Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury

Abstract

Spinal cord injury (SCI) results in loss of oligodendrocytes demyelination of surviving axons and severe functional impairment. Spontaneous remyelination is limited. Thus, cell replacement therapy is an attractive approach for myelin repair. In this study, we transplanted adult brain-derived neural precursor cells (NPCs) isolated from yellow fluorescent protein-expressing transgenic mice into the injured spinal cord of adult rats at 2 and 8 weeks after injury, which represents the subacute and chronic phases of SCI. A combination of growth factors, the anti-inflammatory drug minocycline, and cyclosporine A immunosuppression was used to enhance the survival of transplanted adult NPCs. Our results show the presence of a substantial number of surviving NPCs in the injured spinal cord up to 10 weeks after transplantation at the subacute stage of SCI. In contrast, cell survival was poor after transplantation into chronic lesions. After subacute transplantation, grafted cells migrated >5 mm rostrally and caudally. The surviving NPCs integrated principally along white-matter tracts and displayed close contact with the host axons and glial cells. Approximately 50% of grafted cells formed either oligodendroglial precursor cells or mature oligodendrocytes. NPC-derived oligodendrocytes expressed myelin basic protein and ensheathed the axons. We also observed that injured rats receiving NPC transplants had improved functional recovery as assessed by the Basso, Beattie, and Bresnahan Locomotor Rating Scale and grid-walk and footprint analyses. Our data provide strong evidence in support of the feasibility of adult NPCs for cell-based remyelination after SCI.

Figure 1.

Isolated YFP-adult NPCs show nestin immunoreactivity in vitro. A, YFP-NPCs isolated from the subventricular zone of adult transgenic mice expressing YFP were grown as free-floating neurospheres in an uncoated tissue culture flask. The neurospheres were dissociated weekly into single cells and passaged for expansion. B, Dissociated cells from passages 3–4 were transplanted into the injured spinal cord of rats. C–E, When the dissociated YFP cells were plated as a monolayer on Matrigel-coated multichamber glass slides, they acquired an elongated shape with long but unbranched processes. Immunocytochemistry on these cultures at 24–48 h after plating showed uniform expression of nestin in the majority of these cells.

Figure 2.

Differentiation pattern of adult YFP-NPCs in vitro. Dissociated YFP-NPCs were cultured as a monolayer on Matrigel-coated multichamber glass slides. For the first 2 d, they were maintained in serum-free growth medium containing EGF and bFGF. On the third day, growth factors were withdrawn from the medium and replaced with 1% FBS. After 6 d in this condition, immunocytochemistry revealed a heterogeneous morphology of the YFP cells. A–C, A number of YFP cells (5%) showed the antigenic properties of oligodendrocyte precursor cells (identified by PDGF-αR). D–F, The majority of the differentiated cells (60%) showed immunoreactivity for GFAP and was considered as astrocytes. G–I, A population of YFP cells differentiated into mature oligodendrocytes (20%, identified by CNPase). J–L, Among the cultured cells, a smaller number of the cells (10%) were also positive for MAP-2, a marker for mature neurons.

Figure 3.

YFP-NPCs in the spinal cord of a subacutely injured rat 8 weeks after transplantation. A, A confocal image from a longitudinal section of an injured spinal cord taken from the dorsal spinal cord of a transplanted rat above the central cavity. A low-magnified image shows the extent of YFP-NPC survival within the injured spinal cord 8 weeks after transplantation. Grafted YFP-NPCs (green) were dispersed along the rostrocaudal axis of the spinal cord ∼5 mm away from the implantation sites (*). YFP-NPCs also migrated to the contralateral site of the spinal cord to a lesser extent. Double labeling with the neuronal marker βIII tubulin (Tuj1) showed that YFP-NPCs reside predominantly in the white-matter area (A–D). Our histological data showed no signs of tumor formation in the spinal cord. E, Confocal image of a transverse section of the spinal cord from a transplanted rat (8 weeks after transplantation) showing the distribution of YFP-NPCs in the lateral columns. F, G, YFP cells mainly showed multipolar morphology and extended numerous branches in the white-matter tissue along the length of axons. WM, White matter; GM, gray matter.

Figure 4.

Proliferative rate of grafted NPCs is low after transplantation. BrdU labeling of a transplanted spinal cord revealed very few proliferative profiles among the YFP-NPCs at 3 and 6 weeks after transplantation (data shown at 3 weeks).

Figure 5.

Differentiation of adult NPCs after transplantation. Confocal immunohistochemistry on longitudinal sections of the injured spinal cord of rats 6 weeks after transplantation is shown. A–C, Our histological examination at 2, 3, and 6 weeks after transplantation showed the lack of nestin-positive YFP cells, suggesting that the grafted cells had already adopted a lineage fate. Although we were able to observe some nestin-positive cells among the host spinal cord cells, probably reactive astrocytes, YFP cells were nestin negative. D–F, Presence of some NPC-derived astrocytes identified by GFAP immunoreactivity. G–I, In contrast to our in vitro observations, no neuronal profiles (Tuj1 or MAP-2; data shown for Tuj1) were found among the YFP-positive cells after transplantation. J, K, Immunostaining with p75 indicated the lack of YFP-NPC-derived Schwann cells in the spinal cord. Although we observed the presence of some p75-positive cells in both the transplanted and nontransplanted injured spinal cord, probably showing the invasion of endogenous Schwann cells into the cord, no coclocalization of p75 with YFP-derived cells was found.

Figure 6.

YFP-NPCs mainly differentiated along an oligodendrocyte lineage. Confocal immunohistochemistry on longitudinal sections of transplanted rats is shown. A–C, Grafted YFP-NPCs (green) displayed the antigenic properties of oligodendrocyte precursors (identified by PDGF-αR). D–I, Colocalization of YFP-positive cells with APC protein (a marker for mature oligodendrocytes). J, The majority of differentiated progenies express oligodendroglial markers. Our quantitative analysis in three transplanted rats at 6 weeks after transplantation showed that ∼53% of YFP-positive cells had been differentiated toward an oligodendrocyte lineage: oligodendrocyte precursor (18.7 ± 2.5%) cells as well as mature oligodendrocytes (32.7% ± 3.5). Our quantification showed that only ∼5.6 ± 2.08 of the grafted cells differentiated into astrocytes. No neuronal progenies were observed among the YFP-positive cells. The grafted YFP cells also did not show any nestin immunoreactivity. Approximately 43 ± 4.5% of YFP-positive cells did not show any colocalization with the cell markers that we used in this study. Error bars indicate SD.

Figure 7.

YFP-NPC-derived oligodendrocytes generate MBP and ensheath the injured axons of the spinal cord. A–C, Confocal images of longitudinal sections of an injured spinal cord 8 weeks after transplantation. The area grafted with YFP-NPCs (green) display a robust expression of MBP (red) in white matter of an injured spinal cord. Cell bodies of donor cells are surrounded with MBP. Triple-labeling experiments on longitudinal (D–G) and cross (H–K) sections of spinal cord white matter showed that MBP-expressing YFP-NPCs ensheathed the injured axons (identified by NF200; blue). These images (D–G) clearly show the oligodendrocyte morphology of one grafted YFP cell (arrowheads) that extends its processes and expresses MBP around an injured axon and the close proximity of these cells with newly myelinated axons. L, M, Images taken by deconvolution confocal microscopy show a higher-magnification image confirming axonal ensheathment of MBP-expressing YFP-NPCs around the injured axons.

Figure 8.

Evidence of remyelination of injured spinal cord white matter by grafted NPC 8 weeks after transplantation. A–C, Cross sections of osmium tetroxide-fixed semithin sections of the spinal cord stained with Toluidine Blue 8 weeks after transplantation are depicted for the plain injured, injured control, and NPC-transplanted groups, respectively, at low (A_I–C_I) and higher (A_II–C_II) magnification. The enlargement of the microscopic fields in the boxed areas in A_I–C_I shows the examples of the myelin profiles present in spinal cord white matter of the plain injured, injured control, and NPC-transplanted groups, respectively. As observed in C_II, the NPC-transplanted group showed more extensive oligodendrocyte-myelinated profiles in the area that was occupied by YFP-NPCs. The presence of YFP-NPCs was confirmed by fluorescence microscopy in the adjacent sections. D, E, Myelin index measurements on the three groups showed a significant increase in the MR in the NPC-transplanted group, indicating enhanced myelination in this group compared with the plain and control injury groups (D; p < 0.001; Kruskal-Wallis one-way ANOVA on ranks and Dunn's method postanalysis). The myelin was significantly thicker in the NPC-transplanted animals (p < 0.001; 0.544, 0.429, and 0.651 μm) compared with the plain injured (0.214, 0.160, and 0.258 μm) and control injured (0.226, 0.160, and 0.295 μm) animals (median: 25 and 75%, respectively). The enhanced myelination with NPC transplantation was further apparent by plotting the frequency distribution of MRs (E; p < 0.001; one-way ANOVA, followed by Tukey's post hoc analysis). This plot (E) illustrates a rightward shift toward enhanced myelination with NPC transplantation. Error bars indicate SD. F_I, G_I, Immunoelectron micrographs are depicted, which provide evidence for the spatial overlap of YFP expression and myelin formation around axons in the NPC-transplanted spinal cords. Labeling of the peroxidase reaction product was seen in the YFP cell cytoplasm (F_I, arrows) as well as the processes (G_I, arrows). F_II, G_II, Higher magnification of the boxed areas in F_I and G_I clearly shows the presence of peroxidase reaction product in the cytoplasm and cell processes of YFP cells. H_I, YFP-positive processes from myelinating donor-derived cells were seen in close association with an axon. H_II, Enlargement of the boxed area in C_I showed the presence of peroxidase reaction product in the cytoplasm of a myelinating YFP cell as well as in myelin membrane around the axons.

Figure 9.

Subacute transplantation of YFP-NPCs resulted in a significant locomotor recovery compared with injured rats in the control group. A, BBB rating scale showed a significant improvement in the locomotor BBB score in transplanted rats at 3 weeks after transplantation compared with the plain injured and control groups (n = 5 for plain injured group and n = 8 for other groups). B, Using grid-walk analysis, transplanted rats also showed fewer errors in hindlimb placements at 5 and 6 weeks after transplantation compared with the plain injured and control groups (n = 5 for plain injured group and n = 8 for other groups). C, Representative footprints of normal, plain injured, control, and grafted rats (n = 5 for plain injured group and n = 8 for other groups) shows improvement in interlimb coordination as well as angle of rotation in the transplanted group compared with the plain injured and control groups. D, F, Footprint analysis revealed that transplantation with adult NPCs significantly improved interlimb coordination and reduced the hindlimb angle of rotation at 5 and 6 weeks after transplantation. The data show the mean ± SEM. *p < 0.05.