Overexpression Of Chemokine Receptors On Menstrual Blood-derived Endometrial Stem Cells And Its Therapeutic Effect In EAE Mice

BackgroundMultiple Sclerosis（MS）is an autoimmune mediated neurodegenerative disease of the central nervous system（CNS）.At present,there is no effective clinical cure,and available treatments,such as traditional immunosuppressive drugs cannot improve the disease condition or repair/regeneration of the damaged nerve tissue in patients.Recent reports suggested that stem cell transplantation might be a new treatment strategy for treating MS.Our previous results also confirmed that menstrual blood-derived mesenchymal stem cells（MenSCs）showed a therapeutic effect on experimental autoimmune encephalomyelitis（EAE）mice.However,transplanted stem cells（MenSCs）were not detected in the brain and spinal cord of experimental encephalomyelitis mice.We speculated that the transplanted MenSCs failed to migrate into the nervous system to differentiate and repair damaged neural tissue.Therefore,in this study we genetically modified MenSCs by overexpressing specific chemokine receptors to improve migration of MenSCs into CNS and effectively ameliorate inflammation and promote repair and/or regeneration of damaged nerve tissue in EAE mice.Objectives1.Construct human C-C（CCR2 and CCR6）and C-X-C（CXCR2 and CXCR3）chemokine receptor gene lentiviral expression vectors,and verify their effects on MenSCs（human menstrual blood-derived endometrial stem cells）biological characteristics in vitro;2.Establishment of chemokine-overexpressing cell lines MenSC^CCR2,MenSC^CCR6,MenSC^CXCR2 and MenSC^CXCR3;3.To study the chemotactic ability of MenSC^CCR2,MenSC^CCR6,MenSC^CXCR2 and MenSC^CXCR3 overexpressing chemokine receptors in vitro and in vivo;4.To clarify the effect of MenSC^CCR2,MenSC^CCR6,MenSC^CXCR2 and MenSC^CXCR3transplantation on EAE mice.Methods1.Using enzyme-linked immunosorbent assay（ELISA）to detect the expression levels of chemokines CCL2,CCL20,CXCL1 and CXCL10 in the peripheral and central nervous system of EAE mice;2.The full-length human CCR2,CCR6,CXCR2 and CXCR3 genes were prepared using PCR and packed into plasmids and then incorporated into the lentiviral vector pCDH-CMV-MCS-EF1-GFP-Puro to construct the lentiviral expression plasmids of these genes（referred to as pCDH-CCR2,pCDH-CCR6,pCDH-CXCR2 and pCDH-CXCR3）.Viruses were packaged by calcium phosphate,liposome(using Lipo Fiter^TM transfection reagent)and PEI（polyethyleneimine）methods,and among them the best virus packaging method was selected and optimized;3.Optimize the conditions of lentivirus infection of MenSCs,construct MenSC^CCR2,MenSC^CCR6,MenSC^CXCR2 and MenSC^CXCR3 cell lines overexpressing CCR2,CCR6,CXCR2 and CXCR3 genes separately;extract the total RNA of these different cells,reverse transcription and PCR amplification of CCR2,CCR6,CXCR2 and CXCR3 genes was performed,the expression of genes was detected using agarose electrophoresis,q PCR and flow cytometry simultaneously;4.The in vitro migration ability of MenSC^CCR2,MenSC^CCR6,MenSC^CXCR2 and MenSC^CXCR3 cells was detected by transwell assay;the number of cells migrated to the inflammatory site of CNS was detected by flow cytometry;5.C57BL/6 mice were immunized with synthetic myelin oligodendrocyte glycoprotein peptide fragment 35-55(MOG_35-55),and then randomly divided into control group and MenSC^pCDH control group（pCDH represents empty vector-packaged lentivirus）and MenSC^X（X represents four chemokine receptors）experimental groups,8 mice in each group.On the 6th day,MenSC^pCDH,MenSC^CCR2,MenSC^CCR6,MenSC^CXCR2 and MenSC^CXCR3 cells were injected through the tail vein(MenSC^pCDH control group and MenSC^X experimental group were each injected with 400μL of d PBS cell suspension containing 1×10~6corresponding MenSC^X,EAE model group was injected with 400μL d PBS),the mice score were recorded for disease after transplantation,and the therapeutic effects of MenSC^Xand ordinary MenSC^pCDH on EAE in mice were observed.Results1.ELISA results showed that in the EAE mice at different time points（day 6,day 10and day 19）the chemokines CCL2,CCL20,CXCL1 and CXCL10 were significantly increased compared to the unimmunized Na?ve group mice.Most of the central nervous system（brain and spinal cord）and some peripheral immune organs（draining lymph nodes and spleen）showed an elevated trend;and the expression of four chemokines in the spinal cord and brain of EAE mice at day 19 were significantly higher than the Na?ve control group,especially in the spinal cord,along with the higher disease score,the concentration also increased;2.Lentiviral expression plasmids pCDH-CCR2,pCDH-CCR6,pCDH-CXCR2 and pCDH-CXCR3 successfully packaged overexpressed chemokine receptor CCR2,CCR6,CXCR2 and CXCR3 and empty vector lentivirus,we found the liposome method is the most efficient for virus packaging;3.Comparing the infection efficiency of ultracentrifugation,PEG8000 precipitation method and directly using virus concentrate to MenSCs,it was found that the infection efficiency of directly using virus concentrate can reach upto 90%,which can meet the needs of subsequent experiments.Through flow cytometry and q PCR identification,the results showed that the corresponding chemokine receptors were successfully overexpressed in MenSCs;the results of laser confocal microscopy also confirmed that the overexpressed chemokine receptors were localized on the cell surface membrane;4.Transwell experiments and flow cytometry experiments showed that the ability of MenSC^X overexpressing chemokine receptors to migrate to the corresponding chemokines in vitro was significantly improved.After transplantation into EAE mice,the corresponding overexpression was detected in the central nervous system.MenSCs expressing chemokines;5.Animal experiments results demonstrated that the experimental group mice transplanted with MenSC^CCR2,MenSC^CCR6,MenSC^CXCR2 and MenSC^CXCR3 cells showed lower disease scores than the control group mice transplanted with MenSC^pCDH cells,which significantly reduced the severity of EAE in mice,and MenSC^CXCR2 increased therapeutic effect.ConclusionBased on the lentiviral vector pCDH-CMV-MCS-EF1-GFP-Puro,the lentiviral expression plasmid of four human chemokine receptors CCR2,CCR6,CXCR2 and CXCR3were successfully constructed.This lentiviral expression plasmid can efficiently mediate CCR2,CCR6,CXCR2 and CXCR3 genes are expressed on the surface of MenSCs,and they present characteristics of stem cells in vitro.Compared with wild-type MenSCs,MenSCs expressed with CCR2,CCR6,CXCR2 and CXCR3 can migrate to EAE inflammation sites in vivo,and significantly reduced the disease scores in EAE mice.