Journal: PLoS Biology

Article Title: Neural stem cells traffic functional mitochondria via extracellular vesicles

doi: 10.1371/journal.pbio.3001166

Figure Lengend Snippet: (a) TEM data showing size particle analysis and quantification of mitochondria found in the EV preparations (purple dots) compared to non-mitochondrial EVs (grey dots). Data are mean values (± SEM) from N = 2 biological replicates. (Data available in ). (b) Representative NanoFCM density plot of EVs and EVs Mito_depl. labelled with the canonical EV marker CD63 and the mitochondrial dye MitoTracker red. (Data available in ). (c) Representative cryo-TEM image of a free mitochondria labelled with anti-TOMM20 (red arrows) antibody conjugated to 10-nm gold NP. Inset: magnified ROI pseudocolored (or not) to highlight the 2 mitochondrial membranes (white arrowheads). Scale bars: 200 nm. (d) Representative cryo-TEM image of NSC EVs treated with saponin and labelled with anti-CD63 (red arrowheads) and anti-TOMM20 (red arrows) antibodies conjugated to 10 nm and 20 nm gold NP, respectively. Inset: magnified ROI pseudocolored (or not) to highlight the 3 membranes (white arrowheads). Scale bar: 200 nm. (e) Mitochondrial membrane potential of NSCs, EVs, and Mito preparations treated (or not) with the mitochondrial uncoupler CCCP. * p ≤ 0.05. Data are mean values (± SEM) from N ≥ 2 independent experiments. (Data available in ). (f) Representative mitochondrial respiration of permealised EVs detected by HRR. Representative plot showing O 2 concentration changes over time upon serial additions of selected mitochondrial complexes substrates, inhibitors, and uncouplers, including CI substrates (G+M: ADP), CI inhibitor (rotenone: Rot), CII substrate (succinate: Succ), cytochrome c (Cyt c) to compensate for a possible loss due to outer membrane disruption, CIII inhibitor (antimycin A: AA), CIV electron donor ( N , N , N′ , N′ -tetramethyl- p -phenylenediamine: TMPD) and CIV inhibitor (potassium cyanide: KCN). (Data available in ). (g) Complex respiratory rate in EVs. Data are mean values (± SEM) from N = 3 independent experiments. (Data available in ). (h) Representative image of mitochondria respiratory chain native complexes separated by BN-PAGE showing the presence of structurally intact respiratory complexes (CI–V) in NSCs, released EVs, and isolated Mito preparations. (i) Representative image of in situ gel activity of CI-II-IV in NSCs, EVs, and Mito obtained from BN-PAGE gel incubation for 24 hours. ADP, adenosine diphosphate; BN-PAGE, blue native polyacrylamide gel electrophoresis; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; cryo-TEM, cryo-transmission electron microscopy; EV, extracellular vesicle; G+M, glutamate and malate; HRR, high-resolution respirometry; NanoFCM, nano flow cytometry; NP, nanoparticles; NSC, neural stem cell; ROI, region of interest; TEM, transmission electron microscopy.

Article Snippet: After the first ultracentrifugation at 100,000 x g for 70 minutes at 4°C using an Optima XPN-80 ultracentrifuge with SW 32 Ti swinging rotor (Beckman Coulter), EVs derived from 108 × 10 6 NSCs were resuspended in PBS and stained at 37°C for 30 minutes with MitoTracker Red CMXRos (Thermo Fisher Scientific, M7512, 300 nM) and Vio Bright FITC, REAfinity anti-mouse CD63 antibody (Miltenyi Biotec, 130-108-897, 1:10).

Techniques: Particle Size Analysis, Marker, Membrane, Concentration Assay, Disruption, Isolation, In Situ, Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, Transmission Assay, Electron Microscopy, Flow Cytometry

Journal: PLoS Biology

Article Title: Neural stem cells traffic functional mitochondria via extracellular vesicles

doi: 10.1371/journal.pbio.3001166

Figure Lengend Snippet: (a) In vivo experimental setup of EV and NSC treatment in EAE mice. At PD, mice received a single ICV injection of either fGFP + /MitoDsRed + NSCs, EVs derived from fGFP + /MitoDsRed + NSCs (EVs), or fGFP + /MitoDsRed + EVs depleted of mitochondria (EVs Mito_depl. ), or fGFP + /MitoDsRed + EVs depleted of CD63+ EVs (EVs CD63_depl. ). Behavioural analysis was carried out daily until the end of the experiment. Neuropathology was performed at 55 dpi. (b) Behavioural outcome showing significant amelioration of the EAE score in mice treated with EVs ( N = 5) and NSCs ( N = 5), but not in EAE mice treated with EVs Mito_depl. ( N = 4) or EVs CD63_depl. ( N = 5) vs. PBS ( N = 8). Data are mean values (± SEM). * p < 0.05. (Data available in ). (c) Percentage of fGFP - /MitoDsRed + particles co-localising with GFAP + astrocytes, NeuN + neurons, Olig2 + oligodendrocytes, CD3 + T cells, or F4/80 + mononuclear phagocytes. fGFP + /MitoDsRed + NSCs were injected ICV into EAE mice (white bars) and in nonimmunised control mice (hatched bars). Data are mean values (± SEM) from N = 4 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. (Data available in ). (d) Representative pictures of mitochondrial transfer events detected with confocal imaging (maximal projection). Transfer of MitoDsRed + particles (arrowheads) is shown between fGFP + /MitoDsRed + NSCs and GFAP + astrocytes (cortex), NeuN + neurons (cortex), Olig2 + oligodendrocytes (corpus callosum), CD3 + T cells (meninges), or F4/80 + mononuclear phagocytes (IV ventricle). Long processes of NSCs can be seen in green, while nuclei are stained with DAPI (blue). Scale bars: 20 μm. DAPI, 4′,6-diamidino-2-phenylindole; dpi, days post immunisation; EAE, experimental autoimmune encephalomyelitis; EV, extracellular vesicle; ICV, intracerebroventricular; NSC, neural stem cell; PD, peak of disease.

Article Snippet: After the first ultracentrifugation at 100,000 x g for 70 minutes at 4°C using an Optima XPN-80 ultracentrifuge with SW 32 Ti swinging rotor (Beckman Coulter), EVs derived from 108 × 10 6 NSCs were resuspended in PBS and stained at 37°C for 30 minutes with MitoTracker Red CMXRos (Thermo Fisher Scientific, M7512, 300 nM) and Vio Bright FITC, REAfinity anti-mouse CD63 antibody (Miltenyi Biotec, 130-108-897, 1:10).

Techniques: In Vivo, Injection, Derivative Assay, Control, Imaging, Staining