Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Into Insulin-producing Cells And Analysis Of Its LncRNA Expression Profile

Objective:In this study,human umbilical cord mesenchymal stem cells(HUC-MSCs)were induced to differentiate into insulin-producing cells(IPCs,i.e.?-like cells)by small molecule compounds,LncRNAs and mRNAs at each stage of induced differentiation(S0,S1,S2,S3,S4)were obtained by high-throughput sequencing,and their dynamic expression changes in the process of induced differentiation were analyzed to search for LncRNAs that may be involved in the development and regulation of ?-cell function of pancreas.This study provides a theoretical basis for further investigation into the specific mechanism of LncRNA regulating the development process of pancreatic beta cells,and thus provides ideas for improving the efficiency of inducing differentiation of stem cells into IPCs.Methods:Umbilical cord primary mesenchymal stem cells were obtained by tissue adhesion method.The surface markers of HUC-MSCs were identified by flow cytometry,and their differentiation ability was identified.The HUC-MSCs were induced to differentiate into IPCs by using small molecular compounds,the morphological changes of cells in each stage were observed by inverted microscope,the expression of marker genes in each stage was detected by real-time fluorescence quantitative PCR,the expression of key proteins in each stage was induced by immunofluorescence detection,IPCs in S4 stage was identified by dithizone staining,and the function of IPCs in S4 stage was detected by human insulin ELISA kit.The FPKM values of LncRNA and mRNA expression in each induction stage(S0,S1,S2,S3,S4)were obtained by illuminating PE150 double-ended sequencing.edge R software was used to screen RNA-seq data and differential genes(DEGs).According to Fold Change(FC>=2or<=0.5)and False Discovery Rate(FDR<=0.05),the results were analyzed to determine whether the genes were differentially expressed.GO enrichment analysis of differential genes by KOBAS2.0.Genes involved in pancreatic ? cell development and regulation of ? cell function were screened in the enrichment pathway,so as to find out the LncRNA co-expressed with them.Results:1.under inverted microscope,umbilical cord primary mesenchymal stem cells showed long spindle shape,and differentiation in vitro showed that HUC-MSC successfully differentiated into adipocytes,osteocytes and chondrocytes.The results of flow cytometry showed that the positive rates of CD90,CD44,CD73 and CD105 in HUC-MSCs were all over 90%,while the positive rates of CD34,CD45 and HLA-DR were less than 5%.2.This is shown under an inverted microscope that during the process of cell differentiation,the morphology of cells gradually gathered into pieces from long spindle shape to conglomeration.The q RT-PCR showed that Foxa2 and Sox17 were significantly higher in S1 stage than other stages,Pdx1,Nkx6.1 and Ngn3 were significantly higher in S3 stage than other stages,and Glut2,MAFA and Insulin were significantly higher in S4 stage than other stages(P<0.05).Immunofluorescent protein showed positive expression of Pdx1 protein in S3 and S4 stage,and positive expression of C peptide and insulin protein in S4 stage.IPCs dithizone staining showed flake scarlet in S4 stage.ELISA showed that IPCs had insulin release function in S4 stage.3.The results show that by high-throughput sequencing:1)comparing S0 with S1,S2,S3,S4,S3 with S1,S2,S3,S3 with S1,S2 and S2 with S1,the number of LncRNA and mRNA differentially expressed between them showed a decreasing trend,indicating that cell development gradually matured.2)Comparing S0 with S1,S2,S3 and S4,the enrichment pathways of differentially up-regulated and down-regulated mRNA and mRNA co-expressed with differential LncRNA show that the up-regulated genes are mainly enriched in translation-related pathways,such as translation initiation,extension and termination.It suggests that a large number of protein are translated in the process of gradually inducing the development of cells,thus preparing for development.On the contrary,down-regulated genes are mainly concentrated in the process of cell adhesion and cell connection,which indicates that the adhesion and connection between cells are gradually weakened during cell development,suggesting that cells are preparing for differentiation at this stage.concentrated in cell adhesion and cell connection processes,indicating that cell-cell adhesion and cell connection are gradually weakened during cell development,suggesting that cells are preparing for differentiation at this stage.3)Comparing S4 with S1,S2 and S3 respectively,the enrichment pathways of mRNA co-expressed with up-regulated LncRNA show that mRNA co-expressed with up-regulated LncRNA is mainly enriched in NF-KB signaling pathway and MAPK signaling pathway,while down-regulated genes were mainly concentrated in the HIPPO signaling pathway,PI3K-Akt signaling pathway,p53 signaling pathway and other pathways related to insulin signal transduction in ?-cells.Both the up-regulated and down-regulated differential genes above indicated that the functional pathways of these genes were closely related to ?-cell function.4)Two types of mRNAs with different changing trends in S1,S2,S3 and S4 were identified by K-means analysis.One was the mRNAs whose expression level gradually increased with the progress of cell induction development,and these mRNAs were enriched in the signaling pathway of cell differentiation,that is,with the progress of cell development,the process of cell differentiation became more and more active.On the contrary,the other type of mRNAs showed an opposite trend,mainly concentrated in the negative regulation pathway of cell proliferation,that is,the ability to inhibit cell proliferation became weaker,that is,the cell differentiation tended to be active.JARID2 and PROX1 genes related to pancreatic ?-cell development were screened from these two types of genes.P53 gene involved in the regulation of ?-cell function,and to find out the LncRNA co-expressed with JARID2:AC009014.3,GS1-72M22.1,LncRNA coexpressed with PROX1:CTBP1-AS2,LncRNA co-expressed with p53 :XLOC?050969,LINC00883,XLOC?050981,XLOC?050925,MAP3K14-AS1,rp11-148K1.12,CTD2020K17.3.Conclusion:1.HUC-MSCs were successfully induced and differentiated into IPCs by combining small molecule compounds NA,Exendin-4,b FGF,Actinvin A,EGF,etc.2.S4 was compared with S1,S2 and S3 for enrichment analysis of the genes co-expressed by the differentially up-regulated and down-regulated lnc RNAs,and it was found that many target genes were involved in the NF-k B signaling pathway,MAPK signaling pathway,Hippo signaling pathway,PI3K-Akt signaling pathway,p53 signaling pathway and other pathways related to ?-cell insulin signaling transduction.These differential lnc RNAs may regulate the function of pancreatic beta cells through the regulation of multiple signaling pathways.3.LncRNAs:AC009014.3,GS1-72M22.1 and CTBP1-AS2 may be involved in the development of pancreatic beta cells.LncRNA XLOC?050969,LINC00883,XLOC?050981,XLOC?050925,MAP3K14-AS1,Rp11-148K1.12,CTD2020K17.3may be involved in the regulation of pancreatic ?-cell function.