Figure 6: FRα-expressing exosomes penetrate into the brain parenchyma in vivo and are internalized by astrocytes in vitro.

(a) Exosomes were purified from the cell culture supernatant of transfected Z310 cells and labelled with the dye PKH26 (red). Ten microlitres of the exosome solutions were then injected in mice with surgical implanted intraventricular cannulas 4 h before the mice were killed. Immunofluorescent staining of the mouse brain sections shows that exosomes derived from FRα-transfected Z310 cells (FRα+) cross the ependymal cell layer and are distributed deeply in the brain parenchyma distal from the injection site. In contrast, exosomes derived from vector-transfected cells (FRα-) cannot penetrate the parenchyma but are retained at the ependyma. Nuclei were counterstained with DAPI (blue). Upper scale bar, 100 μm, lower scale bar, 20 μm. (b) Immunostaining of the mouse brain sections with antibodies against GFAP and NeuN reveal that exosomes (red) derived from FRα-transfected Z310 cells colocalize with GFAP-positive astrocytes and NeuN-positive neurons. Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. (c) Purified exosomes from Z310 cells were labelled with the dye PKH26 (red), incubated with FA-FITC (green) and added to primary mixed cortical cultures on days in vitro (DIV) 7, consisting of mouse oligodendrocytes, astrocytes, cortical neurons and microglia. After incubation with exosomes for 2 h at 37 °C, the cells were fixed and analysed by immunofluorescence microscopy. Oligodendrocytes, astrocytes, neurons and microglia were recognized by antibodies against Nogo-A, GFAP, Iba1 and NeuN. The primary antibodies were recognized with secondary biotin-conjugated antibodies and subsequently labelled with streptavidin-coupled Pacific blue (shown in grey for better visualisation). FRα-containing exosomes (FRα+) are selectively taken up by GFAP-positive astrocytes. Partial colocalization of FA-FITC and PKH26 is also observed in these cells. Exosomes purified from the culture medium of vector-transfected Z310 cells (FRα-) are not taken up by astrocytes but are internalized by Iba1-positive microglia. Note that none of the cell types was found to internalize free FA-FITC. Scale bar: 20 μm.