Pre-conditioned Canine Bone Mesenchymal Stem Cells For Treatment Of Ischemic Stroke

Part ?:G-CSF and hypoxic condition improve bone marrow mesenchymal stem cell's proliferation,neural differentiation and migrationObjective:Recent studies have indicated that transplantation using bone marrow mesenchymal stem cells(BMSCs)is emerging as a potential regenerative therapy after ischemic attacks in the brain.However,it has been questioned because very few transplanted BMSCs are detected homing to and survived in the ischemic region.Improving the cell viability and migration ability under the complex ischemic condition seems very important.The aim of our study is to identify new strategy which can improve the cell survival and migration rate of transplanted cells.Methods:BMSCs were isolated from bone marrow obtained from humerus of dogs by density-gradient separation.Confluent cells at passage 3 were used for all experiments.Hypoxic treatment of cells were cultured in hypoxia incubator and the O2 concentration in the chamber was maintained at 1%.Cells of normoxia control were subjected to the same procedures except that they 'were exposed to the 21%O2 incubator.Cell apoptosis,cell viability,cell migration and neuronal differentiation ability were evaluated after hypoxic condition.Results:No significant apoptosis was observed in hypoxic preconditioning(HP)group,our study confirmed that HP improves BMSCs proliferation and migration.Meanwhile,neural induction of BMSCs under hypoxic condition exhibited significant superior results than normoxic condition.Additionally,the addition of G-CSF in HP-BMSCs culture media promoted HP efficiency on BMSCs.Conclusion:These findings shed light on novel efficient strategy on the prosperity of BMSCs.Hypoxic preconditioning and cultured with G-CSF may become a promising therapeutics for cell-based therapy in the treatments of ischemia stroke.Part II:Efficiency of genetically modified canine bone mesenchymal stem cells labeled with superparamagnetic iron oxide differentiating into neural-like cellsObjective:Bone mesenchymal stem cells(BMSCs)are potential therapeutic material for many intractable diseases,such as ischemic stroke.To explore the potential of genetically modifi,ed canine bone mesenchymal stem cells for improving neural function in canine models with ischemic stroke.Methods:Canine bone marrow-derived mesenchymal stem cells(BMSCs)were initially labeled with SPIO and transfected with brain-derived neurotrophic factor(BDNF)gene.The percentage of iron contented cells was calculated according to the positively stained cell numbers.The optimized MOI was calculated by multiplication of the percentage eGFP positive cells by the average cellular survival rate.Then the BMSCs were induced to differentiate into neuron-like cells.Neural markers were evaluated by real-time PCR analysis and immunofluorescence.Results:Our results confirmed the optimized Multiplicity of Infection of lentivirus for canine BMSCs and showed that BDNF modified BMSCs labeled with SPIO can be induced into neuron-like cells with high efficiency and minimally affecting cell viability.Additionally,after neural differentiation,the double-labeled cells expressed significantly higher target gene BDNF and neural markers.Conclusion:The overexpression of BDNF may contribute to BMSCs' neural differentiation,and may have potential benefit for further BMSC based therapy in vivo.Part ?:Comparison of arterial and venous whole blood and plasma clots by by thromboelastography and magnetic resonance imaging in vitroObjective:To compare arerial and venous whole blood and plasma clots by thromboelastography and magnetic resonance imaging in vitro.Methods:Plasma clot samples were centrifuged from whole blood at 4000r/min for 10 minutes.Samples were grouped as:arterial whole blood group(aw),venous whole blood group(vw),arterial plasma group(ap)and venous plasma group(vp).Thromboelastography(TEG)and magnetic resonance imaging(MSI)were used to test clot strength and signal.Results:TEG assay showed MA value of whole blood groups were significantly higher than that of plasma groups,meanwhile,MA value of vp group was significantly higher than that of ap group(P<0.001,P=0.041,respectively).MRI showed the plasma clots were much more homogeneous than whole blood samples.T2WI value in vp group was higher than that of ap group,but statistical significance were not found(364±17,358±20,P>0.05).Conclusion:Plasma clots were more stable than whole blood clots.Additionally,clots from venous plasma were stronger than those from arterial plasma.Part ?:Genetically modified canine bone mesenchymal stem cells are effective in improving neural function after ischemic strokeObjective:Bone mesenchymal stem cells(BMSCs)are potential therapeutic material for many intractable diseases,such as ischemic stroke.The potential of genetically modified canine bone mesenchymal stem cells to improve neural function was explored in canine models with ischemic stroke.Methods:Male beagle dogs aged 2 years were used as the model of ischemic stroke by autologous clots embolizaion.Brain derived neurotropic factor(BDNF)gene modified BMSCs were acquired by lentivirus transfection.After labeled with superparamagnetic iron oxide(SPIO)nanoparticles,BDNF-BMSCs were injected into beagle dogs via internal carotid artery.Cell tracking,infarction volume and neurological scores were used to assess the repairmen roles of BDNF-BMSCs.Results:Compared to BDNF-and MOCK groups,genetically modified BDNF+ BMSCs showed nearly ten times higher BDNF expression in vitro.After neural induction,the neural markers such as nestin,TUJ1 and GFAP also showed 300 to 600 percent improvement.The canine ischemic stroke model was successfully induced with autologous thrombosis,which was made by venous plasma and showed more stable on MRI and stronger on TEG assay,when compared with whole blood or arterial plasma derived thrombosis.The labeling ratio and MR signal of BMSCs showed time dependent MR imaging could only track labeled BMSCs efficiently within 4 weeks.The ischemic infarction volume measured on 24h-T2WI were 4173.23±603.92 mm3.One week after embolization,the infarction volume on T2WI decreased to 327321±616.05 mm3.Infarction volume in BDNF+ BMSC group and BDNF-BMSC group ware significantly decreased two weeks later after transplatation compared with control group.Moreover,BDNF+ BMSC group still showed decreased infarction volume 4 weeks later compared with BDNF-BMSC group.in BDNF+ BMSC group and BDNF-BMSC group were significantly decreased at first week compared with control group.Moreover,BDNF+ BMSC group still showed decreased neurological scores at second week after transplatation compared with BDNF-BMSC group.Conclusion:The increased BDNF expression may be benefit to neural development.This work would pave the way of genetically modified BMSCs transplantation in ischemic stroke to clinical therapies and researches.