Mesenchymal stem cells derived from peripheral blood protects against ischemia

Abstract

Intravenous delivery of mesenchymal stem cells (MSCs) prepared from bone marrow (BMSCs) reduces infarction volume and ameliorates functional deficits in a rat cerebral ischemia model. MSC-like multipotent precursor cells (PMSCs) have also been suggested to exist in peripheral blood. To test the hypothesis that treatment with PMSCs may have a therapeutic benefit in stroke, we compared the efficacy of systemic delivery of BMSCs and PMSCs. A permanent middle cerebral artery occlusion (MCAO) in rat was induced by intraluminal vascular occlusion with a microfilament. Rat BMSCs and PMSCs were prepared in culture and intravenously injected into the rats 6 h after MCAO. Lesion size was assessed at 6 h, and 1, 3, and 7 days using MR imaging and histology. The hemodynamic change of cerebral blood perfusion on stroke was assessed the same times using perfusion-weighted image (PWI). Functional outcome was assessed using the treadmill stress test. Both BMSCs and PMSCs treated groups had reduced lesion volume, improved regional cerebral blood flow, and functional improvement compared to the control group. The therapeutic benefits of both MSC-treated groups were similar. These data suggest that PMSCs derived from peripheral blood could be an important cell source of cell therapy for stroke.

FIG. 1

May–Giemsa staining of BMSCs (A) and PMSCs (B). Scale bar = 20 μm. Flow cytometric analysis of surface antigen expression on BMSCs (C) and PMSCs (D). The cells were immunolabeled with FITC-conjugated and PE-conjugated monoclonal antibody specific for the indicated surface antigen. Dead cells were eliminated by forward and side scatter.

FIG. 2

Evaluation of the ischemic lesion volume with diffusion-weighted images (DWI). BMSCs or PMSCs were intravenously injected immediately after the initial MRI scanning (6 h after MCAO). Images obtained 6 h, and 1, 3, and 7 days MCAO in medium-injected (A1–4), BMSC-treated (B1–4), and PMSC-treated group (C1–4). Summary of lesion volumes evaluated with DWI in each groups (D). Scale bar = 3 mm. *p < 0.05.

FIG. 3

Evaluation of the ischemic lesion volume with T₂-weighted images (T₂WI). BMSCs or PMSCs were intravenously injected immediately after the initial MRI scanning (6 h after MCAO). Images obtained 6 h, and 1, 3, and 7 days MCAO in medium-injected (A1–4), BMSC-treated (B1–4), and PMSC-treated group (C1–4). Summary of lesion volumes evaluated with T₂WI in each groups (D). Scale bar = 3 mm. *p < 0.05.

FIG. 4

TTC brain section slices stained with 2,3,5–triphenyl tetrazolium chloride (TTC) to visualize the ischemic lesions 7 days after MCAO. TTC-stained brain slices from medium-injected MCAO model rats (A1), following BMSC-treated (A2), and PMSC-treated (A3) groups. The Sections were also stained with hematoxylin and eosin at 7 days post-MCAO. Although a larger number of apparent inflammatory cells were obvious in the lesion without cell transplantation (B1), parenchymal brain tissue was greatly preserved in the BMSC-treated (B2) and PMSC-treated group (B3). Inflammatory cells in the lesion were shown in insert of B1. On the other hand, preserved neurons in the lesion were shown in insert of B2 and B3. Intravenously administrated BMSCs and PMSCs accumulated in and around the ischemic lesion hemisphere. BMSCs and PMSCs were transfected with the reporter gene LacZ. Transplanted LacZ-positive MSCs (blue cells) were present in the ischemic lesion (BMSCs, C2; PMSCs, C3). Brain from control (MSC transplantation without LacZ transfection) injected animals with comparable X-gal staining is shown in C1. Confocal images (BMSCs, D2; PMSCs, D3) demonstrating a large number of LacZ-positive cells in the lesion hemisphere. Confocal image of non-treated group is shown in D1. Scale bar = 3 mm (A, C), 40 μm (B), 10 μm (insert of B), 50 μm (D). (Color images can be found online at www.liebertpub.com/jon)

FIG. 5

Region of interest (ROI) for dynamic susceptibility contrast-enhanced perfusion-weighted imaging (PWI) analysis (C). PWI analysis was carried out at four regions of interest (ROI) indicated by the boxed numbered areas on the lesion side of the brain (A, B).

FIG. 6

Evaluation of hemodynamic state (rCBF maps) with perfusion-weighted images (PWI). BMSCs or PMSCs were intravenously injected immediately after the initial MRI scanning (6 h after MCAO). Images obtained 6 h, and 1, 3, and 7 days after MCAO in medium-injected (A), BMSC-treated (B), and PMSC-treated group (C). Summary of rCBF evaluated with PWI in each group: ROI-1 (D), ROI-2 (E), ROI-3 (F), and ROI-4 (G). rCBF ratio (ischemic lesion/contralateral lesion) at 6 h, and 1, 3, and 7 days after MCAO is summarized in D–G. Scale bar = 3 mm. *p < 0.05.

FIG. 7

Seven days after MCAO, the angiogenesis in boundary zone was analyzed using a three-dimensional analysis system. (A) Three-dimensional capillary image with systemically perfused FITC-dextran in the normal rat brain. The total volume of the microvessels in the sampled lesion site decreased 7 days after MCAO (B), but was greater in the BMSC-treated group (C) and the PMSC-treated group (D). Scale bar = 100 μm.

FIG. 8

The treadmill stress test demonstrated that the maximum speed at which the rats could run on motor-driven treadmill was faster in the BMSCs and PMSCs rats than control. Velocity is plotted for three times after MCAO induction.