Fig 1.
Removal of EVs from MVEC-CM reduced its effects on cultured OPCs.
OPCs were cultured in MVEC-CM or EV-dep-MVEC-CM for 3 days (or overnight for cell motility assay). As control, OPCs were cultured in fresh serum-free medium. (A) Phase contrast images. Scale bar, 50 μm. (B) OPCs were stained with Hoechst 33342, and the nuclear morphology was observed. The number of pyknotic nuclei (red arrowheads) in MVEC-CM was smaller when compared to that of EV-dep-MVEC-CM. Scale bar, 50 μm. (C) After labeling with 10 μM BrdU for the last 4 h of culture, cells were fixed and stained with an anti-BrdU antibody. Cell nuclei were stained with propidium iodide (PI, red). Many BrdU-positive cells (green) were observed in MVEC-CM. Scale bar, 50 μm. (D) Phase contrast images of OPC migration 16 h after plating (Insets: OPC aggregates 1 h after plating). Scale bar, 200 μm. (E) All nuclei (pyknotic or non-pyknotic) were counted in a field of microscope and the fraction of pyknotic nuclei was determined, and results are shown as mean ± SE (N = 8 in each condition). Treatment with MVEC-CM significantly inhibited apoptotic cell death of OPCs. ***p<0.001 against control. #p<0.05 against EV-dep-MVEC-CM. (F) All cells were counted in a field of microscope and the fraction of BrdU-positive cells was determined, and results are shown as mean ± SE (N = 8 in each condition). The proportion of BrdU-positive cells in MVEC-CM was significantly larger when compared to EV-dep-MVEC-CM. *p<0.05 against control and MVEC-CM. (G) The distance of OPC migration in MVEC-CM was significantly larger when compared to EV-dep-MVEC-CM. Results are shown as mean ± SE (N = 4 in each condition). ***p<0.001 against control. ###p<0.001 against EV-dep-MVEC-CM. These experiments were repeated three times, and similar results were obtained each time. Typical experiments are shown here.
Fig 2.
Cultured OPCs incorporated isolated MVEC-EVs.
Presence of exosomal markers (CD63 and CD9), and actin, a cytoskeletal protein, in isolated MVEC-EVs was confirmed by Western blot (A). MVEC-EVs were labeled with fluorescent dye PKH67, and added to OPC culture. PKH67-labeled EVs were immediately taken up by OPCs, and many small vesicles (green) were found in OPCs (B). Scale bar: 20 μm.
Fig 3.
MVEC-EVs promoted OPC survival, proliferation and motility.
EVs isolated from MVEC-CM were suspended in fresh serum-free medium at 50 μg/ml of proteins and added to OPC culture, which were maintained for 2 days (or overnight for cell motility assay). As control, OPCs were cultured in fresh serum-free medium without EVs. (A) Phase contrast images. Scale bar, 50 μm. (B) OPCs were stained with Hoechst 33342, and the nuclear morphology was observed. The proportion of pyknotic nuclei (red arrowheads) in OPCs with MVEC-EVs was smaller when compared to control. Scale bar, 50 μm. (C) After labeling with BrdU for the last 4 h of culture, cells were fixed and stained with an anti-BrdU antibody. Cell nuclei were stained with propidium iodide (PI, red). Many BrdU-positive cells (green) were observed in the presence of MVEC-EVs. Scale bar, 50 μm. (D) Phase contrast images of OPC migration 16 h after plating (Insets: OPC aggregates at 1 h after plating). Scale bar, 200 μm. (E) The proportion of pyknotic nuclei in the presence of MVEC-EVs was significantly smaller when compared to control. Results are shown as mean ± SE (N = 8 in each condition). ***p<0.001. (F) The proportion of BrdU-positive cells in the presence of MVEC-EVs was significantly larger when compared to control. Results are shown as mean ± SE (N = 8 in each condition). ***p<0.001. (G) The distance of OPC migration in the presence of MVEC-EVs was significantly larger when compared to control. Results are shown as mean ± SE (N = 4 in each condition). ***p<0.001. These experiments were repeated three times, and similar results were obtained each time. Typical experiments are shown here.
Fig 4.
MVEC-EVs promoted OPC survival, proliferation, and motility in a dose-dependent manner.
EVs isolated from MVEC-CM were suspended in fresh serum-free medium at 0, 3, 12.5, 50, and 200 μg/ml proteins and added to OPC culture, which were maintained for 2 days (or overnight for cell motility assay). (A) The proportion of pyknotic nuclei decreased at doses ≥ 50 μg/ml. Results are shown as mean ± SE (N = 8 in each condition). **p<0.01 and ***p<0.001. (B) The proportion of BrdU-positive cells increased at doses ≥ 50 μg/ml. Results are shown as mean ± SE (N = 8 in each condition).***p<0.001. (C) The distance of OPC migration gradually increased in a dose-dependent manner. Results are shown as mean ± SE (N = 4 in each condition). **p<0.01 and ***p<0.001. These experiments were repeated three times, and similar results were obtained each time. Typical experiments are shown here.
Fig 5.
EC-derived EVs promoted OPC survival, proliferation and migration.
EVs were isolated from conditioned media of various types of endothelial cells (ECs) or Rat-1 fibroblastic cells, suspended in fresh serum-free medium and added to OPC cultures (50 μg/ml of proteins), which were maintained for 2 days. As control, OPCs were cultured in fresh serum-free medium without EVs. (A) Phase contrast images. Scale bar, 50 μm. (B) OPCs were stained with Hoechst 33342, and the nuclear morphology was observed. The proportion of pyknotic nuclei (red arrowheads) in OPCs with EC-derived EVs was smaller when compared to Rat-1-EVs. Scale bar, 50 μm. (C) After labeling with BrdU for the last 4 h of culture, cells were fixed and stained with an anti-BrdU antibody. Cell nuclei were stained with propidium iodide (PI, red). Many BrdU-positive cells (green) were observed in HUVEC-EV. Scale bar, 50 μm. (D) Phase contrast images of OPC migration 16 h after plating (Insets: OPC aggregates 1 h after plating). Scale bar, 200 μm. (E) The proportion of pyknotic nuclei in the presence of EC-derived EVs (except for bEnd.3-EVs) was significantly smaller when compared to those in the presence of Rat-1-EVs. Results are shown as mean ± SE (N = 8 in each condition). ***p<0.001 against control. ###p<0.001 against Rat-1-EVs. (F) The proportion of BrdU-positive cells in the presence of EC-derived EVs (except for HMVEC-EVs) was significantly larger when compared to Rat-1-EVs. Results are shown as mean ± SE (N = 8 in each condition). ***p<0.001 against control and Rat-1-EVs. (G) EVs derived from ECs significantly promoted OPC migration, when compared to control and Rat-1-EVs. Results are shown as mean ± SE (N = 4 in each condition). ***p<0.001 against control and Rat-1-EVs.