Therapeutic Benefit Of Transplantation Of Marrow Mesenchymal Stem Cells Over-expressing CXCR4 In Stroke Rat

Ischemic stroke remains a leading cause of adult disability and no pharmaco- logical treatment is presently available to protect brain tissue from the injury that are arosed by ischemia and reperfusion. Accumulated studies suggest that bone marrow mesenchymal stem cells (MSCs) participate in neuroprotection following Stroke. Increasing evidences suggest that systemically transplanted MSCs can survive, migrate toward injured tissue, and promote recovery of neurological function following cerebral infarction. It seems that the injured tissues can attract MSCs and mediate their migration behavior. However, the mechanisms regulating MSCs migration and accumulation in the injured brain remain to be revealed.Stromal cell-derived factor-1(SDF-1) and its unique receptor, CXCR4 play an important role in stem cell migration, chemotaxis, expression of adhesion molecules, engraftment, proliferation, and survival. Recent studies have shown that SDF-1 is expressed in the ischemic boundary zone of the brain and plays an important role in the migration of the transplanted cells. Due to low native levels of CXCR4 expression in MSCs, they migrate sluggishly toward injured tissue. We hypothesized that CXCR4 gene-modified MSCs will promote stem cell recruitment and injured brain tissue regeneration.In this study, we constructed the lentiviral vectors (LV)carrying the CXCR4 and genetically engineered rMSCs overexpressing CXCR4, and focused on that over-expression of CXCR4 in rMSCs will enhance their engraftment and protect brain subjected to MCAO. This study consisted of four parts.Part1:Construction and identification of the lentiviral vector carrying CXCR4Materials and Methods1) The total RNA was isolated from SD rat's liver with Trizol. 2) The CXCR4 gene was amplified by RT-PCR.3) CXCR4 was inserted into the transfer vector of lentivirus after being digested with restriction endonuclease.4) Then the product pNL-CXCR4-IRES2-EGFP was confirmed by sequencing and being digested with restriction endonuclease.Results1) The result of restriction enzyme digestion and sequencing showed that the full-length fragment of CXCR4 gene was successfully cloned into the transfer vector of lentivirus.Conclusions1) The lentiviral vectors carrying the CXCR4 gene were successfully constructed.Part2:Construction of rMSCs over-expressing CXCR4 Materials and Methods1) pNL-CXCR4-IRES2-EGFP was cotransfected along with pHELPER and pVSVG into 293T to package lentivirus particles.2) According to the enhanced green fluorescent protein (EGFP) expression,the functional titer was determined by flow cytometry after transduction into 293T cells.3) Lentiviral transduction was carried out to over-express either CXCR4/EGFP (CXCR4-rMSCs group), siRNA targeting CXCR4/EGFP (siRNA-rMSCs group) or EGFP alone (null-rMSCs group) in rMSCs. The rMSCs were selected for stable integrants by using EGFP reporter gene.4) The expressing of CXCR4 gene in rMSCs was evaluated with RT-PCR, Western blotting, cellular immunofluorescence and flow cytometry.Results1) Lentiviral vector can be packaged in 293T cells by cotransfection.2) The rMSCs from CXCR4-rMSCs group, siRNA-rMSCs group or null-rMSCs group strongly expressed EGFP.3) The result of FCM showed that CXCR4 expression was significantly higher in CXCR4-rMSCs group as compared with that of null-rMSCs group, and was lowest in siRNA-rMSCs group. The result was confirmed by RT-PCR, Western blotting and cellular immunofluorescence.Conclusions1) The rMSCs overexpressing the CXCR4 gene were successfully constructed. Part3:In vitro migration assay of rMCSs induced by SDF-1Materials and Methods1) In vitro migration of rMSCs in response to SDF-1αwas assessed in a 48-well microchemotaxis chamber using polycarbonate membranes with 8μm pore size. The rMSCs were added to the upper chambers. SDF-1αwas added to the lower wells in different concentrations. The migrated cells were counted and the migration index was calculated to show the difference of migration.2) The rMSCs were incubated by anti CXCR4 monoclonal antibody then the chemotaxis assay was performed..Results1) The result showed that exposure of rMSCs to SDF-1αcaused a robust cell migration in a concentration dependent manner.2) The number of migrated rMSCs was significantly higher in CXCR4 -rMSCs group than that in null-rMSCs group. However, the migration of the rMSCs from the siRNA-rMSCs group in response to SDF-1αwas blocked.3) The migration of the rMSCs incubated by anti CXCR4 monoclonal antibody in response to SDF-1αwas blockedConclusions1) Over expression of CXCR4 enhances the ability of rMSCs to respond to SDF-1 induced chemotaxis.Part4:Migration, differentiation, neuroprotection and angiogenesis of donor rMSCs in stroke ratsMaterials and Methods1) The healthy adult male SD rats were divided into four groups: CXCR4-rMSCs group, siRNA-rMSCs group, null-rMSCs group and PBS group.Rats were received rMSCs or PBS transplantation via femoral vein injection 24 hours after the left middle cerebral artery occlusion (MCAO).2) 7 days after MCAO, the distribution of EGFP+ cells was observed under the fluorescent microscope.3) The total infarct volumes were calculated by 2,3,5- triph-enyltetrazolium chloride (TTC) stain.4) Behavioral tests ( modified Neurological Severity Score [mNSS]) was performed 1 and 7 days after MCAO.5) The expressing of CXCR4, neuronal marker NSE, astrocytic marker GFAP, and vascular phenotypes (vWF) in EGFP+ rMSCs was evaluated with immunohistochemical fluorescence.6) The volumes of the microvessels were analysed with a laser scanning confocal imaging system .Results1) The EGFP-positive cells were found in multiple areas of the ipsilateral hemisphere including cortex, striatum, and few cells were observed in the contralateral hemisphere. The number of migrating rMSCs to damaged brain area especially in ischemic boundary zone was significantly higher in CXCR4-rMSCs group as compared with null-rMSCs group. However, the number of EGFP-positive cells was significantly reduced in siRNA-rMSCs group as compared with null-rMSCs group.2) The average infarct volumes were significantly reduced in the CXCR4-rMSCs and null-rMSCs groups compared with those of the PBS and siRNA -rMSCs groups. The infarct volume was significantly decreased in the CXCR4 -rMSCs group as compared with the null-rMSCs group. There was no significant difference found between the infarct volumes of the siRNA -rMSCs and the PBS group..3) On 7th day, the mNSS scores of the null-rMSCs and CXCR4-rMSCs groups were significantly decreased compared with those of the siRNA-rMSCs and PBS groups. In addition, the score of the CXCR4-rMSCs group was significant decrease compared with that of the null-rMSCs group. There was no significant difference between the sores of the siRNA-rMSCs and PBS group. 4) Double-label fluorescence immunohistochemistry of brain sections of CXCR4-rMSCs group revealed that a number of EGFP-positive cells in the cerebral cortex, were neuronal marker NSE and astrocytic marker GFAP positive, and several EGFP positive cells showed vascular phenotypes (vWF) positive. Most of EGFP positive cells were CXCR4 positive.5) The result of three-dimensional image acquisition of brain slices indicated that the capillary vascular volume ratios were significantly higher in both null-rMSCs and CXCR4-rMSCs groups as compared with those in the PBS and siRNA-rMSCs groups, and the ratio in the CXCR4-rMSCs group was significantly higher than that in the null-rMSCs group. There was no significant difference found between the siRNA-rMSCs and the PBS group.Conclusions1)The interaction of locally produced SDF-1αand CXCR4 expressing on the rMSCs surface plays an important role in the migration of transplanted cells to infarcted brain.2)Overexpressing of CXCR4 in rMSCs was extremely effective in their engraftment in the infarcted brain for post-infarction recovery of neural function.