Herein, the interaction between these cells needed further investigation. Conclusions Overall, although the number of EVs secreted from hp-MSCs Rocuronium was not changed by 5 Gy -ray exposure, EVs from irradiated hp-MSCs caused damage to HUVEC and H9c2 cells. indicated many upregulated or downregulated miRNAs in irradiated MSCs EVs. In vitro experiments using HUVEC and H9c2 cells showed that irradiated MSC-EVs decreased cell proliferation ( 0.01), but increased cell apoptosis and DNA damage. Moreover, irradiated MSC-EVs impaired the HUVEC tube formation and induced calcium overload in H9c2 cells. Conclusions EVs released from irradiated MSCs show altered miRNA profiles and harmful effects on heart cells, which provides new insight into the mechanism of radiation-related heart disease risks. at 4C. Cells treated with 3% formaldehyde in a buffer for 30 min were included as a positive control. The cell pellets were suspended with 100 l cold D-PBS and then added 5 l of Annexin V-FTIC solution, and 2.5 l dissolved PI were added as described in the manual (Beckman Coulter). The samples were kept on ice and incubated for 10 min in the dark. Finally, 400 l ice-cold 1 binding buffer was added to the samples for further experiments. Flow cytometry analysis was performed using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). The acquired data were analyzed using Cell Quest software (Becton Dickinson). Tube formation Corning? Matrigel? Matrix (356230) was thawed Rocuronium overnight on the ice at 4C according to the guidelines in the manual. All pipets and procedures were previously kept on ice. Then, 289 l chilled Corning Matrigel? matrix into 24-well culture plates to avoid air bubbles. Plates were incubated at 37C for 30C60 min. The medium remaining was removed carefully without disturbing the matrix layer, and the plates were ready to use. HUVEC cells were previously cocultured with 10 g/ml non-irradiated-EVs or irradiated-EVs for 48 h ( 0.05. Results Characterization of hp-MSCs and hp-MSC EVs Primarily expanded hp-MSCs exhibited a fibroblast-like morphology (Fig. ?(Fig.1A)1A) and were identified as the biological properties of MSCs according to their expression pattern on the cell surface markers CD44, CD105, CD90, CD73, CD45, and CD34 (Fig. ?(Fig.11 B, C). To investigate the impact of IR on the EVs secretion, hp-MSCs (passaged 2C5) were exposed to 5 Gy -rays and the medium was collected 48 h later for EVs isolation by ultracentrifugation. The successful isolation of EVs was confirmed by electron microscopy (Fig. ?(Fig.11 D), nanoparticle track analysis (Fig. ?(Fig.1E),1E), and Western blot analysis of the expression of membrane markers of CD63 and TSG101 (Fig. ?(Fig.1F).1F). The size distribution (Fig. ?(Fig.1E)1E) and protein concentration (Fig. ?(Fig.1G)1G) were not obviously different between the EVs from the non-irradiated and irradiated MSCs. These data indicated very limited changes in the amount and size distribution of EVs from hp-MSCs within 48 h after exposure to 5 Gy -rays. Open in a separate window Fig. 1 Characterization of hp-MSCs and hp-MSC EVs. A Human placental tissue-derived mesenchymal stem cells (hp-MSCs) displayed identical fibroblast morphology. Representative images are shown. Scale bar: 200 m. Representative histograms (B) and quantitative data (C) of flow cytometry analysis of the expressions of CD44, CD105, CD90, and CD73, but not CD45 and CD34 in hp-MSCs from two passages. D Representative images from electron microscopy Rocuronium show EVs (white arrow) from non-irradiated and irradiated hp-MSCs (EVs isolated from conditioned medium of non-irradiated hp-MSCs EVs isolated from conditioned medium of irradiated hp-MSCs Table 2 The top 20 miRNAs that were downregulated in EVs from irradiated hp-MSCs (versus non-irradiated hp-MSCs) EVs isolated Rabbit Polyclonal to EPHB6 from conditioned medium of non-irradiated hp-MSCs Rocuronium EVs isolated from conditioned medium of irradiated hp-MSCs Uptake of EVs by HUVEC and H9c2 cells Then, we evaluated the biological effects of EVs from the non-irradiated and irradiated hp-MSCs on endothelial cells and cardiomyocytes. By culturing HUVEC and H9c2 cells with the supplement of PKH26-labeled EVs (10 g/ml) in the medium, the uptake of EVs by cells was observed using a confocal microscope. Red fluorescence was clearly detectable in the cytoplasm at 3 h and further enhanced after 24 h (Fig. ?(Fig.22 A, B). However, the uptake of EVs from either non-irradiated or irradiated hp-MSCs Rocuronium was quite similar by HUVEC and H9c2 cells, demonstrating that EVs from non-irradiated or irradiated hp-MSCs could be internalized by the HUVEC and H9c2 cells. Open in a separate window Fig. 2 The uptake of hp-MSC-derived EVs by HUVEC and H9c2 cells. Confocal images of HUVEC (A) and H9c2 cells (B) with internalized PKH26 labeled EVs from non-irradiated/irradiated hp-MSCs after 3 or 24 h of coculture ( 0.5, * 0.5, ** 0.1, *** 0.01, **** 0.001 Cell apoptosis was also evaluated after 48 h of coculture using an Annexin-V flow cytometry assay (Fig. ?(Fig.4A,4A, D). The non-irradiated hp-MSC EVs.