Induced Pluripotent Stem Cells Improve The Brain Microenvironment In Ischemic Stroke

ObjectiveThis study aimed to evaluate the therapeutic effect of induced pluripotent stem cells?i PSCs?in rats with ischemic stroke,and explore the dynamic and long-term changes of protein expression in brain microenvironment after i PSCs transplantation.MethodsMale Sprague-Dawley rats were randomly assigned to one of the following four groups:Sham group,phosphate-buffered saline?PBS?group,i PSCs group,and neural stem cells?NSCs?group.On Day 0,all rats underwent middle cerebral artery occlusion?MCAO?surgery except the rats in Sham group.On Day 3,all rats were injected with i PSCs,NSCs?2.0×10⁶,20?L?or PBS of the same volume,except the rats in Sham group.On Day 1,7 and 14,rats were subjected to ¹⁸F-FDG PET scans followed by neurological tests for evaluating the glucose metabolism and neurologic function.On Day 7 and 14,a part of rats from i PSCs group and PBS group were sacrificed for quantitative proteomics and western blot analysis.In addition,¹¹C-methionine?MET?PET scans were performed on Day 14 in order to detect tumor formation.Finally,the rest rats were sacrificed for hematoxylin-eosin?HE?staining and immunofluorescent staining.ResultsSemiquantitative assessments of ¹⁸F-FDG uptake showed no significant increase in i PSCs and NSCs groups compared with PBS group on Day 7;on Day 14,significantly increased ¹⁸F-FDG uptake was observed in i PSCs group compared with PBS group?P=0.010?,whereas NSCs group not.On Day 7,the neurological scores in i PSCs group were significantly higher than those in NSCs group?P=0.024?as well as PBS group?P<0.001?,and the NSCs group had higher scores than PBS group?P=0.029?;On Day14,the scores in i PSCs group were significantly increased compared with PBS group?P=0.047?,and no significant difference was observed between NSCs group and PBS group.The immunofluorescence showed transplanted stem cells could survive and migrate to the ischemic area,then differentiate into nerve cells and angiogenic cells.HE staining showed the damage of the cells was milder with less vacuoles and more regular arrangement in i PSCs group compared with PBS and NSCs groups.According to the¹¹C-MET PET imaging,no tumor formation was detected within 2 weeks.Quantitative proteomics identified a total of 39 differentially expressed proteins between i PSCs group and PBS group on Day 7 and 14,and these proteins were related to neuronal survival,axonal remodeling,mitochondrial function,and anti-oxidative stress.The western blot results of representative protein showed similar trends with the corresponding i TRAQ results.ConclusionPET imaging and neurological tests showed more significant effects of i PSCs on promoting glucose metabolism and neurological function in cerebral ischemic rats than NSCs.The differentially expressed proteins in i PSCs transplanted rats identified by quantitative proteomics were related to neuronal survival,axon remodeling,oxidative stress and mitochondrial function,indicating the positive microenvironmental changes in ischemic area,and thus provided new insights into the pathophysiology of ischemic stroke and its therapeutic strategies.