Evaluation of the pro-angiogenic effect of nanoscale extracellular vesicles derived from human umbilical cord mesenchymal stem cells

2.1. Cell culture and nanoscale extracellular vesicle preparation

HUVECs and hUC-MSCs were harvested and cultured as previously described [7,8]. After hUC-MSCs were put in contact with nanoscale particle-depleted culture medium for 48 h, nanoparticles were isolated from conditioned medium as reported before [9]. The size and concentration of nanoscale particles were assessed by nanoparticle tracking analysis (NTA, NanoSight^® LM10). Then, the nanoparticles were characterized as nEVs by flow cytometry with specific antibodies for CD9, CD63 and CD81 (BD Pharmingen) as shown elsewhere [9].

2.2. Stimulation with nanoscale extracellular vesicles derived from hUC-MSCs

HUVECs were cultured in EBM™-2 (Lonza) supplemented with 10% FBS (Fetal Bovine Serum). This condition was used as control. HUVECs were also cultured with 10% nEV-depleted FBS and stimulated by nEVs ( 10 9 or 10 10 particles/ml) or not. The uptake of nEVs by HUVECs was confirmed with confocal microscopy after the labelling of nEVs ( 10 9 particles/ml) with PKH67 and HUVECs nuclei with To-Pro^®-3. Then, in vitro capillary-network formation was monitored by performing tube-formation assays (extracellular matrix [ECM] gel solution, ScienceCell). HUVEC tube formation (number of nodes, junctions, segments and branches) was quantified by using Angiogenesis Analyzer by Gilles Carpentier for Image J (NIH).

2.3. Statistical analysis

Results are expressed by means ± SD ( n = 3 ). Differences between control and stimulated cells were analysed by ANOVA test and were considered significant with p < 0.05 .

3.1. Characterization of nanoscale extracellular vesicles

NTA has shown that the particles contained in the medium are in the size range of nEVs (30–150 nm) with a concentration higher than 10 7 particles/ml (Fig. 1(A)). The nanoparticles revealed to be nEVs by expressing the membrane markers CD9, CD63 and CD81 (Fig. 1(B)).

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Fig. 1.

Physical (A) and biological (B) characterization of nanoscale extracellular vesicles (nEVs) secreted by human umbilical cord mesenchymal stem cells. (A) Representative NanoSight^® plot and corresponding table for nanoparticle size and concentration. (B) Expression evaluated by flow cytometry of nEVs markers (CD9, CD63 and CD81) on the surface of nanoparticles. Results are means ± SD ( n = 3 ).

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Fig. 2.

(A) Representative confocal microscopy picture of nanoscale extracellular vesicles (nEVs) uptake by human umbilical vein endothelial cells (HUVECs). HUVECs nuclei were labelled with To-Pro^®-3 and nEVs with PKH67 (some of them are displayed here with white arrows). After 24 h of stimulation, nEVs were found inside HUVECs cytoplasm. (B) Effects on angiogenesis of nEVs stimulation ( 10 9 and 10 10 particles/ml) on HUVECs compared to control. Results are means ± SD, n = 3 , ∗: p < 0.05 , relative to control.

HUVECs and nEVs labelling has permitted to observe that nEVs derived from hUC-MSCs have interactions with HUVECs and are found in their cytoplasm after 24 h of stimulation (Fig. 2(A)). After 4 h of culture on ECM gel solution, the formation of capillary-like tubes by HUVECs evaluated by node, junction, segment and branch formation, seems to be increased in the presence of nEVs derived from hUC-MSCs. The lower nEVs concentration seems to promote node formation (Fig. 2(B)).