Therapeutic benefits by human mesenchymal stem cells (hMSCs) and Ang-1 gene-modified hMSCs after cerebral ischemia

Abstract

Transplantation of human mesenchymal stem cells (hMSCs) prepared from adult bone marrow has been reported to ameliorate functional deficits after cerebral artery occlusion in rats. Although several hypotheses to account for these therapeutic effects have been suggested, current thinking is that both neuroprotection and angiogenesis are primarily responsible. In this study, we compared the effects of hMSCs and angiopoietin-1 gene-modified hMSCs (Ang-hMSCs) intravenously infused into rats 6 h after permanent middle cerebral artery occlusion. Magnetic resonance imaging and histologic analyses revealed that rats receiving hMSCs or Ang-hMSCs exhibited comparable reduction in gross lesion volume as compared with the control group. Although both cell types indeed improved angiogenesis near the border of the ischemic lesions, neovascularization and regional cerebral blood flow were greater in some border areas in Ang-hMSC group. Both hMSC- and Ang-hMSC-treated rats showed greater improved functional recovery in the treadmill stress test than did control rats, but the Ang-hMSC group was greater. These results indicate the intravenous administration of genetically modified hMSCs to express angiopoietin has a similar effect on reducing lesion volume as hMSCs, but the Ang-hMSC group showed enhanced regions of increased angiogenesis at the lesion border, and modest additional improvement in functional outcome.

Figure 1

Flow cytometric analysis of surface antigen expression on primary hMSC (A) and Ang-hMSC (B). The cells were immunolabeled with FITC-conjugated monoclonal antibody specific for the indicated surface antigen. Dead cells were eliminated by forward and side scatter. Angiopoietin-1 production of hMSC, Ang-transfected hMSC were summarized (C). Levels of Ang-1 in the supernatant were shown.

Figure 2

Evaluation of the ischemic lesion volume with DWIs and T₂WIs. Human mesenchymal stem cells or Ang-hMSCs were intravenously injected immediately after the initial MRI scanning (6 h after MCAO). Diffusion-weighted image obtained 6 h MCAO in medium-injected (A-1), hMSC-treated (A-2), and Ang-hMSC-treated group (A-3). T2-weighted images obtained 6 h, 1, 3, and 7 days MCAO in medium-injected (B-1-1 to -4), hMSC-treated (B-2-1 to -4), and Ang-hMSC-treated group (B-3-1 to -4). Bar=5 mm. (C) A summary of lesion volumes evaluated with MRI (T₂WI) were obtained 6 h, 1, 3, and 7 days after MCAO in rats treated with medium (control), hMSC, or Ang-hMSC.

Figure 3

TTC Brain sections slices stained with TTC to visualize the ischemic lesions 7 days after MCAO. 2,3,5-Triphenyl tetrazolium chloride-stained brain slices from (A) mediuminjected MCAO model rats, (B) following hMSC-treated, and (C) Ang-hMSC-treated groups. (D) Seven days after MCAO, there was a reduction in the lesion volume assayed using TTC staining for both hMSC and Ang-hMSC groups. Bar=3 mm.

Figure 4

Seven and twenty-eight days after MCAO, the angiogenesis in boundary zone was analyzed using a three-dimensional analysis system. (A-1) Three-dimensional capillary image with systemically perfused FITC-dextran in the normal rat brain is shown. The total volume of the microvessels in the sampled lesion site decreased (A-2) 28 days after MCAO, but was greater in (A-3) the hMSC-treated group, and (A-4) the Ang-hMSC-treated group. These results are summarized in (B) (7 days after MCAO) and (C) (28 days after MCAO). The ratio (ispilateral/contralateral) was significantly higher in both the hMSC- and the Ang-hMSC-treated groups as compared with the medium-treated group. Also the ratio of the Ang-hMSC-treated group was significantly higher than the ratio of the hMSC-treated group. Bar=150 μm.

Figure 5

Intravenously –administered Ang-hMSCs accumulated in and around the ischemic lesion hemisphere. Angiopoietin-1 gene-modified human mesenchymal stem cells were transfected with the reporter gene LacZ. (A) Transplanted LacZ-positive Ang-hMSCs (blue cells) were present in the ischemic lesion. (B) Confocal images demonstrating a large number of LacZ-positive cells in the lesion hemisphere. Ang-hMSCs differentiated into the endothelial lineage. (C-1 and C-2) Confocal images show the transplanted cells (C-2: LacZ in red) and endothelial cells (C-1: vWF in green). Panel C-3 confirms the co-labeling of LacZ/vWF in the cells. The cell density of vWF-positive cells are shown in D. Panel E demonstrates the cell density of vWF-positive cells co-localized with LacZ. Bar: (A)—3 mm; (B)—150 μm; and (C)—50 μm.

Figure 6

Evaluation of hemodynamic state (rCBF maps) with PWIs. Human mesenchymal stem cells or Ang-hMSCs were intravenously injected immediately after the initial MRI scanning (6 h after MCAO). Images obtained 6 h, 3, and 7 days MCAO in (A) medium-injected, (B) hMSC-treated, and (C) Ang-hMSC-treated group. (D–G) Summary of rCBF evaluated with PWI in each groups. (D) ROI-1, (E) ROI-2, (F) ROI-3, and (G) ROI-4. Regional cerebral blood flow ratio (ischemic lesion/contralateral lesion) at 6 h, 3, and 7 days after MCAO are summarized in D–G. Bar=3 mm, *P<0.05.

Figure 7

The treadmill stress test demonstrates that the maximum speed at which the rats could run on a motor-driven treadmill was faster in the hMSC- and the Ang-hMSC-treated rats than control. One day after MCAO, rats that received Ang-hMSC showed higher running speed compared with the medium alone and the hMSC group. From 3 days on after MCAO, the hMSC and Ang-hMSC groups attained higher velocities but the Ang-hMSC group was greater. The highest level of functional improvement was attained at 7 days.