Tree shrew neural stem cell transplantation promotes functional recovery of tree shrews with a hemi‑sectioned spinal cord injury by upregulating nerve growth factor expression

Abstract

The aim of the present study was to determine the effect of implanted neural stem cells (NSCs) on the functional recovery of tree shrews (TSs) subjected to hemi‑sectioned spinal cord injury (hSCI), and to investigate the possible mechanism involved. NSCs (passage 2), derived from the hippocampus of TSs (embryonic day 20), were labeled with Hoechst 33342 and transplanted intraspinally into the hSC of TSs at thoracic level 10 in the acute (immediately after injury) and chronic (day 9 post‑injury) stages. The Basso‑Beattie‑Bresnahan (BBB) score was recorded from days 1 to 16 post‑injury, and the survival, migration, differentiation and neurotrophic factor (NTF) expression in vivo were detected. In vitro and in vivo, the expanded NSCs were able to differentiate into neurons and astrocytes, and secreted a variety of NTFs, including ciliary NTF, transforming growth factor‑β1, glial cell line‑derived NTF, nerve growth factor (NGF), brain‑derived NTF and insulin‑like growth factor. Following transplantation, the BBB score in the TSs with chronic‑stage transplantation exhibited a statistically significant increase, while there was no significant difference in the acute group, compared with the control group. This corresponded with the marked upregulation of NGF indicated by reverse transcription‑quantitative polymerase chain reaction. In conclusion, the transplantation of NSCs into the hSC in the chronic phase, but not the acute stage, of hSCI in non‑human primate TSs is effective and associated with upregulated NGF expression. These findings may provide novel strategies for the treatment of SCI in clinical patients.

Figure 1

Morphology of TS NSCs cultured in vitro. (A) TS NSCs were observed under a microscope without dye processing on days 1, 3 and 5 of primary culture (P0), and subculture (P1 and P2). Scale bar, 100 μm. (B and C) The number of NSCs in the different culture stages (P0, P1 and P2) was compared at different culture times (days 3 and 5). Data are presented as the mean ± SD (n=5). The area of NSCs in the different culture stages (P0, P1 and P2) was compared at different times (days 3 and 5). Data are presented as the mean ± SD (n=6). TS, tree shrew; NSC, neural stem cell; P, passage; d, day; SD, standard deviation.

Figure 2

Identification and differentiation of the cultured TS NSCs. (A) Immunofluorescent staining of Nestin (red, left) for identifying the TS NSCs, DAPI with blue staining was shown in middle and the merge picture showed Nestin positive rate was 99.58% (right). (B and C) Immunofluorescent staining of NeuN and GFAP for confirmation of the differentiation of the cultured NSCs into neurons and astrocytes (red, left). DAPI stained the nuclei with blue florescence (middle). Merged images show the NeuN- and GFAP-positive cells, from which expression rates of 97.23 and 46.99%, respectively, were obtained (right). White arrows represent the positive cells. Scale bar, 50 μm. Cells from five fields in each well were collected, and each detection in vitro was prepared for 6 plates (6-pore plate) of cells. (D) Representative bar graph for the rate of positive cells. TS, tree shrew; NSC, neural stem cell; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; NeuN, neuron-specific nuclear protein.

Figure 3

Expression of NTFs in the NSCs of tree shrews. Immunofluorescent staining of CNTF, TGF-β1, GDNF, NGF, BDNF and IGF is shown in the left of (A-F) (red), and cell nuclei were redyed by 4′,6-diamidino-2-phenylindole (DAPI) (blue) in vitro (A-F, middle). The merged images show positive CNTF (B; right), GDNF (C; right) and IGF (D; right) expression, with a determined mean rate of 100%, BDNF (A, right) was 50%, NGF was 89.96% (E, right) and TGF-β1 was 96.67% (F, right). Scale bar, 50 μm. White arrows represented the positive cells. Cells from five fields in each well were collected, each detection in vitro was prepared for six plates (six-pore plate) of cells. NTF, neurotrophic factors; NSCs, neural stem cells; CNTF, ciliary NTF; TGF-β1, transforming growth factor-β1; GDNF, glial cell line-derived NTF; NGF, nerve growth factor; BDNF, brain-derived NTF; IGF, insulin-like growth factor.

Figure 4

Analysis of functional recovery measured by BBB scores following NSC transplantation. (A) The BBB scores in hSCI tree shrews treated with NSCs immediately after hSCI did not differ from those of the controls. (B) The chronic group exhibited significantly better functional recovery than the chronic control group at day 16 post-injury. Data are presented as the mean ± standard deviation (n=10). ^*P<0.01 vs. control group. Arrowheads indicate the transplantation time. Chronic, NSC transplantation at day 9 post-injury; acute, NSC transplantation immediately after injury; TS, tree shrew; BBB, Basso-Beattie-Bresnahan; NSC, neural stem cell; hSCI, hemi-sectioned spinal cord injury.

Figure. 5

Transplanted TS NSCs survived and differentiated into neurons and glia-like cells in vivo. (A) NSCs labeled with Hoechst (blue fluorescence, left) survived and migrated at day 16 post-injury in vivo. The control exhibited no positive reactivity (right). (B) Hoechst⁺ grafted NSCs (blue florescence, middle) differentiated into NeuN⁺ neurons (green florescence, left), and GFAP⁺ astrocytes (red, left) in the chronic groups, with merged images shown in the right panels. Scale bar, (A) 100 μm and (B and C) 50 μm. White arrows indicate the positive cells. The images were captured 1 cm below the lesion. (D) Representative bar graph for the rate of NeuN- and GFAP-positive cells. NSC, neural stem cell; TS, tree shrew; NeuN, neuron-specific nuclear protein; GFAP, glial fibrillary acidic protein.

Figure. 6

Expression of NTFs in vivo after tree shrew NSC transplantation. (A) Analysis of CNTF mRNA expression in the spinal cord tissues caudal to the injured site (20-mm long containing the injury and injection sites) among the sham, hSCI and NSC transplantation groups in the chronic phase at day 16 post-injury. (B) Analysis of BDNF mRNA expression in the tissues aforementioned. (C) Analysis of NGF mRNA expression in the tissues aforementioned. *P<0.05 vs. NSC group. Data are represented as the mean ± SD (n=10). NSC, neural stem cell; hSCI, hemi-sectioned spinal cord injury; NTF, neurotrophic factor; CNTF, ciliary NTF; BDNF, brain-derived NTF; NGF, nerve growth factor.