Extracellular Vesicles Derived from Wharton's Jelly Mesenchymal Stem Cells Prevent and Resolve Programmed Cell Death Mediated by Perinatal Hypoxia-Ischemia in Neuronal Cells

Abstract

Hypoxic-ischemic (HI) insult in the perinatal phase harbors a high risk of encephalopathy in the neonate. Brain cells undergo apoptosis, initiating neurodegeneration. So far, therapeutic approaches such as cooling remain limited. Transplantation of mesenchymal stem cells (MSCs) exhibits therapeutic success despite the short-time survival in the host brain, providing strong evidence that their beneficial effects are largely based on secreted factors, including extracellular vesicles (EVs). The aim of this study was to investigate the effects of human Wharton's jelly MSC (hWJ-MSC)-derived EVs on neuroprotection and neuroregeneration, using an in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) mimicking HI injury in the mouse neuroblastoma cell line neuro2a (N2a). hWJ-MSC-derived EVs were isolated from cell culture supernatants by multistep centrifugation and identified by endosomal marker expression and electron microscopy. OGD/R significantly increased DNA fragmentation and caspase 3 ( Casp3) transcription in N2a cells relative to undamaged cells. OGD/R-mediated DNA fragmentation and Casp3 expression could be prevented as well as resolved by the addition of hWJ-MSC-derived EV before and after OGD, respectively. hWJ-MSC-derived EV also tended to increase the phosphorylation of the B cell lymphoma 2 (Bcl2) family member Bcl-2-antagonist of cell death (BAD) in N2a cells, when added prior or post OGD, thereby inactivating the proapoptotic function of BAD. Fluorescence confocal microscopy revealed the close localization of hWJ-MSC-derived EVs to the nuclei of N2a cells. Furthermore, EVs released their RNA content into the cells. The expression levels of the microRNAs (miRs) let-7a and let-7e, known regulators of Casp3, were inversely correlated to Casp3. Our data suggest that hWJ-MSC-derived EVs have the potential to prevent and resolve HI-induced apoptosis in neuronal cells in the immature neonatal brain. Their antiapoptotic effect seems to be mediated by the transfer of EV-derived let-7-5p miR.

Keywords: apoptosis; extracellular vesicles (EVs); let-7-5p; neuroprotection; neuroregeneration; oxygen–glucose deprivation/reoxygenation (OGD/R).

Fig. 1.

Characterization of human Wharton’s jelly mesenchymal stem cells (hWJ-MSC)-derived extracellular vesicles (EVs). (A) Representative Exo-Check antibody array of isolated hWJ-MSC-derived EVs. The antibody array has been done with 4 different EV preparations. (B) Western blot analysis of GRP94 expression in hWJ-MSC lysates and hWJ-MSC-derived EVs. Anti-β-actin staining was used as loading control, even though it is more abundant in cells than in EVs. To prove absent GRP94 expression in EVs, the membrane was overexposed. Western blot analysis has been done for 3 different EV preparations. (C) Representative electron microscopy image of hWJ-MSC-derived EVs, (D) revealing a mean vesicle diameter of 34.34 nm. Electron microscopy has been performed with 2 different EV preparations.

Fig. 2.

Prevention of oxygen–glucose deprivation/reoxygenation (OGD/R)-triggered apoptosis in neuro2a (N2a) cells by human Wharton’s jelly mesenchymal stem cells (hWJ-MSC)-derived extracellular vesicles (EVs). N2a cells were either left undamaged or subjected to 6 h of oxygen–glucose deprivation (OGD), followed by 24 h or 48 h of reoxygenation, respectively. To test the neuroprotective capacity of hWJ-MSC-derived EVs, 0.1 µg/mL (ca. 2 pg/cell) or 1 µg/mL EVs (ca. 20 pg/cell) were added to the cells 24 h or 1 h before OGD induction. (A) Experimental outline. (B to H) After the pretreatment with EVs 1 h before OGD and 24 h of reoxygenation, N2a cells were evaluated by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) test. TUNEL-positive cells are marked by arrows. N2a nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images of undamaged N2a cells (B), N2a cells subjected to OGD/R (C), and N2a cells pretreated with 1 µg/mL EVs before OGD initiation (D). (E) Differences in TUNEL-positive cells relative to DAPI. Bars illustrate mean ± standard error of mean (SEM) of 6 different images. *P < 0.05, **P < 0.01. Student’s t-test. (F and G) Real-time polymerase chain reaction analysis of caspase 3 and B cell lymphoma 2-associated antagonist of cell death (Bad) transcription post 48 h of reoxygenation. Bars illustrate mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01. Analysis of variance with post hoc Holm-Sidak test. (H) Western blot analysis for phosphorylation of BAD at the serine at position 112 site expression.

Fig. 3.

Reversion of oxygen–glucose deprivation/reoxygenation (OGD/R)-triggered apoptosis in neuro2a (N2a) cells by human Wharton’s jelly mesenchymal stem cells (hWJ-MSC)-derived extracellular vesicles (EVs). N2a cells were either left undamaged or subjected to 6 h of oxygen–glucose deprivation (OGD), followed by 24 h or 48 h of reoxygenation, respectively. To test for the neuroregenerative capacity of hWJ-MSC-derived EVs, 0.1 µg/mL (ca. 2 pg/cell) or 1 µg/mL EVs (ca. 20 pg/cell) were added after 6 h of OGD. (A) Experimental outline. (B to G) After the treatment with EV post-OGD, followed by 24 h of reoxygenation, N2a cells were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. TUNEL-positive cells are marked by arrows. N2a nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Representative images of undamaged N2a cells (B), N2a cells subjected to OGD/R (C), and N2a cells treated with 1 µg/ml EVs after OGD (D). (E) Differences in TUNEL-positive cells relative to DAPI. Bars illustrate mean ± standard error of mean (SEM) of 6 different images. *P < 0.05, **P < 0.01. Student’s t-test. (F) Real-time polymerase chain reaction (PCR) analysis of caspase 3 transcription post 48 h of reoxygenation. Bars illustrate mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.01. Analysis of variance with post hoc Holm-Sidak test. (G) Western blot analysis for phosphorylation of BAD at the serine at position 112 site expression.

Fig. 4.

Human Wharton’s jelly mesenchymal stem cells (hWJ-MSC)-derived extracellular vesicles (EVs) interact with and deliver their RNA into neuro2a (N2a) cells. hWJ-MSC-derived EVs were either labeled with the membrane dye chloromethyl (CM)-Dil (red) or the RNA dye Exo-Red and cocultured with N2a cells. The nuclei of N2a cells were counterstained with 4-6-diamidino-2-phenylindole-dihydrochloride (DAPI) (blue). (A) Representative fluorescent confocal microscopy image after the coculture of CM-Dil-stained EV and N2a cells for 24 h. (B) Three-dimensional visualization of the coculture of CM-Dil-stained EV and N2a cells by fluorescent confocal microscopy. (C) Representative fluorescent microscopy image after 2 h of coculture of Exo-Red-stained EV RNA and N2a cells. (D) Real-time polymerase chain reaction (PCR) analysis of let-7a and let-7e expression in N2a cells after 6 h oxygen-glucose deprivation (OGD) and 24 h of reoxygenation with and without the pretreatment with 1 µg/mL EVs (ca. 20 pg/cell) 24 h or 1 h before OGD initiation, relative to undamaged N2a cells. (E) Real-time PCR analysis of let-7a and let-7e expression in N2a cells after 6 h OGD and 24 h of reoxygenation with and without the treatment with 1 µg/mL EVs after 6 h of OGD, relative to undamaged N2a cells. Bars illustrate mean ± standard error of mean (SEM) of 3 independent experiments; *P < 0.05, **P < 0.01. Analysis of variance with post hoc Holm-Sidak test.