The Safety, Efficacy And Mechanism Of Neural Stem Cell Transplantation In The Treatment Of Ischemic Stroke In Cynomolgus Monkey Model

Section One:Bibliometric Analysis of Stem Cell Transplantation for Treatment of Nervous System DisordersObjective:Ischemic stroke and Parkinson's disease(PD)are two research hotspots in treating neurological diseases with stem cell transplantation.Bibliometrics is used to combine mathematical and statistical methods to analyze bibliographic information quantitatively,and it becomes increasingly popular in the field of medicine.In this study,we used bibliometrics to perform an intuitive and quantitative analysis of stem cell transplantation to treat neurological diseases to demonstrate the landscape of the development of this research field and provide a basis for formulating stem cell-related strategies.Methods:In this study,the Web of Science core collection database was used as the data source,and the time was limited from 1999 to 2018.The literature search on stem cell treatment of ischemic stroke and PD was conducted.HistCite,VOSviewer,and Medical Text Indexer were used to analyze the changes in literature characteristics and medical subject headings in different periods.Results:In the field of stem cell treatment of ischemic stroke,the bibliometric findings were as follows:the annual output of publications kept rising in a fluctuating manner;Stroke was the most influential journal in this research field because it ranked first in the number of publications and co-citations;Michael Chopp and Cesario V.Borlongan were the top two researchers with the most significant number of publications and the most potent academic influence;the United States and China were the top two countries with the most academic contributions and close cooperation,and the top ten productive research institutions were all from these two countries;neural stem cells(NSCs)and mesenchymal stem cells(MSCs)were the most studied cell types,and the number of studies on endothelial progenitor cells rapidly increased.In the field of stem cell therapy for PD,the bibliometric findings were as follows:the annual number of publications was gradually increasing;Harvard University,Karolinska Institute,Kyoto University,and Lund University were among the most active research institutions;the number of publications from the United States ranked first in the world,far surpassing others countries;the number of China's publications has increased significantly since twenty years ago,and China is currently the second most productive country in the world;induced pluripotent stem cells(iPSCs),NSCs and embryonic stem cells(ESCs)were the most widely studied cell types,iPSCs were in a leading position in clinical translation,the preclinical data of NSCs has accumulated rapidly,but the prospect of clinical use of ESCs was hindered due to ethical issues and risk of teratoma formation.Conclusion:In two hot research areas of stem cell treatment of ischemic stroke and PD,the United States has the most research contributions.China has made significant progress in the past two decades.In the field of ischemic stroke,MSCs and NSCs are the most widely explored cell types;in the area of PD,iPSCs and NSCs are the most investigated cell types,both of which have brilliant prospects of clinical translation.Section Two:Behavioral and Imaging Studies of Cynomolgus Monkey Models of Isc-hemic StrokeObjective:The evolutionary relationships between humans and primates are close.Non-human primates are highly recommended as experimental animals for ischemic stroke studies.The ischemic stroke can be induced by photochemical methods,which have advantages of minimal invasiveness and good reproducibility.In this study,we evaluated the rationality of the primate models of ischemic stroke made by photochemical methods in behavior assessment and functional imaging changes.Methods:In this study,adult male cynomolgus monkeys were used as experimental animals,and focal cortical infarction was induced by photochemical methods.The cynomolgus monkeys were scored according to the modified Kito scale and received multimodal MRI scans including 3D-T1,3D-T2,and BOLD sequences on the 1st and 3rd day after induced stroke.The behavioral and imaging data of experimental animals in different periods of ischemic stroke were analyzed.Results:According to the results of the modified Kito scale,cynomolgus monkeys showed apparent neurological dysfunction on the 1st and 3rd day after modeling,and the motor system score on the 3rd day was significantly higher than that on the 1 st day(P=0.024).On the 1st day after modeling,the lesion volume was positively correlated with facial sensory score(r=0.791,P=0.034),hand movement score(r=0.805,P=0.029)and total score(r=0.786,P=0.036);on the 3rd day after stroke,the lesion volume was positively correlated with consciousness score(r=0.749,P=0.005),sensory system score(r=0.637,P=0.026)and total score(r=0.777,P=0.003).The bilateral F1 areas were selected as regions of interest in the seed-based functional connectivity analysis.On the 1st day after stroke,the functional connectivity strength between area F1 of left side and the cerebellum of right side decreased;the functional connectivity strength between area F1 of right side and area V1 of right side as well as Brodmann area 5 of right side was weakened,and the functional connectivity strength between area F1 of right side and area F7 of right side increased.On the 3rd day after stroke,the functional connectivity strength between left area F1 and right area F1 and the functional connectivity strength between right area F1 and left substantia nigra were weakened.Conclusion:In the hyperacute and acute phases of ischemic stroke,the significant correlation between the Kito scale score and the lesion volume can be observed.The functional imaging results suggest that the animal models can present changes such as crossed cerebellar diaschisis and weakened functional connectivity of bilateral primary motor areas.The validity of the cynomolgus monkey models of ischemic stroke is proved in this study.Section Three:Preclinical Studies of Neural Stem Cell Transplantation in the Treatment of Ischemic StrokeObjective:The preclinical data of stem cell therapy in animal models are vital for future clinical trials.There was no preclinical study using primate models to determine the safety and efficacy of neural stem cells(NSCs)in ischemic stroke treatment.In this study,we evaluated the safety and efficacy of the domestically produced NSCs in treating ischemic stroke cynomolgus monkey models,aiming to provide preliminary evidence for future clinical studies.Methods:The experimental animals were divided into four groups:stereotactic intracerebral injection group,trans-nasal submucosal injection group,intra-carotid artery injection group,and control group.We used critical parameters from blood routine,liver and kidney function,tumor markers,head MRI scan,and histologic examination to evaluate the safety.We compared the modified Kito scale score and lesion volume between groups after NSCs treatment to assess the efficacy.Results:Compared with the control group,the body weight of the experimental animals in the NSCs treatment group did not show significant decrement within three months,and the blood routine,liver and kidney function,and tumor markers were within the reference ranges within nine months.The histologic examinations of the liver,spleen,lung,kidney,and testis showed no tumor formation.No tumor was found on the brain MRI.Experimental animals showed a certain degree of improvement in neurological function after receiving NSCs treatment.However,there was no significant difference in the modified Kito scale scores and lesion volumes when comparing the treatment groups and the control group.Conclusion:NSCs transplantation is safe and feasible for the treatment of ischemic stroke.Preliminary results demonstrate improved neurological function in animal models.Section Four:Potential Mechanism of Neural Stem Cell Transplantation in the Treatment of Ischemic Stroke Cynomolgus Monkey ModelsObjective:The applications of exosome and multimodal brain imaging technology in early diagnosis,prognosis prediction,and therapeutic targets receive more and more attractions.We aimed to explore the potential mechanisms of neural stem cell(NSCs)transplantation in treating ischemic stroke through exosomal miRNA sequencing and functional MRI scans.Methods:In this study,plasma-derived exosomal miRNA sequencing was performed six weeks after NSCs transplantation,and the differentially expressed miRNAs were found in comparison with the control group.Then GO enrichment analysis and KEGG pathway enrichment analysis were performed.We selected bilateral motor system cortex(area F1 to F7)as regions of interest in the seed-based functional connectivity analysis.The strength of functional connectivity was compared between the NSCs treatment groups and the control group.Results:We selected the top 20 differentially expressed miRNAs according to fold changes and did thorough literature reviews.The majority of exosomal miRNAs were not found to be associated with neuroprotection except the down-regulation of miR-182 in the stereotactic injection group,the down-regulation of miR-92a-3p in the trans-nasal injection group,and the down-regulation of miR-143-3p in the trans-arterial injection group.GO analysis revealed that target genes of differentially expressed miRNAs showed decreasing enrichment trends in cell killing,viral components,and electron carriers and increasing trends in cell aggregation,extracellular matrix,and collagen trimer.KEGG pathway analysis showed that the target genes of differentially expressed miRNAs were mainly involved in signaling pathways of endocytosis and regulation of actin cytoskeleton.The functional connectivity between the right area F5 and the right area 23b as well as left area F6 and left area 24c'was enhanced in the trans-nasal injection group,and the functional connectivity between multiple brain areas in the trans-arterial injection group was enhanced.Conclusion:The down-regulation of exosomal miR-182,miR-92a-3p and miR-143-3p expressions after NSCs transplantation may be important factors for neuroprotection.NSCs transplantation may promote the recovery of neurological function by enhancing the functional connectivity between the motor system cortex and other brain areas.