Regulation Of OCT4A Alternative Splicing On The Self-renewal Capacity Of Human Dental Pulp Stem Cells

Stem cells are used to repair defects of the organ which have became a research hotspot of regenerative medicine.Human dental pulp stem cells(hDPSCs)is a tissue engineering seed cells with high application value which is due to its rich resource and small trauma.However,the amplification and proliferation ability of hDPSCs were lower than that of embryonic stem cells which limited its application in tissue engineering.It was reported that the multipotency of dental pulp stem cells were decreased with serial passage in vitro[1].Therefore,it was urgent to find multipotency regulation targets and maintaining mechanism of hDPSCs,which will contribute to solve the bottleneck problems of the hDPSCs tissue engineering application.Our previous reports found that the expression of OCT4 A alternative splicing and the pluripotent characteristic of stem cells were reduced gradually with continuous passage[1,2].Besides,the expression of OCT4 A was lower in hDPSCs than that in ESCs,which showed us that OCT4 A alternative splicing may play a regulatory role in maintaining hDPSCs self-renewal ability.OCT4 transcription factor as the pluripotent marker of stem cells palys an important role in maintaining pluripotency of ESCs and ASCs[3].The pre-m RNA of OCT4 could be alternative spliced into OCT4 A,OCT4B and OCT4B1,and OCT4 A regulates the pluripotency of stem cells[4,5].TIP110(squamous cell carcinoma antigen recognized by T-cells 3)as an splicing factor takes an important part in regulating splicing of OCT4 A m RNA[6].In this study,the OCT4 A regulation role in maintaining pluripotency of hDPSCs was studied by shRNA and overexpressing lentivirus expression vector.Meanwhile,The expression of OCT4 A in hDPSCs whether was regulated by TIP110 was detected.The effects of TIP110 on the proliferation and self-renewal ability of hDPSCs were investigated yet.Finally we verify the OCT4 A and TIP110 regulation role in hDPSCs by nude mouse transplantation experiment.The study was expected to lay a foundation for further study on the regulation of OCT4 A splicing by TIP110 and its effect on the pluripotency,self-renewal ability of hDPSCs.Furthermore,it provide a novel idea and theoretical foundation for investigating the maintaining pluripotency mechanism of hDPSCs and tissue engineering clinical application.The experimental method and result are as follows:1.Isolation,cultivation and identification of hDPSCsTissue block combined with enzymatic digestion methods were used to isolate and culture primary hDPSCs.Flow Cytometry was used to identify the cell surface antigen.Then the osteogenic and adipogenic ability of hDPSCs were identified.All above results confirmed that we successfully culture hDPSCs.2.Regulation of OCT4 A on the self-renewal capacity of hDPSCsHDPSCs was cultured in vitro and studied by shRNA and overexpressing lentivirus expression vector at P3.Then the proliferation,differentiation ability,cloning ability and the expression of pluripotent transcription factor of each group were detected.The result are as follows:(1)The proliferation ability of hDPSCs was significantly inhibited after silencing OCT4 A which was detected by CCK-8.However,the proliferation ability of hDPSCs increased after overexpression OCT4 A.(2)Cell colony forming unit(CFU)was used to detect the clone formation ability of each group.The results showed that the cloning ability of hDPSCs could be inhibited after silencing OCT4 A,while the cloning ability was promoted after overexpression OCT4 A.(3)Real-Time PCR was used to detect the expression of Klf4,Sox2,Nanog and CMyc in hDPSCs.It was found that the expression of pluripotent transcription factors in hDPSCs decreased after silencing OCT4 A and up regulated after overexpressed OCT4 A.(4)The results of adipose differentiation test showed that the lipid droplets formed in the experimental group were significantly lower than those in the control group after silencing OCT4 A and the expression of PPAR? was significantly inhibited which was found by Real-Time PCR.However,the adipogenic ability of hDPSCs and expression of PPAR? was increased after overexpression OCT4 A.(5)The alizarin red staining and Real-Time PCR were introduced to evaluate the odonto/osteogenic differentiation ability of human DPSCs.The results of mineralization test showed that the staining intensity of experimental group was significantly weaker than control group,and significantly stronger after overexpression OCT4 A.The formation of calcified nodules and the expression of osteogenic/odontogenic related genes DSPP,BSP,OCN and ALP was inhibited after silencing OCT4 A.The opposite result was showed after overexpression of OCT4 A.In vivo transplantation experiments showed that hDPSCs were induced to be mineralized for 2 weeks and then implanted on hydroxyapatite(HA-TCP)scaffolds for implantation in nude mice.After 8 weeks,Masson and immunohistochemistry staining showed that the osteogenic/odontogenic differentiation was inhibited after silencing OCT4 A,while the osteogenic/odontogenic differentiation was promoted after overexpression OCT4 A.In vivo and in vitro results are consistent.3.Regulation of TIP110 on the self-renewal capacity of hDPSCsThe difference between proliferation,differentiation,clonal formation and pluripotent molecules was observed after silencing TIP110 in hDPSCs.The results showed that:(1)Real-Time PCR was used to detect the expression of pluripotent transcription factors in hDPSCs after silencing TIP110.The expression of Klf4,Sox2,Nanog and C-Myc was inhibited after silencing TIP110.(2)CCK-8 test results show that the proliferation ability of hDPSCs decreased after silencing TIP110.(3)CFU test results showed that the number of clones and clone formed volume in the experimental group was significantly lower than that in the control group after silencing TIP110,which indicate the clone formation ability was inhibited.(4)The osteogenic/odontogenic and adipogenic differentiation ability of hDPSCs in vitro was studied.It was found that the differentiation ability of hDPSCs significantly decreased after silencing TIP110 which was detected by alizarin red,oil red O staining and Real-Time PCR.(5)Real-Time PCR was used to detect the expression of OCT4 alternative splicing(OCT4A,OCT4 B and OCT4B1)in hDPSCs after silencing TIP110.The result shows that the expression of OCT4 A was inhibited,but the expression of OCT4 B and OCT4B1 was not changed after silencing TIP110.In summary,the results of this study confirmed that OCT4 A plays an important role in the pluripotency maintenance of hDPSCs.TIP110 regulates OCT4 A expression in hDPSCs and regulates the self-renewal ability of hDPSCs too.The next step of this study will further research that TIP110 is involved in the regulation of hDPSCs' self-renewal ability by regulating the expression of OCT4 A.This study was designed to solve the bottleneck problem of hDPSCs in tissue engineering applications and provide a theoretical basis for the application of pulp injury and repair therapy.