PTBP1 Mediated Conversion Of Human Umbilical Cord Mesenchymal Stem Cells To Neurons

The central nervous system(CNS)has a limited capacity for repair and regenerate after injury,and damaged neurons cannot be replaced after death.Thus,CNS injury is usually severe and often irreversible.In recent years,the rise of stem cell therapy has opened new avenues for the treatment of neurological injuries.Human umbilical cord derived mesenchymal stem cells(HUCMSCs)have been used in clinical treatment in several fields because of their convenient materials,low immunogenicity,and suitability for autologous transplantation.HUCMSCs have the potential of neurogenic transdifferentiation.The research of HUCMSCs as seed cells for neurogenic transdifferentiation can lay a foundation for the clinical treatment of nervous system diseases.MicroRNAs(miRNAs)play an important role during neural maturation,and miRNAs are endogenous noncoding single stranded small RNAs containing 20 to 24 nucleotides that generally induce target mRNA degradation or inhibit translation by binding messenger RNA(mRNA)3'untranslatedregions(3'UTRs)to achieve post-transcriptional.regulation of target genes.Recent studies have shown that miR-9 and miR-124 regulate neural development.High expression of miR-9 and miR-124 can transdifferentiate some non-neural cells into neurons.During embryonic development,polypyrimidine tract binding protein(PTB)is involved in the regulation of neuronal genesis and maturation.PTB is a typical inhibitor of pre-mRNA cleavage that binds to pyrimidine-rich regions of nucleic acid sequences and is involved in the regulation of pre-mRNA cleavage.In the mammalian genome,the PTB family consists of three members,PTBP1,which is expressed in most cell types,PTBP2,which is expressed only in the nervous system,and PTBP3,which is mainly expressed in immune cells.Recent studies have shown that non-neural cells can be transdifferentiated into neurons by interfering with PTBP1.TargetScan target gene prediction software analysis showed that PTBP1 may be the target gene of miR-9 and miR-124.Using dual-luciferase reporter assays,we were able to determine whether miR-9 and miR-124 directly target PTBP1.In our laboratory,we previously constructed lentiviral vectors with high expression of both miR-9 and miR-124 to investigate the relationship between miR-9 and miR-124 and neurogenic transdifferentiation of HUCSMCs.HUCMSCs were infected with high miR-9 and miR-124 lentivirus for 3 days and then changed to N3 medium to induce transdifferentiation for 7 days,resulting in Tujl-positive cells,while PTBP1 expression was decreased in the Tuj1 positive cells obtained by transdifferentiation.Therefore,whether interfering with PTBP1 is able to induce the transdifferentiation of neurons from HUCMSCs and how to improve their transdifferentiation efficiency,became our other major research content.To demonstrate whether miR-9 and miR-124 directly target PTBP1,we cloned a PTBP1 3'UTR fragment containing one miR-9 conserved binding site and two miR-124 conserved binding sites,on the basis of which miR-9 and miR-124 inactive 3'UTR fragments were obtained by PCR site-directed mutagenesis,and the obtained fragments were ligated into a dual-luciferase reporter plasmid vector to obtain wild-type as well as mutant PTBP1 3'UTR dual-1uciferase reporter vectors.Dual-luciferase reporter assays were performed using the vectors described above,and the results indicated that miR-9 and miR-124 were able to regulate PTBP1 expression by targeting the 3'UTR region of PTBP1.We infected HUCMSCs by interfering with PTBP1 lentivirus to reduce PTBP1 mRNA levels by 85%and protein levels by 90%in the cells,and switched to N3 medium for 7 days after virus infection to obtain Tujl-positive neurons with a transdifferentiation efficiency of approximately 20%.The expression of cell related genes on the 3rd day of infection and 7 days of culture in N3 medium was detected,respectively,and the results showed that the expression of PTBP1 decreased,PTBP2 first increased and then decreased,and the expression of miR-9 and miR-124 gradually increased,both of which were consistent with the law of neuronal maturation;therefore,we speculated that the process of neurogenic transdifferentiation of HUCMSCs was the result of the balance and mutual restriction of miR-9,miR-124,and PTBP1.To improve the transdifferentiation efficiency,we cocultured HUCMSCs 3 days after infection with the virus with astrocytes,and the transdifferentiation efficiency could be increased to 32%.To further improve the differentiation efficiency,this experiment used a protocol that interfered with PTBP1 in combination with high expression of miR-9 and miR-124 to induce the transdifferentiation of HUCMSCs into neurons,and Tuj1 positive neurons were obtained after 7 days of transdifferentiation,with a transdifferentiation efficiency of approximately 45%.In summary,PTBP1 is a common target gene of miR-9 and miR-124,and interference with PTBP1 can induce the transdifferentiation of HUCMSCs into neurons,and both astrocyte co-culture and interference with PTBP1 in combination with high expression of miR-9 and miR-124 improve neuronal transdifferentiation efficiency,laying a theoretical foundation for the use of HUCMSCs-derived neurons in clinical therapy.