Chemokine SDF-1 And Fractalkine Improve The Migration Of Intravenously Grafted Human Bone Marrow Stromal Cells Toward Ischemic Brain Lesions In Rats

Ischemic stroke has become one of major diseases to harm the health and life quality of the aged. Stem cell transplantation can promote the repair of brain damage and improve neurological function. It has good prospects in clinical applications. As ideal donor cells, transplanted bone marrow stromal cells (BMSCs) have been demonstrated to have protective effects by many experimental studies of cerebral ischemia. The specific mechanisms involved in their migration to lesions are still to be fully elucidated. Chemokines form a superfamily that shares a common structure and function-related peptide. The basic function of a chemokine is to induce the directional chemotaxis of cells with corresponding receptors. It has been reported that chemokines and their receptors may be involved in the promotion of directional migration of transplanted BMSCs. Stromal cell-derived factor-1 (SDF-1) and fractalkine are the only chemokines constitutively expressed in the central nervous system (CNS). Studies have shown up-regulated expression of SDF-1 and fractalkine in select CNS lesions, such as those caused by inflammation, ischemia and hypoxia. Through interaction with their receptors, CXCR4 and CX3CR1 respectively, they promots the activation of microglial cells and their migration to the lesions, elevates the number of mononuclear cells, natural killer cells and T lymphocytes in the blood, plays a critical role in the clearance of necrotic tissue and promotes neurofunction repair. In other situations, it may aggravate the injury. Other studies found that BMSCs expressed CXCR4 and CX3CR1. These results indicate that SDF-1/CXCR4 and fractalkine/CX3CR1 may be involved in the induction of directional migration of transplanted BMSCs to injured brain regions. But there is still a lack of in vivo studies have reported. This study aimed to explore whether SDF-1/CXCR4 and fractalkine/CX3CR1 play an important role in the induction of directional migration of transplanted hMSCs to ischemic brain tissue. Furthermore, we aimed to explore the possible mechanisms involved in BMSCs migration.Methods1.The migration of intravenously grafted human bone marrow stromal cells toward ischemic brain lesionshMSCs were isolated from human bone marrow by combination of gradient centrifugation and different adherent time method. Cell surface markers were tested by flow cytometer. The transient middle cerebral artery occlusion (MCAO) was induced using a method of intraluminal vascular occlusion. At 24 hours after the onset of cerebral ischemia, model animals received 2×106 hMSCs transplantation. At 1, 3, and 7 days after cell transplantation, the directional migration of transplanted hMSCs to the damaged region was observed through detection of mAb1281 positive cells.2.SDF-1 and fractalkine expression in the infarcted brain after transient middle cerebral artery occlusionA rat model of MCAO was established. At 2, 4 and 8 days after cerebral ischemia, the model animals were sacrificed, the tissues were processed, and the expression of SDF-1 and fractalkine in the ischemic brain was determined by real time PCR and immunohistochemistry.3.CXCR4 and CX3CR1 expression in human bone marrow stromal cells hMSCs were isolated, purifIed and amplified. The CXCR4 and CX3CR1 expression were detected by real-time PCR , western blotting and immunocytochemistry. Then, after short-term exposure of hMSCs to 3% oxygen, the changes of CXCR4 and CX3CR1 expression were detected.4.The impact of down-regulation of CXCR4 or CX3CR1 expression to the migration of transplanted human bone marrow stromal cells to the damaged brainhMSCs were transduced with CXCR4 or CX3CR1 shRNA construct by lentivirus-mediated gene transfer at a multiplicity of infection (MOI) of 20. Transduction efficiency was measured by determining the frequency of green ?uorescent protein (GFP)-positive cells using flow cytometry. Cells were harvested 5-7 days following transduction, CXCR4 or CX3CR1 expression was determined by real-time PCR and western blotting. The MCAO rats were divided into 4 groups: rats in Group 1 (n=18) received 2×106 CXCR4 shRNA transduced hMSCs; rats in Group 2 (n=18) received CX3CR1 shRNA transduced hMSCs; rats in Group 3 (n=18) received 2×106 control-transduced hMSCs; rats in Group 4 (n=18) received PBS as a control. Rats were sacrificed at 1, 3, and 7 days after transplantation, and hMSCs distribution were analyzed by immunohistochemistry.Results1.The migration of intravenously grafted human bone marrow stromal cells toward ischemic brain lesionsAll of the BMSCs had a fibroblast-like morphology in culture and were uniformly positive for CD105 (99.38%) and CD29 (99.13%) and negative for CD34 (0.78 %) and CD45 (0.40 %), as determined by flow cytometry. MCAO was induced using a method of intraluminal vascular occlusion. Once the animals awoke, behavioral changes were evaluated according to the standard described by Longa et al. Rats with scores of 2-3 were included in the next experiments. The brains were stained with TTC 24 hours after MCAO. Normal brain (gray matter) tissue typically stains with TTC, but infarcted lesions show no or reduced staining. At 1, 3, and 7 days after cell transplantation, the transplanted hMSCs were mainly distributed in the ischemic hemisphere. Only a few of mAb1281 positive cells scattered in the heart, liver, spleen, lung and kidney.2.SDF-1 and fractalkine expression in the infarcted brain after transient middle cerebral artery occlusionSDF-1 mRNA expression was found to be up-regulated in the injured hemisphere on days 2, 4 and 8, in comparison with the normal control tissues (2.285, 2.543, and 1.710 times respectively). fractalkine mRNA expression was 1.154, 2.453, and 1.341 times higher at 2, 4, and 8 days respectively (P<0.05). Similarly, SDF-1 and fractalkine expression was dramatically up-regulated in the injured hemisphere at 2, 4, and 8 days after ischemia, as determined by immunofluorescence analysis. Quantification revealed that the density of SDF-1 or fractalkine-immunoreactive cells in the ischemic hemisphere was significantly (p<0.01) increased at 2, 4, and 8 days after ischemia.3.CXCR4 and CX3CR1 expression in human bone marrow stromal cellsThe result of immunocytochemistry revealed the localization of CXCR4 and CX3CR1 expression on the membranes and in the cytoplasm. In vitro study using real-time PCR and western blotting revealed that CXCR4 and CX3CR1 were expressed in normal cultured hMSCs. Exposure of hMSCs to 3% oxygen increased expression of the CXCR4 and CX3CR1, both as mRNA and as protein.4.The impact of down-regulation of CXCR4 or CX3CR1 expression to the migration of transplanted human bone marrow stromal cells to the damaged brainTransduction efficiencies were more than 90%, as indicated by the frequency of GFP-positive cells by ?ow cytometry. Real-time PCR analysis revealed that, the CXCR4 mRNA level in cells expressing the CXCR4 shRNA construct were about 80-fold lower (P<0.01) than those of non-transduced and control-transduced cells, and the CX3CR1 mRNA level in cells expressing the CX3CR1 shRNA construct were about 70-fold lower (P<0.01) than those of non-transduced and control-transduced cells. Also, in the hMSCs transduced with CXCR4 or CX3CR1 siRNA, a reduced level of CXCR4 or CX3CR1 protein was detected by western blotting. GFP-labeled cells were more abundant (P<0.01) in the ischemic hemisphere of rats injected with control-transduced hMSCs, than in that of rats injected with CXCR4 knock-down or CX3CR1 knock-down hMSCs at 1, 3 and 7 days after cell transplantation.Conclusion1.Intravenously grafted bone marrow stromal cells can directionally migrate to ischemic brain lesion.2.SDF-1 and fractalkine expression was up-regulated in the ischemic brain.3.Bone marrow stromal cells expressed CXCR4 the receptor of SDF-1and CX3CR1 the receptor of fractalkine.4.CXCR4-knockdown or CX3CR1-knockdown dramatically decreased the migration of transplanted human bone marrow stromal cells to the ischemic brain.5.The interactions of SDF-1 with its specific receptor CXCR4 and fractalkine with its receptor CX3CR1 were involved in the directional migration of transplanted bone marrow stromal cells to the ischemic damaged brain region.