Functional recovery and neural differentiation after transplantation of allogenic adipose-derived stem cells in a canine model of acute spinal cord injury

Abstract

In this study, we evaluated if the implantation of allogenic adipose-derived stem cells (ASCs) improved neurological function in a canine spinal cord injury model. Eleven adult dogs were assigned to three groups according to treatment after spinal cord injury by epidural balloon compression: C group (no ASCs treatment as control), V group (vehicle treatment with PBS), and ASC group (ASCs treatment). ASCs or vehicle were injected directly into the injured site 1 week after spinal cord injury. Pelvic limb function after transplantation was evaluated by Olby score. Magnetic resonance imaging, somatosensory evoked potential (SEP), histopathologic and immunohistichemical examinations were also performed. Olby scores in the ASC group increased from 2 weeks after transplantation and were significantly higher than C and V groups until 8 weeks (p < 0.05). However, there were no significant differences between the C and V groups. Nerve conduction velocity based on SEP was significantly improved in the ASC group compared to C and V groups (p < 0.05). Positive areas for Luxol fast blue staining were located at the injured site in the ASC group. Also, GFAP, Tuj-1 and NF160 were observed immunohistochemically in cells derived from implanted ASCs. These results suggested that improvement in neurological function by the transplantation of ASCs in dogs with spinal cord injury may be partially due to the neural differentiation of implanted stem cells.

Fig. 1

Adipogenic and osteogenic differentiation of canine adipose-derived stem cells (ASCs). A: ASCs cultured in DMEM + 10% FBS media (control media), not stained by Oil red O. B: Oil red O stained after 3 weeks incubation at adipogenic media. C: ASCs cultured in control media, not stained with Alizarin red S. D: Intense Alizarin red S stained after 3 weeks incubation at osteogenic media and confirmed calcium deposition. A and B: Oil red O stain, C and D: Alizarin red S stain, ×100.

Fig. 2

Green fluorescence protein (GFP) labeled canine adipose-derived stem cells (ASCs). (A) Typical morphological feature of canine ASCs. (B) Green fluorescence was identified in ASCs at 48 h after transfection. ×100.

Fig. 3

Flow cytometric analysis of surface-marker expression on ASC. The seventh passage of ASCs expressed CD44, CD90 and CD105, and were negative for CD14, CD34, CD45. The overwhelming majority (> 95%) of cASC expressed the mesenchymal cell surface markers CD90 and CD105.

Fig. 4

Olby scores during the 9 week post-SCI study period. The scores in ASC group were significantly higher than these in the other two groups at 5 and 9 weeks after spinal cord injury (^*p < 0.05).

Fig. 5

Images of the spinal cord injury lesion. A: control group with no ASCs transplantation. B: vehicle group with PBS. C and D: ASC group with transplantation with ASCs. Sagittal image: a, b, c and d. T1-weighted MR image at 5 weeks (a), T2-weighted MR image at 5 weeks (b), T1-weighted MR image at 9 weeks (c), T2-weighted MR image at 9 weeks (d). White arrowhead indicated the cranial, center and caudal portions of the transverse image. Transverse, T1-weighted MR image at 5 weeks (T1) and transverse, T2-weighted MR image at 5 weeks (T2). Black arrow indicated cavitation. The hyperintense lesions in T2-weighted MR image at 5 and 9 weeks after spinal cord injury were not different among groups.

Fig. 6

Percentage of luxol fast blue staining positive areas in the transverse sections at the epicenters of injured spinal cords. Luxol fast blue positive areas in the control group and vehicle group were smaller than those in the ASC group. ^*p < 0.05 compared to control gruop, ^†p < 0.05 compared to vehicle group.

Fig. 7

Histopathological findings 9 weeks after spinal cord injury. A and D: group C; B and E : group V; C, F-H: group ASC. All groups showed extensively damaged tissues. F: Positive areas for Luxol fast blue staining were observed at injured sites in the ASC group (circles). G and H: These showed structural consistency with nerve cell. A, B and C: H&E stain, D-G: Luxol fast blue and cresyl violet stain, H, H1, H2 and H3: Immunofluorescence staining. Scale bars = 50 µm.

Fig. 8

Immunofluorescence staining of ASC group; A1-A3: Glial fibrillary acidic protein (GFAP), B1-B3: Neurofilament M (NF160), C1-C3: Neuronal class III beta tubulin (Tuj-1), D1-D3: Oligodendrocyte, Green fluorescence protein (GFP)- labeled lentiviral vector inserted stem cells were positive with GFAP, NF 160, Tuj-1, and oligodendrocytes in injured lesions (arrows). Scale bars = 50 µm.