Intravenous administration of glial cell line-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in the adult rat

Abstract

Intravenous administration of human mesenchymal stem cells (hMSCs) prepared from adult bone marrow has been reported to ameliorate functional deficits after cerebral artery occlusion in rats. Several hypotheses to account for these therapeutic effects have been suggested, and current thinking is that neuroprotection rather than neurogenesis is responsible. To enhance the therapeutic benefits of hMSCs potentially, we transfected hMSCs with the glial cell line-derived neurotrophic factor (GDNF) gene using a fiber-mutant F/RGD adenovirus vector and investigated whether GDNF gene-modified hMSCs (GDNF-hMSCs) could contribute to functional recovery in a rat permanent middle cerebral artery occlusion (MCAO) model. We induced MCAO by using intraluminal vascular occlusion, and GDNF-hMSCs were intravenously infused into the rats 3 hr later. MRI and behavioral analyses revealed that rats receiving GDNF-hMSCs or hMSCs exhibited increased recovery from ischemia compared with the control group, but the effect was greater in the GDNF-hMSC group. Thus, these results suggest that intravenous administration of hMSCs transfected with the GDNF gene using a fiber-mutant adenovirus vector may be useful in the cerebral ischemia and may represent a new strategy for the treatment of stroke.

Fig. 1

May-Giemsa staining of primary hMSCs (A) and GDNF-hMSCs (B). C: Secreted GDNF levels in supernatant of hMSCs (ng/10⁵ cells/48 hr) transfected with AxCAhGDNF-F/RGD (GDNF-hMSC) at MOI 300, 1,000, and 3,000 pu/cell. Scale bars = 20 μm.

Fig. 2

Evaluation of the ischemic lesion volume with MRI diffusion-weighted images (DWI). hMSCs or GDNF-hMSCs were intravenously injected immediately after the initial MRI scanning (3 hr). Images obtained 3, 6, 24, 72 hr and 7 days after MCAO in medium-injected (A1–5), hMSC-treated (B1–5), and GDNF-hMSC-treated (C1–5) groups. Summary of lesion volumes evaluated with MRI diffusion-weighted images at 3, 6, 24, 72 hr and 7, 14, 28 days in each group (D). *P < 0.05, **P < 0.01. Scale bar = 3 mm.

Fig. 3

Evaluation of the ischemic lesion volume with MRI T₂-weighted images (T₂WI). hMSCs or GDNF-hMSCs were intravenously injected immediately after the initial MRI scanning (3 hr). Images obtained 6, 24, 72 hr and 7, 14, 28 days after MCAO in medium-injected (A1–6), hMSC-treated (B1–6), and GDNF-hMSC-treated (C1–6) groups. Summary of lesion volumes evaluated with MRI T₂-weighted images in each group (D). *P < 0.05, **P < 0.01. Scale bar = 3mm.

Fig. 4

A—C: Brain slices stained with TTC to visualize lesions. TTC-stained brain slices from medium injection (A) and following intravenous delivery hMSCs and GDNF-hMSCs 3 hr after MCAO are shown in B and C, respectively. D: Lesion volumes in each group. *P < 0.05, **P < 0.01. E: GDNF in vivo levels assayed with ELISA. Levels of GDNF were significantly increased in the ischemic hemisphere of hMSC and GDNF-hMSC compared with nontreated rats. In addition, GDNF levels were greater in the ischemic hemisphere of GDNF-hMSC-transplanted compared with rats that received hMSC. Assays were obtained 7 days after MCAO induction. *P < 0.01, **P < 0.005. Scale bars = 3 mm.

Fig. 5

Intravenous administrated GDNF-hMSCs transfected with the reporter gene LacZ accumulated in the ischemic lesion hemisphere as observed 1 week postinjection. A: Vibratome section (1.0 mm) showing β-galactosidase reaction product for LacZ-positive cells (blue cells) in the ischemic hemisphere. Confocal images (B) demonstrating LacZ-positive cells in the penumbra in the lesion hemisphere. Scale bars = 3 mm in A; 100 μm in B.

Fig. 6

Treadmill stress test demonstrates that the maximum speed at which the rats could run on a motor-driven treadmill was greater in the hMSC- and the GDNF-hMSC-treated rats than in control. Moreover, the greatest velocity was achieved in the GDNF-hMSC group. Velocity is plotted for 1, 3, 7, 11, 15, 19, 23, 27, and 31 days after MCAO induction. *P < 0.05, **P < 0.01.