Figure 1.
Radiation-induced demyelination in the dorsal funiculus of cervical spinal cord.
(A) LFB/H&E staining showed normal spinal cord without irradiation. (B) Four months after irradiation, there was a focal demyelinated zone in the dorsal funiculus. (C) Six months following injury, focal necrosis was seen in the dorsal funiculus. (D) Immunostaining for MBP and NF, enlargement of framed area in A, depicted that non-irradiated myelin surrounding the axons (arrows). (E) Four months after irradiation, most of the axons lost myelin (arrows). (F) At six months, axons began to show necrosis. (G) Electron microscopy confirmed the normal structure of myelin. g, Higher magnification of the tissue in the box in G, indicated the compact myelin sheath. (H) Four months after irradiation, most of axons remained demyelinated (asterisk). (I) Six months following damage, axons necrosis was found (asterisk). Bar, 200 µm (A–C); bar, 50 µm (D–F); bar, 1 µm (G, H); bar, 100 nm (g); bar, 2 µm (I).
Figure 2.
In vitro differentiation of Olig2-GFP-mouse mESCs into Olig2+-GFP+- OPCs.
(A) Schematic procedure for differentiation of Olig2-GFP-mESCs into Olig2+-GFP+-OPCs. (B) Phase contrast photograph of mESCs colonies on mouse embryonic fibroblast. (C) mESCs remained undifferentiated and stained positive for Oct4/Sox2. (D) Phase contrast photograph of Olig2+-GFP+ spheres at day 12. (E) GFP+ spheres expressed Olig2. (F) At day 12, the percentage of GFP+ cells was 76.79%±1.35%, sorted by FACS method. At day 14, all purified OPCs expressed GFP as well as the OPC markers NG2 (G) and PDGFRα (H). At day 18, plated cells adopted a typical oligodendrocyte morphology characterized by multiple branches and expressed GFP and MBP (I). (J) Quantification of immunostaining: 81.69±8.16% of cells expressed NG2, 78±4.31% of cells expressed PDGFRα, and 68.47±5.59% of cells expressed O4. Data in J are expressed as the mean±SEM, n = 3 independent experiments, 6 total replicates. Bar, 200 µm (B–D); bar, 50 µm (E–H); bar, 30 µm (I).
Figure 3.
Distribution and morphology of Olig2+-GFP+-OPCs in irradiated spinal cord at four weeks after transplantation.
(A) GFP+ cells were observed and distributed in the damaged spinal cord. (B) At higher magnification of the boxed area from (A), the GFP+ cells exhibited typical bipolar OPC morphology. (C) Many grafted Olig2+-GFP+-OPCs co-expressed the OPCs marker NG2. Bar, 250 µm (A); bar, 100 µm (B); bar, 20 µm (C).
Figure 4.
Olig2+-GFP+-OPCs primarily differentiate along the oligodendrocyte lineage.
At eight weeks after transplantation, a certain number of grafted GFP+ cells still expressed NG2 (A–C, arrows) and most of the grafted GFP+ cells became APC-positive mature oligodendrocytes (D–F, arrows). Double staining for GFP and MBP in cross-sections further confirmed that GFP-immunoreactive rings were composed of MBP+ myelin (G–I, arrows). The grafted GFP+ cells did not co-expressed GFAP (J–L) or P75 (M–O). (P) Quantification of GFP+ cell populations in the spinal cord. Data are expressed as the mean number of cells/spinal cord±SEM (n = 6). GFP+ cells expressed NG2 (15.1±5.53%), APC (54.6±10.5%) and MBP (40.5±3.8%). Bar, 50 µm (A–C); bar, 35 µm (D–O).
Figure 5.
Transplantation of Olig2+-GFP+-OPCs results in a delayed demyelination and cavitation.
(A) LFB/H&E staining showed smaller cystic cavitations in the transplanted cord than those in the sham and the control group in the dorsal funiculus. (B) Optical densities of dorsal funiculus in spinal cord was significantly increased in rats grafted with Olig2+-GFP+-OPCs, as compared to the sham and the control group (*p < 0.05, n = 12). (C) Enlargement of framed area in part a, illustrated immunostaining of MBP and NF, showing axons (green arrow) with or without myelin (red arrow). (D) Electron microscopy confirmed the structure of axons (asterisk). Bar, 200 µm (A); bar, 35 µm (C); bar, 0.5 µm (D).
Figure 6.
Forelimb locomotion is improved after transplantation of Olig2+-GFP+-OPCs.
Locomotion function, as defined using the clinical grade score, was significantly reduced in Olig2+-GFP+-OPCs grafted animals at six to eight weeks after transplantation, as compared to the control group (*p < 0.05, n = 12).