The Experimental Study On The Role And Mechanism Of Neuron Stem Cell Derived Exosomes Promoting Neuronal Differentiation

Objective: Neural tube defects(NTDs)are the most common congenital defects in the central nervous system.Surgical treatment can only repair the defects,but not nerve damage.Cell therapy with bone marrow mesenchymal stem cells(BMSCs)can both cover defects and repair spinal cord damage.The higher neuronal differentiation promotes the better neuron repair and functional recovery.In order to obtain better effect of cell therapy,we explore approaches to increase the neuronal differentiation rate of BMSCs.Exosomes are small vesicles containing components of the derived cells and thus showing the same functional characteristics with secreted cells.Neural stem cells(NSCs)have the ability to self-renew and differentiate into neurons and glial cells.We hypothesize that neural stem cell derived exosomes contain certain proteins that can induce neuron differentiation of BMSCs.Neural stem cell derived exosomes would be used to promote the neuron differentiation and then improve the therapeutic effect of BMSCs.Methods: Lable free mass spectrometry was used to detect the protein profiles of neural stem cell derived exosomes,NSCs and BMSCs.GO analysis was used to select proteins with neuronal differentiation.The expression difference of candidate proteins in neural stem cell-derived exosomes,NSCs,and BMSCs was compared.Differential proteins were verified by Western Blot analysis.NSCs differentiation was induced by 500ng/ml Netrin1,and immunofluorescence staining and Western Blot analysis were used to detect the expression of neuronal markers Map2,NF and ?3-Tubulin.GSE54107,the Netrin1 related dataset,was retrieved from the GEO database.Molecules that are up-regulated and promote neuronal differentiation were selected from the dataset and verified.Neural stem cell derived exosomes and Netrin1(500ng/ml)were respectively added into the BMSCs medium and the expressions of Map2,NF and ?3-Tubulin were detected.And BMSCs and BMSCs mixed with neural stem cell derived exosomes were injected into amniotic cavity of spinal bifida rat embryos respectively.The percentage of?3-Tubulin positive cells in BMSCs was detected.All values are presented as the arithmetic mean ± SD.A two sided t-test was used for the comparison of 2 groups and ANOVA was used for that of multiple groups.P < 0.05 was considered statistically significant.Results:? The protein profiles of neural stem cell derived exosomes,NSCs and BMSCs from Lable free mass spectrometry? The proportion of extracellular matrix components in neural stem cell derived exosomes was higher than that of NSCs.? The results of GO enrichment analysis of up-regulated proteins in neural stem cell derived exosomes compared with NSCs show enrichment in stem cell differentiation.? 30 proteins are positive for neuronal differentiation in GO annotation(GO: 0045666).During these 30 proteins,Netrin1,Nedd4 l,Ptprz1 and Fn1 in neural stem cell derived exosomes showed higher expression level than those of NSCs.The expressions of Pafah1b1,Camk2 d,Gsk3b,Prpf19,Netrin1,Rufy3,Camk2 b,Ptprz1,Reelin in neural stem cell derived exosomes were higher than those of BMSCs.? Western Blot analysis showed the enriched expression of Netrin1 and Nedd4 l in neural stem cell exosomes(p = 0.0007,p < 0.0001),and the expression level was higher than BMSCs(p = 0.0004,p < 0.0001).? Netrin1 induced the differentiation of NSCs into neurons.? The percentage of ?3-Tubulin positive cells in NSCs with 500ng/ml Netrin1 was higher than that in the control group(p < 0.0001).And the expressions of Map2,NF and?3-Tubulin protein were up-regulated(p = 0.0068,p = 0.0112,p = 0.002).? A RNA sequencing dataset GE54107 about Netrin1 was retrieved in GEO database.The bioinformatics analysis screened three downstream molecules,Phox2 b,Hand2 and Wnt10 a,which were related with the neural crest differentiation(GO: 0014033).PPI analysis showed the interaction between Hand2 with Phox2b.? The expressions of Phox2 b and Hand2 were increased in the neural stem cells with Netrin1(500ng/ml),(p = 0.0013,p = 0.0023).Compared with the NC control,Map2,NF and ?3-Tubulin were down-regulated by Phox2 b knocking down and Netrin1(500ng/ml),(p = 0.0001,p = 0.0003,p = 0.0002).? Neural stem cell derived exosomes and Netrin1 induced BMSCs neuronal differentiation respectively.? The percentage of ?3-Tubulin positive cells in Neural stem cell derived exosomes group was higher than that in control group(p = 0.0138).The expression levels of Map2,NF and ?3-Tubulin in BMSCs treated with neural stem cell derived exosomes were up-regulated(p = 0.0033,p = 0.0009,p = 0.028).? The percentage of ?3-Tubulin positive cells in Netrin1(500ng/ml)group was higher than that in control group(p < 0.0001).The expression levels of Map2,NF and?3-Tubulin in BMSCs treated with Netrin1(500ng/ml)were up-regulated(p = 0.0001,p= 0.0064,p = 0.0061).? In the rat model of neural tube malformation in whole embryo culture,the positive rate of ?3-Tubulin in BMSCs with neural stem cell exosomes was higher than that in BMSCs group(p < 0.0001).Conclusion:1.The neural stem cell derived exosomes carry 30 neuron-differentiating proteins and appear enrichment effects on Netrin1 and Nedd4 l.2.Netrin1 promotes the differentiation of neural stem cells into neurons by up-regulating Hand2 and Phox2 b.3.Both neural stem cells derived exosomes and Netrin1 can induce the differentiation of BMSCs into neuron.Neural stem cells derived exosomes also improve the neuronal differentiation efficiency of BMSCs during NTDs cell therapy.