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    Neural stem cell-derived exosome as a nano-sized carrier for BDNF delivery to a rat model of ischemic stroke
  • Figure 1|The features of hNSCs and engineered exosomes (Exo). 

    hNSCs grow in cell suspension and can form large neurospheres when cultured for 5–7 days. We used immunofluorescence to observe free single cells and neurospheres and confirmed that the NSC markers nestin and Sox2 were expressed (Figure 1A). Under induction with differentiation medium, NSCs were observed to differentiate into oligodendrocytes (identified by the marker MOG), neurons (Tuj1), and astrocytes (GFAP). 


    BDNF protein was encapsulated into neural stem cell-derived exosomes. Pure nanoparticles from the culture supernatant of hNSCs (hNSC-Exo and BDNF-hNSC-Exo) were evaluated by TEM, NTA, and western blotting. TEM revealed that the nanoparticles showed the typical cup-shaped exosome morphology (Figure 1B). NTA indicated that the diameter of the particles ranges from 50 to 150 nm (Figure 1C). Western blotting revealed the expression of typical exosome markers such as CD81, HSP70, and TSG101, whereas the calnexin negative control was not expressed (Figure 1D). Therefore, these isolated vesicles were considered to be hNSC-Exo. Notably, the diameter of exosomes did not change after encapsulating BDNF. These results suggest that BDNF loading does not affect the characteristics of exosomes derived from hNSCs. We also used exosomes for cell tracing in vitro. PKH67-labeled exosomes accumulated around the nucleus of target cells, and the entrapment of BDNF did not affect the uptake into target cells (Figure 1E).


    Figure 2|BDNF-hNSC-Exo promotes the survival and differentiation of neural stem cells in vitro.  

    Oxidative stress is one of the key pathogenic mechanisms of nerve apoptosis and neurological dysfunction in ischemic stroke (Belayev et al., 2017; Lai et al., 2020). We used H2O2 to induce oxidative damage in NSCs. We transfected BDNF-hNSC-Exo into NSCs treated with H2O2, and western blotting confirmed the expression of BDNF in target cells transfected with BDNF-hNSC-Exo (Figure 2A). 
    qRT-PCR, western blotting, and immunofluorescence staining showed that both BDNF-hNSC-Exo and hNSC-Exo promoted the differentiation of NSCs into neurons, and the differentiation ability of the BDNF-hNSC-Exo group was better than that of the hNSC-Exo group (Figure 2B–D). 


    CCK8, TUNEL, and western blotting were performed to evaluate the effect of exosomes on NSCs treated with H2O2. CCK8 assay showed that BDNF-hNSC-Exo exhibited a higher cell viability under oxidative stress (Figure 2E). Similar results were found using TUNEL staining. As shown in Figure 2F, the TUNEL-positive rate was lower in both hNSC-Exo and BDNF-hNSC-Exo groups compared with the PBS control group. However, BDNF-hNSC-Exo could greatly enhance the survival of NSCs compared to hNSC-Exo alone. The expression of Bax and cleaved-caspase-3 was detected by western blotting to assess apoptosis. The BDNF-hNSC-Exo-treated cells showing lower apoptosis in response to H2O2 and reduced induction of Bax and cleaved caspase 3 (Figure 2G).


    Figure 4|BDNF-hNSC-Exo inhibit neuroinflammation and promote neurogenesis in the peri-infarct zone of rats. 

    The rate of BrdU/Tuj1 and BrdU/GFAP double-positive cells was evaluated in the peri-infarct zone. Immunofluorescence staining showed that there were more BrdU/Tuj1 double-positive cells around the infarct area in the BDNF-hNSC-Exo and hNSC-Exo groups compared with the PBS group, indicating that more functional neurons were produced (Figure 4A). The percentage of BrdU/GFAP double positive cells around the ischemic area was lower in the hNSC-Exo group and the BDNF-hNSC-Exo group than in the PBS group (Figure 4B). To evaluate the inflammatory response of BDNF-hNSC-Exo to the brain after cerebral ischemia, the rate of Iba-1-positive cells near the injury site was evaluated using immunofluorescence. The results confirmed that BDNF-hNSC-Exo significantly inhibited the activation of microglia (Figure 4C). 



  • 发布日期: 2022-07-20  浏览: 143
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