Differentiation Of Human Umbilical Cord Wharton’s Jelly-derived Mesenchymal Stem Cells Into INS-producing Cells Modified By MAFA And PDX1

Objective:The purpose of this study is to explore the possibility of Human Umbilical Cord Mesenchymal Stem Cells（Hu MSCs）,modified by MAFA and PDX1,differentiated into insulin-producing cells（IPCs）,providing a new cell source for stem cell treatment of diabetes.Methods:（1）Hu MSCs were isolated,cultured and expanded by tissue adherence culture method,identified by flow cytometry（FCM）.（2）We constructed,packaged and concentrated MAFA-PDX1 lentivirus expression vectors.（3）We infected Hu MSCs with MAFA-PDX1 lentivirus expression vectors,with different concentration gradients of multiplicity of infection（MOI）.After 72 hours of culture,the infection efficiency was detected by inverted fluorescence microscope and flow cytometry,and the most suitable MOI was selected for subsequent experiments.（4）Compare the efficiency and potentiality of Hu MSCs differentiated into IPCs with three methods by cell morphology,Real Time Quantitative PCR（RT-qPCR）,and dithizone staining（Program A:Simple lentivirus group,Program B:Drug induction followed by lentivirus group,Program C:Lentivirus and drug induction at the same time group）.Results:（1）Hu MSCs were successfully isolated and cultured from umbilical cord tissue,and enough cells were expanded to meet the needs of subsequent experiments.Hu MSCs is high expression of surface markers CD73,CD 90,and CD105 and absence of CD11b,CD 19,CD 34,CD 45,and human leukocyte antigen-DR（HLA-DR）characterized by FCM.（2）MAFA-PDX1 lentivirus expression vectors was constructed and a high-concentration lentiviral stock solution was acquired successfully.（HBLV-Zs Green-PURO:3*10^8TU/m L;HBLV-h-MAFA-PDX1-3xflag-Zs Green-PURO:1*10^8TU/m L）.（3）When MOI=5,the infection rate of lentivirus is 52.6%.The infection rate of lentivirus is99%,100%,and 100%respectively when the MOI is 10,20,and 30.The most suitable MOI value is to select the minimum MOI value when the infection rate is higher than 90%and the cells are in good condition.Therefore,MOI=10 was selected to infect Hu MSCs in the follow-up operation of this experiment.（4）The result of inducing Hu MSCs modified by MAFA and PDX1 into IPCs with three protocols.（1）Morphological change of cells:the morphology of post-induced cells lost the typical fibroblast shape,began to appear round or polygonal changes,and then slowly appeared cell clusters,which enlarged gradually and finally changed into pancreatic islet cells.At day 11,the Hu MSCs induced by protocol B showed the most cell clusters among the three protocols,appearing aggregated islet-like cell clusters.（2）Islet related genes expression detected by RT-qPCR:Protocol A（Lentivirus alone group）:On the 5^th day of induction,the expression of MAFA,PDX1,GCG,GLUT2,and NKX6.1 genes increased（P<0.05）,while the expression of INS and NEUROG3 genes were statistically insignificant.The expression of MAFA,PDX1,GCG and NKX6.1 genes increased on the 11^th day（P<0.05）,while the expression of INS,GLUT2 and NEUROG3 genes have no meaning.Comparison of islet related gene expression results on the5^th and 11^thday of induction:the expression of gene MAFA,PDX1,GCG and NKX6.1 increased both on the 5^th and 11^th day,and the expression level on the 5^th day of induction was higher than that on the 11^th day.The expression level of GLUT2 gene increased on the 5^th day of induction,but had no significant change on the 11^th day.Protocol B（Drug induction followed by lentivirus group）:On the 5^th day of induction,the expression of MAFA,GCG,INS,and NKX6.1 genes increased（P<0.05）,while the expression of PDX1,GLUT2 and NEUROG3 genes were statistically insignificant.On the 11^th day of induction,the expression of MAFA,PDX1,GCG,INS and GLUT2 genes increased（P<0.05）,while the expression of NKX6.1 and NEUROG3 genes have no sense.Comparison of islet related gene expression results on the 5^th and 11^th day of induction:the expression of genes MAFA,GCG and INS increased both on the 5^th and 11^th day,and the expression of MAFA and INS genes on the 11^th day was significantly higher than that on the 5^th day,with GCG was the opposite.The PDX1 and GLUT2 genes were not significantly expressed on the 5^th day while increasing on the 11^th day.NKX6.1 gene increased on the 5^th day of induction,but had no obvious expression on the 11th day.Protocol C（Lentivirus and drug induction at the same time group）:On the 5^th day of induction,the expression of MAFA,PDX1,GCG and INS genes increased（P<0.05）,while GLUT2,NKX6.1 and NEUROG3 gene expression showed no statistically significant difference.On the 11^th day of induction,the expression of MAFA,PDX1 and INS genes increased,GLUT2gene decreased（P<0.05）,and there was no significant difference in GCG,NKX6.1 and NEUROG3 gene expression.Comparison of the expression results of islet related genes on the5^th and 11^th day of induction:the expression of MAFA,PDX1 and INS genes increased both at day 5 and 11,with the feature that the expression of those gene at day 5 were much higher than that of day 11.The expression of MAFA and PDX1 at day 5 were higher than that of at day 11,while the gene INS was the opposite.The expression of GCG increased on the 5^th day of induction,but had no obvious expression on the 11^th day.GLUT2 gene was not significantly expressed on the 5^th day of induction,but decreased on the 11^th day.The islet related genes expression difference of Hu MSCs induced by three protocols:the expression of MAFA and PDX1 genes was the highest in protocol C on the 5^th day of induction,and that in protocol B was the highest on the 11^th day.Hu MSCs induced by protocol B has the greatest expression of GCG gene at day 5,INS and GLUT2 genes at day 11.From the above results,protocol B may be the optimal choice.Horizontal comparison of the three schemes at the same induction time point showed that the expression levels of MAFA and PDX1 in scheme C were the highest on the 5^th day of induction,and those in scheme B were the highest on the 11^thday of induction.Plan B had the highest expression of GCG gene on the 5th day of induction,and the highest expression of INS and GLUT2 gene on the 11^th day of induction.Therefore,Plan B has the potential to differentiate into IPCs.（3）Dithizone staining to identify zinc ions:it can be observed under an inverted phase-contrast microscope that part of the post-induced cells were stained brownish red by dithizone at the end of differentiation.The small island cells stained deep in scheme B,While the control group was not stained.Conclusion:（1）The overexpression of MAFA and PDX1 can promote the differentiation of Hu MSCs into IPCs.（2）The combination of MAFA-PDX1 gene modification and drug induction is superior to the single gene modification in inducing differentiation of Hu MSCs into IPCs,and we conclude that the former scheme has the potential to differentiate into IPCs.