Fig 1.
A: Electron micrograph of vesicles in exosome fraction from MSC conditioned media sample on formvar coated grid. B: Histogram of size distribution of 536 presumptive exosomes. Note the average size of the vesicles was 59.09 ± 0.58 nm, consistent with exosomes. C-D: Fluorescence micrographs of intact spinal cord directly injected with exosomes fractions from SytoRNAse (C) and DiR (D) labeled MSCs before perfusion. Note that the presence of detectable levels of 488nm (SytoRNAse) and 650nm (DiR) fluorescence indicates the presence of both RNAs and lipids in the exosome fractions. Scale bar in A indicates 100nm. Scale bars in C &D indicate 1 mm.
Fig 2.
A-C: Two photon confocal micrographs of contused spinal cord 3 hours after IV infusion of DiR labeled exosomes showing the colocalization of regions of high endogenous 488 autofluorescence with 650nm DiR hotspots. D-H: Endogenous DiR wavelength in the spleen (D-E) and lungs (F-H) of contused animals with no exosome infusion. High magnification shows sizes and shapes of DiR hotspots in match those of red blood cells. I-J: Endogenous DiR wavelength fluorescence of paraformaldehyde fixed rat red blood cells isolated from peripheral blood. Scale bar in A indicates 100 μm and applies to A-C. Scale bars in D, F indicate 100 μm and applies to A-C. Scale bars in D, F indicate 100 micrographs of contused spinal cord 3 hours DiR hotspots.
Fig 3.
IV infused DiR labeled MSCexos co-localize with OX42+ macrophages/microglia in the contused spinal cord.
A-B: Low magnification confocal images of a representative region of a contused spinal cord 3 hours after DiR MSCexos infusion stained for GFAP (green) and OX42 (red) and counterstained with DAPI (blue) (A) or DiR fluorescence only (cyan) (B). Large numbers of DiR hotspots can be seen near the margins of the contusion site correlating with high levels of GFAP+ reactive astrocytes (green) and OX42+ macrophages (red). C-E: Higher magnification image of the lesion shown in A showing the relationship between DiR hotspots and OX42 staining. C1-E1: Enlarged and rotated boxed regions of the lesion shown in C-E showing that the location of DiR hotspots within OX42+ cells. F-H: Representative confocal images of contused spinal cord 24 hours after DiR MSCexos (cyan) infusion shown stained for GFAP (green) and OX42 (red) and counterstained with DAPI (blue), (F), OX42 and DiR (G) or DiR fluorescence only (cyan) (H). F1-H1: Enlarged and rotated boxed regions of the lesion shown in F-H. All scale bars indicate 20 μm. Dashed line in A and B indicates the approximate border of the lesion, as defined by the reactive astrocyte boundary, with the lesion to the left and largely intact neural tissue to the right. DiR hot spots appear to be clustered more densely near the borders of the lesioned area, in regions containing both OX42+ macrophages (red) and strongly GFAP+ reactive astrocytes (green).
Fig 4.
DiR hotspots in DiR-MSCexos infused animals co-localize with CD63 exosome marker.
A-E: Representative region of a contused spinal cord 24 hours after DiR MSCexos (cyan) infusion stained with antibodies directed against OX42 (green) and the tetraspanin CD63 (red), and counterstained with DAPI (blue) (A) or DiR fluorescence only (cyan). Images show all wavelengths (A), OX42 & CD63 (B), OX42 & DiR (C), CD63 & DiR (D), DiR only (E). A1-E1: enlarged and rotated sections of boxed regions shown in A-E. Images below B1, C1, and D1 respectively show full 90 degree rotations of boxed regions showing CD63, DiR, and CD63 & DiR fluorescence. Note the strong co-localization between DiR fluorescence and CD63 staining. Scale bar in E = 20 μm E1 = 10 μm.
Fig 5.
IV infused DiR labeled MSCexos localize with CD206+ Type M2 macrophages in the contused spinal cord.
A-E Confocal micrographs of a representative region of a frozen sectioned contused spinal cord harvested 24 hours after IV infusion of DiR labeled exosomes (cyan), immunostained with antibodies directed against Type M1 macrophage marker iNOS (red) and Type M2 macrophage marker CD206 (green) and counterstained with DAPI (blue). Images from left to right show the same area showing florescence channels for (A) iNOS, CD206, DAPI, & DiR, (B) iNOS, CD206, & DiR (C) iNOS, & DiR, (D) CD206, & DiR, and (E) DiR only. Images in a-e show enlarged images of boxed area above rotated and illustrated in 3D. A smaller portion of the regions shown in c-e is further enlarged and rotated below. Note the strong co-localization of DiR florescence with CD206, but not iNOS staining, suggesting that DiR labeled exosomes are taken up by Type 2 macrophages. Scale bars in E and e & d indicate 50 μm and 20 μm and apply to A-E and a-e respectively.
Fig 6.
DiR hotspots not detected in other cell types at the lesion site.
Selected regions contused spinal cords 24 hours after infusion of DiR MSCexos (A-G) or PBS with free DiR (G-H), showing close proximity between DiR hotspots and some endothelial cells (A-C) and pericytes (D-G) within the central scar tissue at the contusion site and paucity of DiR fluorescence signal in DiR only infused animal. A-C: Region near the border of the lesion with antibody staining directed against neurofilament (NDH green), the endothelial cell marker RECA-1 (red) (A), RECA-1 (red) and DiR wavelengths (B), or DiR fluorescence only (cyan) (C). A1-C1, B2: enlarged and rotated boxed region shown in A-C. Note that DiR hot spots are very close to the endothelial cells, but not within them. D-G: Selected region in the center of the contusion site containing high concentrations of pericytes Area is shown with antibody staining directed against neurofilament (green), PDGFR-B (red), DiR wavelength fluorescence (cyan) and DAPI counterstaining (blue) (D), PDGFR-B (red) and DiR wavelength fluorescence (cyan) (E), PDGFR-B (red) (F), or DiR wavelength fluorescence only (cyan) (G) D1-G1: enlarged and rotated boxed region shown in D-G. Note that DiR hotspots are located close to pericytes but are not within the pericytes. H-I: Representative region of the lesion in a contused rat infused with DiR only, without exosomes and stained with antibodies directed against RECA-1 (red), and neurofilament (green) and counterstained with DAPI (G) and the same regions showing DiR wavelength fluorescence only. Dashed lines in A-C and H, I indicate the approximate lesion boundary with the lesioned area above and intact spinal cord tissue below. Scale bars in A, G & I = 100μm. C1 50 μm, E1 = 20 μm.
Fig 7.
DiR hotspots transiently localize to kidney and more persistently to spleen.
A-D: DiR fluorescence (cyan) in kidney (A, C) and spleen (B, D) 3hours (A, B) and 24 hours (C, D) after infusion with DiR-MSCexos. Note the decrease in DiR fluorescence in the kidneys between 3 and 24 hours after DiR-MSCexos infusion, but increase in the spleen during the same time period. E-F: Spleens at 24 hours after DiR-MSCexos infusion in SCI rats immunostained with antibodies directed against CD206 (green) and CD8 (red) DiR fluorescence hotspots. E1-F1: Enlarged rotated images of boxed areas in E and F. Note that DiR hotspots localize to both CD206+ and CD4+ cells in the spleen. Scale bars in E 100 μm and applies to A-D. Scale bars in E&F indicate 20 μm.