Characterization of Extracellular Vesicles from Preconditioned Human Adipose-Derived Stromal/Stem Cells

Abstract

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.

Keywords: adipose-derived stromal/stem cells; extracellular vesicles; hypoxia; mesenchymal stromal/stem cells; proteomics; renal tubular epithelial cells.

Figure 1

Characterization of adipose-derived mesenchymal stromal/stem cells (ASCs) in normoxia and hypoxia. (A,B) Cell morphology after culture in a normoxic (A) or a hypoxic environment (1% O₂) (B) after 48 h (phase contrast microscopy, scale bar = 100 µm). (C,D) Effect of hypoxia on mRNA expression of vascular endothelial growth factor (VEGF) and insulin-like growth factor 2 (IGF2). Expression was quantified by qPCR analysis, normalized to β-actin, and calculated relative to the control using the ΔΔCT method (mean ± SD; n = 5, * p < 0.05).

Figure 2

Characterization of isolated nanoparticle tracking analyses (EVs) from ASCs in normoxia and hypoxia. (A,B) Representative nanoparticle tracking analyses (NTA) of EVs isolated from ASCs after culture in a normoxic (A) or hypoxic (B) environment for 48 h. (C) Calculated absolute average number of isolated EVs measured by NTA using a NanoSight NS500 (n = 13, ASCs cultured under normoxic conditions (nEVs); n = 9 ASCs cultured under hypoxic conditions (hEVs)). No significant differences were detected. (D) Calculated average protein content of isolated EVs (µg total protein normalized to mL conditioned medium used for EV isolation, n = 17 (nEVs), n = 10 (hEVs)). No significant differences were detected.

Figure 3

Analysis of proteomics. Six selected protein samples from EV isolations (3 nEVs versus 3 hEVs samples) were used to perform proteomic analysis via mass spectrometry. (A) Heatmap with hierarchical clustering. Forty-one human proteins were detected with different cargos between nEVs and hEVs (either >1.5 or <0.6 in the ratio hEVs/nEVs). (B,C) Proteomic analysis of hEVs. (B) Bar chart displaying the number of proteins that cluster for selected gene ontology (GO) terms for the cellular component (CC) and biological process (BP). (C) Pie chart showing the percentage of detected proteins that cluster for indicated cellular components (CC GO terms).

Figure 4

PKH26 staining of EVs and incorporation into cultured tubular epithelial cells (TECs). Fluorescence microscopy showed that TECs (nuclei stained with 4′,6-diamidin-2-phenylindol (DAPI)) uptake PKH26-labeled EVs (red). (A,B) Negative control. TECs incubated with a processed PKH26 solution without EVs ((A) nuclei staining, (B) red fluorescence channel). (C) PKH26-labeled EVs on an adhesion slide. (D–F) TECs incubated with PKH26-stained EVs clearly showing incorporation of labeled EVs into the cytosol of TECs ((D) nuclei staining, (E) red fluorescence channel, (F) overlay of (D) and (E)). Scale bar = 10 µm.

Figure 5

Effect of nEVs and hEVs on cultured TECs. (A) Cell viability after treatment with EVs. TECs were cultured with serum-free medium (M199), standard cell culture medium containing 10% fetal bovine serum (FBS) (MF10), or M199 containing nEVs or hEVs (3 × 10⁸ EVs/well, correlating to an estimated 80,000 EV-releasing ASCs) for 48 h. The 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was performed, and optical density was measured in a microplate reader at 492 vs. 650 nm (arbitrary units). Results were calculated as percent versus serum-free M199 as a control (=100%) (mean ± SD, n = 12–16). (B) Measurement of oxidative stress using 2′,7′-dichlorofluorescein diacetate (DCF-DA). TECs were cultured with serum-free medium (M199), standard cell culture medium containing 10% FBS (MF10), or M199 containing nEVs or hEVs (3 × 10⁸ EVs/well, correlating to an estimated 80,000 EV-releasing ASCs) for 48 h. Then, DCF-DA was added for 30 min at 37 °C. Fluorescence of intracellular DCF was measured and normalized to serum-free M199 (=100%; mean ± SD, n = 7–8). *** p < 0.001, * p < 0.05.