The Mechanism Study Of Umbilical Cord Mesenchymal Stem Cells On Experimental Autoimmune Encephalomyelitis In Cynomolgus Monkeys

Objectives:Multiple sclerosis(MS)is an autoimmune disease that affects the central nervous system of young and middle-aged people.It's the most common cause of neurological dysfunction in young people.It is also the second leading cause of disability just next to trauma.The current treatment methods still can't cure MS,but only play a role in reducing the risk of relapse and delaying the disease progression.In our previous research,we found that human umbilical cord mesenchymal stem cells(hUC-MSCs)have shown good immunoregulatory effects and curative effects in patients with MS.However,the specific mechanism is still unclear.Thus,our present study was planned to use hUC-MSCs to treat non-human primates(NHP)model with multiple sclerosis.The animal model of experimental autoimmune encephalomyelitis(EAE)of cynomolgus monkey was used to evaluate the effects of hUC-MSCs on the course of EAE,disease pathology and peripheral immune status.Recent studies have shown that the main pathway for stem cells to exert immunomodulatory effects may be dependent on the secretion of exosomes.Exosomes derived from UCMSCs can participate in immune regulation through the transmission of genetic material and inflammatory molecules.Microglia was innate immune cells in the central nervous system(CNS),which have an M1/M2 phenotypic transformation that participates in the CNS inflammation of MS and affects the process of MS demyelination.Therefore,this study would also explore the effects of UCMSCs-exo on microglial polarization in an inflammatory environment,and study the proteomics of UCMSCs-exo to provide new scientific evidence for how UCMSCs can exert immunomodulatory effects.It also further promoted the progress of UCMSCs as a treatment strategy for MS.Methods:The first part:hUC-MSCs were isolated by tissue attachment method,and the cell surface markers and multidirectional differentiation abilities were detected by flow cytometry.The cells were identified as umbilical cord mesenchymal stem cells.Subsequently,the tumorigenicity of hUC-MSCs were evaluated in nude mice,and cynomolgus monkeys by intravenous injection.The effects of hUC-MSCs on the proliferation and migration of lung adenocarcinoma cell line SPC-A-1 were evaluated in vitro,and the changes in the cytokine profile after co-culture were analyzed.At the same time,in vivo experiments were performed to evaluate the metastatic and proliferative effects of hUC-MSCs on the SPC-A-1 xenograft model.The second part:MOG34-56 peptide mixed with incomplete Freund's adjuvant was injected subcutaneously into the axillary and groin lymph nodes of cynomolgus monkeys for EAE modeling.Magnetic resonance magnetic resonance(Magnatic Resonance Imaging,MRI)was used to detect the demyelination of CNS in cynomolgus monkeys.The pathological examination of the EAE model was performed using HE staining,LFB staining,immunofluorescence,and transmission electron microscopy.Changes of cytokines in cell subpopulations were detected by ELISA in EAE models.The third part:The cynomolgus monkey EAE model was given intravenous infusion of UCMSCs as treatment and physiological saline(NS)as the control.MRI was used to detect the changes in CNS demyelinating lesions.Moreover,HE staining,LFB staining,immunofluorescence detection,and transmission electron microscopy were used to detect changes in demyelinating lesions,inflammatory cell infiltration,and astrocyte activation in EAE models respectively.Cultivation experiments were performed to analyze the changes in PBMC proliferative capacity of UCMSCs compared with NS group after treatment.ELISA was used to detect the changes of 23 inflammatory factors in peripheral blood of the two groups.Flow cytometry was used to detect the proportion of peripheral blood immune cells,and RT-PCR was used to analyze the expression of genes which were specific to cell populations and differential inflammatory factors in brain tissue and PBMC.The fourth part:UCMSCs-derived exosomes were isolated by PEG precipitation combined with density gradient centrifugation,and identified by NTA detection,transmission electron microscopy,flow cytometry,and Western blotting.The proteomics(label-free)was used to detect the proteins contained in the exosomes,and the bioinformatics analysis was used to obtain proteins with high correlation with immunity.The effect of UCMSCs-exo on the proliferation of resting microglia was analyzed by CCK8,and the effect of UCMSCs-exo on the chemotaxis of resting microglia was analyzed by co-culture experiments.Flow cytometry was used to detect the effect of LPS stimulation on the proportion of microglia M1 type.UCMSCs-exo were co-incubated with LPS-stimulated microglia.Flow cytometry and WB methods were used to detect the proportion of M1 and M2 microglia and the expression of marker proteins,respectively.Results:The first part:hUC-MSCs were successfully isolated and obtained numerous stable passaged cells.The morphology,surface markers and multiple differentiation capabilities are consistent with the characteristics of mesenchymal stem cells.It has not been found to have tumorigenicity in vivo experiments.HUC-MSCs haven't promote the proliferation of SPC-A-1,and inhibited the migration and tube-forming ability of SPC-A-1.hUC-MSCs had no tumor-promoting effect and tumor-promoting effect on SPC-A-1 xenograft model.The second part:Autoimmune response were successfully induced in cynomolgus monkeys.MRI showed a large number of white matter demyelinating lesions in the brain of the EAE model.Typical cuff-like changes around blood vessels and inflammatory cell infiltration were seen in HE detection.Demyelination was seen in LFB staining.The immunofluorescence detection showed astrocyte aggregation,the proportion of T cell subsets in the peripheral circulation of the EAE model increased significantly,and only sCD40L and IL-4 were decreased in 23 inflammatory factors which had significant statistical differences.The third part:Compared with the NS group,the UCMSCs treatment group can significantly alleviate the disease progression of the cynomolgus monkey EAE model.MRI tests show that the white matter demyelinating lesions in the brain are reduced after UCMSCs treatment.Inflammatory cell infiltration was reduced in HE results.LFB staining suggested demyelinating lesions reduced and astrocyte activation reduced were showed by immunofluorescence.After treatment with UCMSCs,the proportion of regulatory T cells was significantly increased,the proportion of Thl and Th17 cells was significantly decreased,sCD40L in peripheral blood was significantly increased.Co-culture experiments suggest that UCMSCs can significantly reduce the expression levels of most inflammatory factors.The fourth part:Exosomes were isolated successfully and identified by NTA and transmission electron microscopy.Flow cytometry and WB detection revealed that UCMSCs-exo can express CD3,CD9,CD81,HSC70 and TSG101 on surface.Proteome analysis of UCMSCs-exo revealed that a total of 511 proteins,and 15 types of immune-related proteins were obtained after GO,KEGG,and PPI analysis.UCMSCs-exo had a dose-dependent effect on the proliferation of resting microglia,and it began to significantly promote proliferation at a concentration of 100×108p/mL.UCMSCs-exo had a significant chemotactic effect on resting microglia within 48 hours.After co-incubation of UCMSCs-exo with LPS-stimulated(50ng/mL)microglia,the data from flow cytometry and WB suggested that UCMSCs-exo could inhibit the expression of CD86 and iNOS on the surface of M1 cells,but also promoted the expression of CD206 and Arg-1 in M2 cells.Conclusions:1.UCMSCs were isolated successfully in this study.They had no obvious tumorigenic and tumor-promoting properties,and were safe in preclinical applications.2.The cynomolgus monkey EAE model was highly similar to the clinical manifestations and disease characteristics of MS in the onset symptoms,MRI manifestations and pathological characteristics.Therefore,it was suitable to used to evaluate the efficacy of UCMSCs transplantation in the later stage.3.UCMSCs could alleviate the progress of the cynomolgus monkey EAE model effectively,and also significantly increased the numbers of Treg cells,as well as reduced the ratio of Thl and Th17 cells in the cynomolgus monkey EAE model.UCMSCs played a role in immune modulation through increase sCD40L in serum.Furthermore,UCMSCs inhibited the activation of astrocytes to reduce CNS demyelination and inflammatory infiltration.4.UCMSCs-exo could be extracted and isolated by PEG precipitation with density gradient centrifugation successfully.The mechanism studies showed that some proteins from UCMSCs-exo could regulate the polarization of BV2 cells to M2 type under inflammatory stimulation,which might be belonged to the integrin protein family including ITGA5,ITGAV,ITGB5,and others such as TGFB2,THBS4,etc.