The Application And Mechanism Of Neural Stem Cell Exosome-based Therapy In Ischemic Stroke

Part I The effect of neural stem cell derived exosomes in the treatment of ischemic stroke Objective This study aimed to explore the potential role of hNSC-Exo in the treatment process through the effects of human neural stem cell-derived exosomes(hNSC-Exo)on neural stem cells after hypoxic injury at in vitro and in vivo levels.Methods Human hippocampal neural stem cells(hNSCs)were isolated and cultured,and neural stem cell markers were identified by immunofluorescence.hNSC-Exo were extracted from neural stem cell culture supernatant,and the morphology,size,and surface marker proteins of exosomes were identified using transmission electron microscopy,nanoparticle size analysis(NTA),and western blot.Use PKH67 to label exosomes and observe the uptake of exosomes by target cells.The OGD/R model for neural stem cells(OGD-hNSCs)was set up,and hNSC-Exo was added as an intervention.The apoptosis and differentiation of OGD-hNSCs were evaluated by immunofluorescence staining.The rat middle cerebral artery ischemia-reperfusion injury model(MCAO)was established,hNSC-Exo was stereotaxically placed into the rat brain,and the neurological function of the rat was evaluated by mNSS,Rotarod test,and Postural Reflex test;immunofluorescence was used to evaluate the rat nerve apoptosis and regeneration.The OGDhNSCs were treated with hNSC-Exo and PBS,and miRNA high-throughput sequencing technology was used to find the miRNAs that were expressed differently in the two groups.Results Neural stem cells are neurosphere-like when cultured in vitro,express specific markers like Nestin and SOX2,and can differentiate into neurons,astrocytes,and oligodendrocytes;hNSCExo has typical exosome shape and size,The markers HSP70,TSG101,and CD81 were expressed.In vitro experiments showed that hNSC-Exo could inhibit the apoptosis and astrocyte differentiation of OGD-hNSCs and promote the differentiation of OGD-hNSCs into neurons.In vivo experiments show that hNSC-Exo can significantly improve the neurological function of rats with cerebral ischemia,affect the differentiation of endogenous hypoxic neural stem cells and the apoptosis of peripheral nerve cells in the ischemic area.miRNA high-throughput sequencing and qRT-PCR revealed that miRNA-362-3p in OGD-hNSCs was significantly upregulated after exosome intervention.Conclusions This study preliminarily demonstrated that human neural stem cell-derived exosomes can exert a neuroprotective effect on hypoxic neural stem cells and cerebral ischemia tissue.Part Ⅱ The mechanism of human neural stem cell exosomes promoting endogenous nerve repairObjective This study aimed to investigate the effects of miRNAs in human neural stem cellderived exosomes on neural stem cells and cerebral ischemia rats after hypoxic injury at in vitro and in vivo levels.Methods The differentially expressed miR-362-3p was selected as the research object,miR-362-3p lentivirus overexpression vector and a miR-362-3p stable expression neural stem cell line were constructed,and exosomes(miR-362OE-hNSC-Exo)were extracted.);miR-362OE-hNSC-Exo was intervened on OGD-hNSCs,and qRT-PCR,immunofluorescence,and western blot were used to evaluate cell viability,differentiation,and the effect of the Wnt/β-catenin signaling pathway;and miR-362OE-hNSC-Exo directional transplantation into MCAO rats,the nerve function and nerve reconstruction were evaluated.The downstream target genes regulated by miR-362-3p were predicted by bioinformatics software(starBase,Targetscan,and miRnada)and verified by qRT-PCR;the dual luciferase reporter gene was used to verify the binding ability of miR-362-3p to target genes;the effect of target genes on the Wntβ-catenin signaling pathway was evaluated by western blot.Results miR-362OE-hNSC-Exo and miR-NCOE-hNSC-Exo had typical exosome morphology and size,as well as HSP70,TSG101,and CD81 expression.In vitro experiments showed that miR362OE-hNSC-Exo could inhibit the apoptosis and astrocyte differentiation of OGD-hNSCs,promote the differentiation of OGD-hNSCs into neurons,and activate the Wntβ-catenin signaling pathway.In vivo experiments showed that miR-362OE-hNSC-Exo could improve the neurological function of rats with cerebral ischemia,affect the differentiation of endogenous hypoxic neural stem cells and the apoptosis of peripheral nerve cells in the ischemic area.The downstream target gene of miR362-3p,melanoma cell adhesion molecule(MCAM),was discovered using bioinformatics and western blot.Dual-luciferase reporter gene analysis and qRT-PCR analysis confirmed the interaction between miR-362-3p and MCAM.A western blot confirmed that MCAM knockdown could activate the Wntβ-catenin signaling pathway.Conclusions This study preliminarily demonstrated that miR-362-3p in human neural stem cellderived exosomes affects the apoptosis and differentiation of OGD-hNSCs by activating the Wnt/βcatenin signaling pathway and promotes the recovery of neurological function in rats with cerebral ischemia.Part Ⅲ Neural stem cell-derived exosomes carrying BDNF in the treatment of ischemic stroke Objective This study aimed to investigate the effects of engineered exosomal BDNF-hNSC-Exo on neural stem cells and cerebral ischemia in rats after hypoxic injury.Methods Based on neural stem cell-derived exosomes,the engineered exosomes BDNF-hNSCExo were constructed after transfection with neurotrophic factor(BDNF).Transmission electron microscopy,nanoparticle size analysis(NTA),and western blot were used to identify BDNF-hNSCExo morphology,size,and signature proteins of Exo and hNSC-Exo.The oxidative stress cell model was constructed by hydrogen peroxide,and BDNF-hNSC-Exo was used for intervention.Cell activity and apoptosis were assessed by CCK8,TUNEL,and western blot;qRT-PCR,western blot,and immunofluorescence were used to assess cell differentiation.After directional transplantation of BDNF-hNSC-Exo into MCAO rats,mNSS,Rotarod test and Postural Reflex test were used to evaluate the neurological function of the rats;TTC staining was used to assess the volume of cerebral infarction in the rats;and immunofluorescence was used to assess the rats’ nerve reconstruction.Results Both BDNF-hNSC-Exo and hNSC-Exo had typical exosome shape and size,and expressed markers HSP70,TSG101 and CD81,but not Calnexin protein.In vitro experiments showed that BDNF-hNSC-Exo could inhibit the apoptosis of neural stem cells under stress and promote their differentiation into neurons.In vivo experiments show that BDNF-hNSC-Exo can improve the neurological function of rats with cerebral ischemia,inhibit the expression of microglia,promote the differentiation of endogenous hypoxic neural stem cells into neurons,and reduce the volume of cerebral infarction.Conclusions This study preliminarily confirmed that BDNF-hNSC-Exo can promote nerve regeneration and survival,thereby exerting a therapeutic effect.