syntenin-1, CD63's higher abundance in the lysate suggests its substantial post-secretion uptake. Further studies aimed at elucidating CD63's specific contribution and identifying the domains involved in vesicle trafficking could provide valuable insights into EV bioengineering.

Keywords: CD63; EVs engineering; GFP; Mesenchymal Stromal Cells; Palmitoylation; Syntenin; TSG101; extracellular vesicles; transfection.

Figure 1

Fibroblast-like morphology of c-Ad-MSCs in culture. Contrast-phase microscopy (40× magnification).

Figure 2

Schematic maps of the sequences encoding GFP-tagged proteins. Top: The sequence encoding the palmitoylation signal was ligated into the KpnI restriction site of pTagGFP2-N. cDNA from canine RNA was ligated into the SalI restriction site of pTagGFP2-N. Bottom: Gel electrophoresis of PCR products from the amplification of canine cDNA. Grey arrows: cDNA coding sequences. Green arrows: GFP coding sequence. White arrow: synthetic oligo for Lck palmitoylation motif. MGCSCSSNPE: amino acid sequence of Lck motif. KpnI and SalI restriction enzymes used for cloning process.

Figure 3

Western blotting for the detection of GFP-tagged proteins in c-Ad-MSC lysate and c-Ad-MSC-derived EVs. The anti-GFP immunoblotting assay in the upper panels shows the different molecular sizes of the chimeric proteins. a: untagged GFP (27 kDa); b: palmitoylated GFP (29–30 kDa); c1: CD63-GFP (65–75 kDa), polyubiquitylated forms; c2: CD63-GFP (60–75 kDa), polyubiquitylated forms; d1: Syn-GFP (60 kDa), full length; d2: Syn-GFP (50–60 kDa), full length and clavated or short form; e: untagged/cleaved GFP. Control positive markers of EVs: TSG101 (49 kDa) and Alix (95 kDa). Control negative markers of EVs: Mitofilin (75 kDa). Loading control: b-Tubulin (50 kDa). Palm-GFP: Palmitoylated GFP, Syn-GFP: Syntenin-1-GFP, UT: not transfected, Mw: molecular weight.

Figure 4

Ratio between secreted and intracellular tagged GFP. (a)—Relative amount of GFP content in cell lysates of c-Ad-MSC overexpressing tagged proteins and GFP content in isolated EVs, calculated based on Western blotting. Tagged GFP was normalized to untagged GFP. GFP in cells and EVs was normalized to bTubulin. (b): Graphical representation of the ratio between secreted GFP and intracellular GFP. (Palm-GFP: palmitoylated GFP, Syn-GFP: Syntenin-1-GFP). p-value * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n.s. > 0.05.

Figure 5

CD63-GFP localization. Confocal and TEM micrographs of transfected c-Ad-MSCs overexpressing CD63-GFP. Cells were labelled for lysosomes and other acidic compartments, including early and late endosomes (Lysoview—dark blue), for nuclei (Hoechst—light blue), and for endoplasmic reticulum (ER-ID Red Assay—red). (a) CD63–GFP displayed a focal distribution (green) involving compartments of different sizes. (b) A higher magnification view of the boxed region in (a) reveals large (asterisks) and small (arrowheads) compartments showing CD63–GFP localization. (c) A weak surface distribution of CD63–GFP was also observed (green). (d) A higher magnification view of the boxed region in (c). In (e), a large compartment is shown (asterisk), mainly reactive along the boundary. The inset shows a magnified view of the area outlined by the square. In (f), a late endosome with positive vesicles is visible in the lumen, as shown by the presence of black gold dots. In (g), a small endosome with gold particles mainly observed at the periphery (white arrowhead). In (h), positive vesicles are emerging from the cell surface. Scale bars: (a–c) 50 μm, (b–d) 10 μm, (e) 2 μm, (e) (inner panel) 100 nm, (f–h) 200 nm.

Figure 6

Localization of Syntenin-1-GFP. Confocal and TEM micrographs of transfected c-Ad MSCs overexpressing Syn-GFP. Syn-GFP showed a diffuse distribution in the cytoplasm (a), at the periphery of intracellular vesicular compartments (b), and on cell surface protrusions (c). Panels (b) and (c) show higher magnification views of the boxed regions in (a). Immunogold labelling showed a diffuse distribution in the cytoplasm (d,e) and a superficial focal distribution (f). Scale bars: (a) 50 μm, (b,c) 10 μm, (d) 500 nm, (e,f) 200 nm.

Figure 7

Localization of TSG101-GFP. Confocal micrographs of transfected c-Ad-MSCs overexpressing TSG101-GFP. Nuclei (blue) and endoplasmic reticulum (red) were labelled. TSG101-GFP shows a multifocal pattern in the form of discrete cytoplasmic small spots. Panels (c) and (d) show higher magnification views of the boxed regions in (a) and (b). Scale bars: (a,b) 50 μm, (c,d) 10 μm.

Figure 8

Localization of Palm-GFP. Confocal and TEM micrographs of transfected c-Ad MSCs overexpressing Palm-GFP. Cells were labelled for nuclei (light blue), endoplasmic reticulum (red), and lysosomes (dark blue). Palm-GFP showed a slightly diffuse distribution in the cytoplasm, together with a recurrent strong paranuclear signal, negative for ER or lysosome staining, compatible with the Golgi apparatus (arrowheads) (a,b). Panel (c) shows higher magnification view of the boxed region in (a) showing diffuse distribution of the construct. The same field shown in (b) is presented in (d), highlighting the area outlined by the square and imaged using only the Lysoview filter.Immunogold labelling showed surface-positive protrusions for Palm-GFP (e) and intracellular diffuse distribution (f). Scale bars: (a,b,d) 50 μm, (c) 10 μm, (e,f) 500 nm.